ELSEVIER

Food Research International, Vol. 29, Nos 34, pp. 265-290, 1996 Copyright 0 1996 Canadian Institute of Food Science and Technology

Published by Elsevier Science Ltd Printed in Great Britain

PII: SO963-9969(96)00028-Z 0963-9969/96 $15.00 + 0.00

Modern analyses and binding studies of flavour volatiles with particular reference to dairy

protein products

R. J. Stevenson,” X. D. Chen”* & 0. E. Mills6

aFood Science and Process Engineering Group, Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealand

hSensory Science Section, New Zealand Dairy Research Institute, Palmerston North, New Zealand

Various techniques are used to separate and isolate mixtures of volatile flavour compounds from sample matrices. These include headspace sampling (static and dynamic), distillation followed by liquid-liquid extraction, simultaneous distilla- tion-extraction, solid-phase extraction and new methods of extraction such as solid-phase microextraction and membrane-based systems.

After clean-up and concentration, most mixtures of volatiles are separated and analysed in gas chromatographs using open-tubular columns. Gas chromato- graphic injection techniques, columns and detectors are discussed. Mass spectro- metry coupled with gas chromatography is a major method used to identify volatile flavour compounds.

Establishment of the real flavour profiles perceived by humans is discussed with reference to some appropriate experiments.

Volatiles can be lost from foodstuffs by oxidation, polymerisation, reactions with other components in the foodstuff and evaporation. Effective binding of flavour constituents is important during storage and transportation of foodstuffs. On the other hand, the release of off-flavours from a foodstuff is desirable before consumption or formulation with other foodstuffs. Binding experiments dis- cussed include those in liquid systems, desiccation experiments with dry foods and microencapsulation. Copyright 0 1996 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd

Keywords: flavour volatiles, dairy proteins, analyses, flavour release, binding.

INTRODUCTION

The dairy industry handles large amounts of whey pro- tein powders such as whey protein concentrate powder (WPC; Morr, 1976; Kinsella & Whitehead, 1989; Morr & Foegeding, 1990; Morr & Ha, 1993) during either casein or cheese whey processing. Due to its low moist- ure content (about 5%) and reasonable preservation at low temperatures (Ferretti & Flanagan, 1971a; Walker, 1977; Morr, 1979) it is used as a water binding, foaming or gelling agent in a variety of other food formulations (Morr, 1979). Certain undesired basic flavours or off- flavours (Badings & Neeter, 1980; Morr & Ha, 1991) in WPC could be carried over to the final powdered pro-

duct and, therefore, WPC can have a detrimental impact on the flavour of food it is mixed with in high quantities. A blander flavour of WPC would find greater acceptance in a wider range of other combined foods. WPC powder typically contains about 80% by weight protein, 2-5% residual lactose and 3-7% lipid materials that may be susceptible to reactions respon- sible for the staleness and the off-flavours.

Some authors have already published reviews of dif- ferent aspects of the problems encountered with dairy flavours. For example, Maarse (1993) has outlined methods of analysing taints and off-flavours in general, while Jeon (1993) has reviewed undesirable flavours in dairy products; Morr & Ha (1991) have reviewed the off-flavours found in WPC.

*To whom correspondence should be addressed. Although some groups (Ferretti & Flanagan, 197141;

265

266 R. J. Stevenson, X. D. Chen, 0. E. Mills

Walker, 1977; McGugan et al., 1979; Mills & Solms, 1984; Mills, 1986; Laye et al., 1995a,b) have attempted to ascertain some of the volatile compounds (several hundred different volatile compounds exist in dairy products) responsible for the typical off-flavour in WPC, there remains much work to be done in this area.

compounds from the sample matrix, they all may be divided into two general classes:

1. Analyses of the gas volume on top of a sample (headspace techniques)

2. Classical distillation and extraction techniques

The aim of this review is to outline the modern methods in analytical flavour research (Acme, 1993; Maarse, 1993; Mussinan, 1993; Anon., 1994), which, when applied to the volatiles of dairy protein products, might shed some light on the important flavour/off-flavour constituents.

It has been found that a combination of different methods (Klein et al., 1990; Barbieri et al., 1994; Careri et al., 1994; Xanthopoulos et al., 1994) is usually required to completely isolate and characterise the fla- vour-impact constituents of a sample (either in liquid form or solid form).

REVIEW OF METHODS The above two classes will be reviewed in the follow-

ing content.

Flavour is an important part of food quality, improved quality is the goal of food research and flavour is caused by chemicals in food. What are these flavour chemicals and how do they work? The first part of this question may be answered by chemists and the second part by sensory scientists.

Headspace analysis

Since the first attempts at isolating and characterising flavour compounds in foods and drinks in the second half of the last century, analytical flavour research has made giant strides (due to technological developments in other areas such as computers) in the direction of improved sensitivity and selectivity to arrive at the pre- sent highly sensitive methods. Examples of modern fla- vour analysis are coffee with 800 and cooked beef with 670 identified volatile constituents. These leaps forward have also been happening in dairy research. In a recent investigation by Shiratsuchi et al. (1994a), 187 volatile constituents were identified in spray-dried skim milk powder. These figures indicate the dilemma of present day flavour research; the flood of constituents that have been identified (500 in the 1960s and now more than 6000), or can be identified, is increasing all the time, whereas establishing their importance to the product flavour simply cannot keep pace with it. It is no longer the sole aim of flavour research to identify as many individual substances as possible. Methods are more and more frequently being introduced that permit the flavour-impact of the individual compound discovered to be assessed and, thus, steer flavour research towards the most significant constituents.

Sample injection in gas chromatography (GC) at first seems deceptively simple: a microlitre aliquot is quickly injected into an inlet system and elution and detection follow. For samples such as WPC containing significant amounts of nonvolatile material, one or more sample preparation steps are required in order to separate volatile analytes from non-volatiles that would otherwise contaminate the inlet system and column, eventually leading to reduced chromatographic performance.

\ I Cu chromrtognphy with

W.TCD,ECD,FPD,NPD, MS,IR

Although each project has its own problems and requires careful adaption of experimental details, the funda- mental steps may be summarised as shown in Figure 1.

As previously stated, current research in flavour ana- lysis is almost exclusively concerned with the volatile constituents of the sample matrix. Thus, it is considered that compounds with perceptible odours (volatiles) shape the flavour of a food, whereas the nonvolatiles are responsible for the sweet, salty, sour, bitter or umami (Fuke & Shimizu, 1993) tastes.

I Preparative bolatlon of unkown umtytu for NMR and IR

I

Although there are a considerable number of methods (Majors, 1991, 1992) available for isolating volatile

Fig. 1. The steps in flavour analysis. FID: flame ionisation detector; TCD: thermal conductivity detector; ECD: electron capture detector; FPD: flame photometric detector; NPD: alkali flame ionisation detector; MS: mass spectrometry; IR:

infrared; NMR: nuclear magnetic resonance.

Analyses and binding studies of j?avour volatiles 267

Headspace sampling (Shaw & Wilson, 1982; Hin- shaw, 1990b; Ettre & Kolb, 1991; Vittenberg, 1991; Xanthopoulos et al., 1994) of the vapour above a food product may provide a convenient way around the pre- separation requirements for GC analyses. The foodstuff (liquid or solid) is placed in a sealed container and sometimes heated to a predetermined temperature for a period of time. Volatiles begin to partition between the gas and sample phases in the container. The tempera- ture of the sample container is held for a sufficiently long period to bring the gas-phase and sample-phase analyte concentrates into equilibrium. Thus, the tech- nique is known as equilibrium headspace sampling or static headspace sampling. This method is simple (i.e. draw up to 10 ml of headspace gas into a syringe and inject it into the injection port of a gas chroma- tograph), rapid and only samples what the nose perceives. However, it has the disadvantages of being low in sensitivity, so that it can only determine the highly volatile principal components. Furthermore, milk products are generally bland compared with some other foods due to a lower concentration of flavour producing compounds. Although raising the tem- perature is one way to enhance headspace sampling sensitivity, thermal reactions are known to occur in milk products at elevated temperatures (greater than 60°C for instance). While the use of splitless (Grob & Brem, 1992; Grob & De Martin, 1992; Grob & Frohlich, 1992, 1993) or on-column (McCalley, 1989; Oeggerli & Heinzle, 1994) headspace techniques may improve the sensitivity of the headspace methods, the technique is sometimes not adequate for the analysis of trace volatiles in food. Other disadvantages include possible condensation of volatiles inside the sampling syringe, sorption of volatiles on the septum of the con- tainer, inconsistency of injection sizes and relating headspace volatile concentration in the food itself to the concentration in the headspace. Despite the problems and disadvantages of this method, static headspace sampling has been used frequently to analyse the vola- tile compounds from various milk products (Marsili, 1981; Degorce-Dumas et al., 1986; Ulberth, 1991; Christensen & Reineccius, 1992; Xanthopoulos et al., 1994).

Although static headspace sampling is not an exhaustive extraction technique, quantitative determi- nation of volatiles in air, water and soil has been carried out using multiple headspace extraction GC (Kolb, 1982, 1995; Maggio et al., 1991).

To be able to register trace constituents, an enhance- ment technique must precede the GC analysis. An inert gas is swept over or through the thermostated sample for a period of time sufficient to extract most or all of the volatile components. Because the total extraction gas volume is usually too large for direct injection, a trap is required to concentrate the volatiles into a nar- row band. Hence, this technique is known as purge-and-

trap or dynamic headspace sampling (Hinshaw, 19896, 1990b; Vittenberg, 1991).

The most common methods of isolating and con- centrating volatiles by this method are:

Cryogenic trapping Adsorption on a desorbable sorbent bed On-column vapour traps (cryofocusing; Zlatkis et al., 1973) Whole-column cryotrapping (Pankow, 1987; Pankow & Rosen, 1988; Vercellotti et al., 1988; Buttery et al., 1989; Pankow, 1991; Rechart et al., 1991; Barcarolo et al., 1992; Janicki et al., 1993)

The solutes trapped in different ways, after liberation from the sample matrix, are separated on a column and detected. A variety of trap designs for a range of appli- cations are presented in the literature (Young, 1981; Badings et al., 1985; Mehran et al., 1990; Rivier et al., 1990; Imhof & Bosset, 1991; Jursik ef al., 1991; Kohno & Kuwata, 1991; Oguri et al., 1991; Ishihara & Honma, 1992; Morales et al., 1994; Salinas et al., 1994). Parti- cularly relevant to our dairy product research are a number of reports dealing with milk products (Liardon et al., 1982; Wellnitz-Ruen et al., 1982; Mills, 1986; Urbach, 1990; Cormier et al., 1991; Park & Goins, 1992; Careri et al., 1994; Imhof & Bosset, 19946; Xanthopou- 10s et al., 1994; Arora et al., 1995) where purge-and-trap dynamic headspace GC has been used to analyse the volatile constituents. Cryogenic trapping (Chang et al., 1977; Pankow, 1991) using, for example, liquid nitrogen or dry ice/acetone, involves passing the headspace or purge gas through a series of cold traps resulting in volatile components condensing from the purge gas. Wood et al. (1994) have developed a method of analysis of the volatile components from cheese which reflects the natural profile of its aroma. This involved an effec- tive sample collection technique to trap the volatiles, using liquid nitrogen, prior to GC analysis and mass spectrometry (MS) identification. Unfortunately, water is the most abundant volatile in most foods and, there- fore, the trap condensate is primarily water. Thus, an additional step is usually necessary to extract the vola- tile components with diethyl ether, dichloromethane or some other organic solvent from the water. The solvent extract is then dried (with anhydrous magnesium sulfate or sodium sulfate) and concentrated for GC analysis. Concentration is usually performed with the aid of a Kuderna-Danish apparatus or spinning band distilla- tion setup. Disadvantages include the long sampling time giving opportunity for sample changes during fla- vour isolation, the possibility of solvent impurities dur- ing solvent extraction (artifacts), the efficiency of extraction (uniformity) and flavour losses during solvent concentration. Badings et al. (1985) have developed an on-line system including a pretrap which eliminates the majority of water from the purge gas.

268 R. J. Stevenson, X. D. Chen, 0. E. Mills

The trapping of volatiles on an adsorbent that has minimal affinity for water eliminates the need for sol- vent extraction and associated problems. Activated car- bon was one of the earlier materials used for the trapping of volatiles from headspace vapours (Paillard, 1965; Jennings & Nursten, 1967; Tang & Jennings, 1967; Grob, 1973; Chang et al., 1977; Schaefer, 1981). Although carbon has a large adsorbent capacity (Jennings & Nursten, 1967), disadvantages include arti- fact formation (Palamand et al., 1968; Baigrie et al., 1984) during thermal desorption. The use of microwave desorption (Reineccius & Liardon, 1985; Toulemond & Beauverd, 1985; Liardon & Spadone, 1986; Klein et al., 1990) has seen an increase in the use of carbon traps as the rapid desorption alleviates the need for cryofocusing of the desorbed volatiles.

Synthetic porous polymers (Young, 1981; Mehran et al., 1990; Oguri et al., 1991; Rechart et al., 1991; Ishi- hara & Honma, 1992; Morales et al., 1994; Ndiege et al., 1994) such as Tenax GC, Tenax TA, Tenax GR, Porapak Q, Chromosorb 105 and Carbopak-B/Carbo- sieve, Amberlite resins such as XAD-2, XAD-7 and XAD-9, and reverse-phase Cis-bonded silica are now more commonly used as adsorbents for the trapping of volatile constituents from headspace vapours. A num- ber of reports (Withycome et al., 1978; Olafsdottir et al., 1985; Mehran et al., 1990; Imhof & Bosset, 1991; Mar- inichev et al., 1992; Cao & Hewitt, 1993; Contarini & Leardi, 1994) deal with comparative trapping abilities or optimum performance (Vellejo-Cordoba & Nakai, 1993, 1994a,b) of some of these adsorbents.

Mills (1986) described the evaluation of some of the important parameters affecting the trapping of a head- space including time, nitrogen flowrate through the trapping apparatus and type of porous polymer used as an adsorbent. Ten volatile compounds were identified in the headspace of a lactic WPC solution.

Laye et al. (1995a) have analysed accelerated storage (8 days/55”C) commercial WPC by dynamic headspace using four different adsorbent traps: Tenax TA, Tekmar No. 8, Vocarb 3000 and Vocarb 4000. Nine classes and 33 individual volatile compounds were recovered. Tenax TA was most effective for recovering esters, aliphatic hydrocarbons and aromatic hydrocarbons, while Vocarb 3000 was most effective for recovering alcohols, ketones and furans.

Lee & Morr (1994) compared changes of headspace volatile compounds produced by oxidation in spray- dried whole milk powder (WMP; 26% milkfat), non-fat milk powder (NFMP; 0.8% milkfat), potassium case- inate (PC; (1% milkfat), WPC (7% milkfat), whey protein isolate (WPI; < 1% milkfat) and sweet whey powder (WP; 1% milkfat) by dynamic headspace ana- lysis (Tenax TA adsorbent trap). Volatile compounds expected to arise from milkfat oxidation included alde- hydes and ketones: 2-methyl propanal, butanal, 2- methyl butanal, 3-methyl butanal, hexanal, heptanal,

octanal, nonanal, 2-butanone, 2-heptanone, 2-octanone and 2-nonanone. Esters that were identified included: acetic acid, ethyl ester; ethanethioic acid, S-methyl ester; and thiocyanic acid, methyl ester. Acids and lactones were not detected in the headspace of the stored, dried dairy products.

Monnet et al. (1994) have used automatic headspace GC to assay volatile compounds such as diacetyl and acetoin in fermented milks.

Hall et al. (1985) used both static headspace and dynamic headspace sampling to monitor the flavour changes in WMP during storage. It was found that sta- tic headspace was more suitable for trapping the most volatile compounds while dynamic headspace trapped the less volatile compounds more effectively.

A disadvantage of the purge-and-trap technique is the purging of significant amounts of water vapour along with the analytes. Problems include decrease of the adsorption capacity of the sorbent used for the con- centration of volatiles owing to co-adsorption of water, condensation of water along with the analytes on the walls of the tubes connecting the purge device with the sorbent trap or GC injection port, plugging of traps and GC columns at sub-zero temperatures, degradation of the performance and retention gaps of GC columns, and variations of retention times and responses of the compounds that elute near water. A number of proce- dures including using a hydrophobic sorbent such as Tenax, the use of a desiccant, heating the connection tubes, removal of water by molecular sieves or silica gel, removal of water by passing the moist streams through a short column of glass beads at - 1 O”C, have been used to avoid these adverse effects. A recent method (Janicki et al., 1993) involves the permeation removal of water by using a membrane tube. It was found that water was quantitatively removed from the humidified gas stream and that the analyte compounds were virtually unaffected.

A problem with both adsorbents and cold traps is diffusion or ‘breakthrough’ losses due to substances not retained by the trap. Some workers (Vercellotti et al., 1988; Buttery et al., 1989) have obtained better recovery of volatiles by stripping them from foods under vacuum at lower temperatures. Barcarolo et al. (1992) and Barcarolo & Casson (1995) have developed a technique for performing headspace GC-MS analysis of volatile components of food in which the carrier gas flow is reversed during sampling in order to overcome pro- blems caused by the diffusion of volatiles not retained by a cold trap.

A variation of inert gas stripping called spray vapor- isation (Chriswell, 1977) is relatively simple and is claimed to lead to a significant increase in the con- centration of volatile organic compounds. Water is ato- mised into a high-velocity gas stream using a nebuliser of the type used on perfume bottles and throat sprayers. The very fine water mist which is produced is directed to

Analyses and binding studies ofJavour volatiles 269

impact on a glass surface; there it condenses and coa- lesces on impact and drains into a reservoir. GC volatile organics in the water are carried from the system in the gas stream.

Vercellotti et al. (1992) found that volatiles analysis, by direct GC and olfactory ‘sniffer port’, was improved by an external closed inlet device (ECID) (Legendre et al., 1978) with a wide bore capillary column as both trap and separation medium. A study was made of recovery of standards either directly from vegetable oil or from Tenax GC or Carbopack B/Carboseive SIII. Roasted peanuts were analysed for volatiles by direct GC from the ECID and ‘aromagrams’ were generated.

Recently, Zhang & Phillips (1995) have developed a multiplex GC technique for determining organics in solid samples such as cigarettes, soil and plastic. Direct headspace sampling, which is carried out in a sampler modified from an injector, provides a continuous gas stream for multiplex GC. Volatiles evaporating from the sample are picked up and carried by a carrier gas to a thermal desorption modulator and column. The mod- ulator is simple and effective and is used as both a trap and desorption device. It is possible to determine vola- tiles in solid samples where the solid material is neither volatile nor soluble in a solvent.

Dalla Rosa et al. (1994) have investigated the influ- ence of water content and water activity on the con- centration of volatile compounds in the headspace over starch-water model systems, hard cheese, apple puree and raw ham. For solid foods at intermediate water activity values, the highest volatile concentration in the headspace strongly depended on the availability of water. The headspace volatile concentration decreased rapidly when the water vapour became totally free and the water vapour pressure was close to that of pure water.

Although there are problems using headspace adsorption techniques in volatile flavour analysis, the method is commonly used both in qualitative and quantitative flavour studies.

Voice & Kolb (1994) have compared European and American techniques for the analyses of volatile organic compounds in environmental matrices. They concluded that static headspace analysis is more firmly established in Europe, whereas purge-and-trap is prevalent in the United States.

Most methods for determining volatile profiles in food are based on the analysis of ‘whole foods’, whether by headspace or solvent extraction. While this indicates the profile when food is smelled prior to consumption, the physicofhemical environment may change when food is eaten, and this may affect the volatile profile and perception of aroma. Release of volatiles in the mouth is important in determining the profile perceived by receptors in the nasal cavity and, thus, relates directly to our perception of aroma when food is eaten. Linforth & Taylor (1993), Linforth et al. (1994) and Ingham et al. (1995a,b) have shown that volatile profiles during eating

can be measured by trapping the volatiles from ‘nose- space’ on Tenax traps, followed by desorption and GC- MS analysis. Operators had a soft plastic tube fitted to their nostrils and sampling was effected with the trap inserted through the plastic tube at right angles so that the tip of the trap was in the stream of expired air from the nostrils, whilst either tomatoes or mints were eaten. Air was drawn through the trap by a vacuum pump. Headspace and nosespace profiles were found to differ for both these foodstuffs examined.

Using a similar apparatus to Linforth & Taylor (1993), Linforth et al. (1994), Ingham et af. (1995a,b) and Delahunty et al. (1994) have compared direct headspace volatiles with those released from the mouth for both full-fat and low-fat Cheddar cheeses. GC-MS profiles showed significant differences between full- and low-fat cheeses and between those taken in vitro and from the mouth during mastication.

Van Ruth et al. (1994, 1995a,b) studied flavour release from three rehydrated vegetables (French beans, red bell peppers and leeks) directly in the mouth of 12 assessors (oral vapour) and in 3 mouth-model systems: purge-and-trap and dynamic headspace with and without mastication. Flavour release from the three vegetables in the ‘dynamic headspace and mastication’ model system did not differ significantly from release in the mouth.

Roberts & Acree (1995) have investigated the effects of saliva, temperature, shearing and oil on flavour release with a retronasal aroma simulator using a modified headspace technique on model systems. The device produced different GC-MS profiles than those of dynamic headspace trapping and it should permit foods with many different physical forms to be analysed.

Classical methods of distillation and extraction

For a compound to make a contribution to aroma it must be volatile. Thus, distillation is used to take advantage of the volatility of flavour components and nonvolatility of the bulk of a foodstuff. Steam distilla- tion enables the volatile components to steam distil from the food matrix and condense in cold traps (ice/ water, dry ice/acetone or liquid nitrogen). The steam distillate is a very dilute solution of volatile compounds in water. Extraction (discontinuous or continuous) of these volatiles is done with an organic solvent such as diethyl ether, dichloromethane or pentane. The method of extraction depends on the physical state of the food (solid or liquid), quantity of food and extracting solvent (heavier or lighter than water). The simplest method is multiple-batch extraction using a separating funnel (Reineccius et al., 1972). Hussein et al. (1983) have reported analyses performed by extraction of aqueous mouth rinses with dichloromethane, followed by con- centration and GC analysis. Disadvantages of this method include the fact that it is tedious and labour

270 R. J. Stevenson. X. D. Chen, 0. E. Mills

intensive, and emulsion formation leading to poor phase separation is common.

Continuous liquid-liquid extraction (Figure 2) gen- erally provides more efficient volatile extraction from steam distillates. It usually takes 2-4 h and the appara- tus can be constructed to accommodate solvents which are heavier or lighter than water. Correct design enables continuous extraction with a small volume of solvent that is always being distilled for extraction. A dis- advantage is (if the solvent has a relatively high boiling point) the inevitable thermal stress of the organic extract throughout the entire duration of the extraction process; it may lead to noticeable sensory and analytical changes in the aroma isolated.

In a recent investigation by Mills (1993), volatile compounds were extracted from solutions of WPC (25% w/v) at 40°C by vacuum distillation. Compounds in the distillate were transferred into diethyl ether by continuous liquid-liquid extraction. Compounds identi- fied included saturated and unsaturated aldehydes, methyl ketones, alcohols, alkyl pyrazines and saturated fatty acids.

Moio et al. (1993a,b) used vacuum distillation fol- lowed by liquid-liquid extraction to isolate more than

Fig. 2. Continuous liquid-liquid extraction apparatus.

100 volatiles from bovine, ovine, caprine and water buffalo fresh raw milk. About 30 of these components had not been detected previously in milk.

Badings & Neeter (1980) used vacuum distillation, freeze concentration, extraction and micro-concentra- tion to detect more than 300 GC peaks in both low- pasteurised milk and ultrahigh-temperature heated milk.

Jaddou et al. (1978) developed a spray distillation method for studying the flavour volatiles in heat-treated milk. The headspace over milk was enriched in volatile components by spraying milk into a flask using a nozzle and a pressure differential generated by evacuating the flask with a water-pump. This gave a fine dispersion of milk droplets, with a high surface area to volume ratio. The vacuum and the large surface area in the sprayed milk favoured rapid release of milk volatiles. Nitrogen gas swept the volatiles out of the flask via a condenser (O’C) and into a liquid nitrogen cooled trap. It was considered that this method should minimise the pickup of water, give a true representation of the composition of the headspace over milk and yet avoid the production or introduction of spurious substances.

Sugisawa et al. (1984) have used steam distillation in combination with a Tenax or charcoal trap (distillation- adsorption) to recover volatiles, while Ulberth & Rou- bicek (1995) have used a steam distillation-GC method to monitor oxidative deterioration of milk powder. In a variation of the distillation-extraction method, Umano & Shibamoto (1987) purged a sample with an inert gas and the volatiles were directed into a water trap that was being continuously extracted by a refluxing solvent. Valcarcel et al. (1994) have proposed the use of a GC and a continuous sample pretreatment module based on liquid-liquid extraction for automatic conditioning of samples (pre-concentration, dilution, derivatisation, solvent changeover).

A popular method of isolating volatiles via distilla- tion is to use a simultaneous distillation-extraction @DE) method with a Likens-Nickerson apparatus (Likens & Nikerson, 1964). Steam (containing volatiles) from the aqueous food homogenate and the extracting solvent vapour (either heavier or lighter than water) mix intimately, allowing highly efficient volatile extraction. Several modifications to the original apparatus have appeared in the literature (Picardi & Issenberg, 1973; MacLeod 8z Cave, 1975; Flath & Forrey, 1977; Shultz et al., 1977; Au-Yeung & MacLeod, 1981; Godefroot et al., 1981, 1982; Maignial et al., 1992; Blanch et al., 1993; Seidel & Linder, 1993; Jayatilaka et al., 1995). Some of these are suitable for vacuum operation (Figure 3), thus, allowing lower distillation temperatures which should reduce the formation of thermally induced artifacts.

Au-Yeung & MacLeod (1981) developed a system whereby lipid samples could be steam distilled and extracted. Barbieri et al. (1994) and Careri et al. (1994) used both SDE and dynamic headspace sampling to

Analyses and binding studies ofjlavour volatiles 271

Fig. 3. Vacuum operation apparatus.

identify 167 volatile compounds of Parmesan cheese. With headspace sampling, the most volatile compounds were obtained, whereas SDE was suitable even for long- chain carbonyl derivatives, acids, esters and lactones, which are considered to be important compounds of the flavour. De Frutos et al. (1991) identified about 70 volatile components from six artisanal cheeses using a micro-SDE procedure followed by GC-MS.

Shiratsuchi et al. (1994a,b) identified 187 volatiles of spray-dried skim milk powder by SDE under reduced pressure (80 mmHg). However, they concluded that levels of responsible flavour compounds in the skim milk powder were very low and their composition extremely complicated. Free fatty acids and lactones present in relatively high level were considered to be fundamental contributors to the skim milk flavour. It was considered that aldehydes, aromatic hydrocarbons and some of the heterocyclic compounds, such as indoles or thiazole, seem to participate indirectly in the skim milk flavours.

Both steam distillationextraction and SDE require concentration of the volatile containing organic extract. Loss of volatiles and introduction of artifacts may occur during this step. Water must be removed prior to con- centration, or steam distillation of the volatiles may

result. Oxidation of the flavour components may occur if long times/high temperatures are used. Use of either a Kuderna-Danish apparatus (Figure 4) or a spinning band fractionating column (Coleman & Ho, 1980; Ho & Coleman, 1980) provides effective solvent removal with minimal loss of volatile flavours.

A disadvantage of steam distillation is the possibility of artifact formation from antifoam agents, vacuum greases on glass joints, contaminated water, solvent impurities, rubber tubing and thermally induced chemi- cal changes.

Although diethyl ether is a very common solvent for flavour extractions, a relatively new method involves extraction with supercritical phases as solvent media (Charpentier et al., 1986; Chen et al., 1986; Moyler & Heath, 1986; Floment et al., 1987; Hawthorne et al., 1988; Moyler, 1988; Magashi et al., 1990; Miles & Quimby, 1990). Very gentle treatment of the sample is possible with this method. The critical temperature of CO2 is 31°C so that extraction can take place at a slightly higher temperature. Concentration occurs by expanding the supercritical extraction solution to stan- dard pressure. The dissolving power of the supercritical phase can be regulated by suitable selection of para- meters such as pressure, temperature and co-solvents.

Fig. 4. A Kudema-Danish apparatus.

272 R. J. Stevenson, X. D. Chen, 0. E. Mills

However, specialised equipment is needed for this method.

Dialysis methods (Benkler & Reineccius, 1979; Chang & Reineccius, 1980) have been used as alternatives to steam distillation-extraction for recovery of volatiles from foods containing lipids.

Solid-phase extraction (SPE; Junk & Richard, 1988; Zief & Kiser, 1990) can be an effective alternative to liquid-liquid extraction, and the time required to isolate the volatiles is much shorter. Disadvantages include high blank values, large variation between products offered by different manufacturers and lot variation. The plastic SPE cartridges can adsorb volatiles and, thus, increase interference in the analysis. Coulibaly & Jeon (1992) compared activated carbon, Cis reversed- phase silica, Florisil and silica gel as solid-phase mate- rials in SPE of the less volatile flavour compounds from ultrahigh-temperature processed milk.

A recent review (International Union of Pure and Applied Chemistry, 1994) outlines SPE using silica bonded phases. The synthesis and types of available sorbents are discussed. Practical details of performing SPE are given and method development is considered, with particular attention to selectivity.

New methods of extraction

Most of the research in separation science is concerned with the improvement of derivatisation techniques and injection methods, the preparation of stationary phases, the proper selection of mobile phases, the better under- standing of coating and packing procedures for the appropriate chromatographic column, the application and technical advancement of detectors, the construc- tion of elaborate interface designs for coupling with chromatographic methods to spectroscopic instruments or the mathematical development to process the output of detectors. Many sample preparation practices are based on nineteenth century technology. For example, the commonly used Soxhlet extraction was developed in 1879. Environmental awareness has resulted in interna- tional initiatives to limit the use of organic solvents. An ideal new sample preparation technique should be sol- vent-free, simple, inexpensive, efficient, selective and compatible with a range of separation methods and applications (Pawliszyn, 1995). It should be able to be used simultaneously to separate and concentrate the analytes.

Majors (1995) has outlined four techniques that may be on the verge of widespread acceptance: microwave- assisted extraction, disk-solid extraction, solid-phase microextraction and restricted-access media separation.

A relatively new technique known as solid-phase microextraction (SPME; Belardi & Pawliszyn, 1989; Arthur & Pawliszyn, 1990; Arthur et al., 1992a,b,c,d; Hawthorne et al., 1992; Louch et al., 1992; Potter & Pawliszyn, 1992; Arthur et al., 1993; Buchholz &

Pawliszyn, 1993; Motlagh & Pawliszyn, 1993; Otu & Pawliszyn, 1993; Page & Lacroix, 1993; Zhang & Paw- liszyn, 1993a,b; Boyd-Boland et al., 1994; Buchholz & Pawliszyn, 1994; Homg 8z Huang, 1994; Popp et al., 1994; Potter & Pawliszyn, 1994; Wan et al., 1994; Yang & Peppard, 1994; Zhang et al., 1994; Barnabas et al., 1995; Boyd-Boland & Pawliszyn, 1995; Chen & Pawlis- zyn, 1995; Gorecki & Pawliszyn, 1995; Mindrup, 1995; Wittkamp & Tilotta, 1995; Yong & Bayona, 1995; Zhang & Pawliszyn, 1995) eliminates some of the drawbacks in an aqueous sample. SPME involves extraction onto a chemically modified fused-silica opti- cal fibre which is then thermally desorbed in a GC injection port. An SPME unit consists of a length of fused-silica, coated with a stationary phase such as polydimethylsiloxane, polyimide, polyacrylate, Carbowax- divinylbenzene, polydimethylsiloxane-divinylbenzene or pencil lead (Wan et al., 1994) and bonded to a stainless steel plunger, and a holder which looks like a futuristic microlitre syringe. Solvents are eliminated, blanks are reduced and extraction time can be reduced to a few minutes. The device can be used to concentrate volatile and semi-volatile compounds in either liquid, gaseous or solid matrices, where the analytes partition between the sample matrices and the stationary phase until equili- brium is reached. It provides linear results over a wide range of analyte concentrations and can be used with any GC or GC-MS system. The fibres can be cleaned and reused many times. Detection limits of the head- space SPME technique can be at parts per trillion level when an ion-trap mass spectrometer is used as the detector (Zhang & Pawliszyn, 19936). SPME units (both for manual and automated use) are now available from Supelco (Bellefonte, PA; Figure 5). A recent review (Zhang et al., 1994) outlines the possibilities of SPME.

Although this technique has been studied mainly for analysis of pollutants in environmental water samples, SPME is also a potentially useful method for flavour analysis. Hawthorne et al. (1992) successfully applied SPME with an uncoated fused-silica fibre for determi- nation of caffeine in beverages.

Page & Lacroix (1993) studied the application of SPME to the headspace GC analysis of halogenated volatiles in selected beverages and finely divided dry foods. Their work included study of the headspace responses for milk samples with varying concentrations of butter ‘fat. They concluded that when headspace SPME is applied to foods, increase in the lipid material markedly reduces the method sensitivity, the decrease being greatest for analytes of least volatility. Standard addition is required for quantitation of foods. In water, the much greater response for the less volatile analytes than those of greater volatility compliments headspace GC with gas sampling.

Yang & Peppard (1994) examined liquid and head- space SPME sampling in a test solution containing 25

Analyses and binding studies ofJIavour volatiles 273

Plunger retaining screw y

Hub-viewing window .

Fibre attachment

z slot

Adjustable needle guide/depth

, gauge

Septum piercing needle

needle c Coated SPME fused-silica tlbre

Fig. 5. Solid-phase microextraction device produced by Supelco (Bellefonte, PA).

common flavour components and applied this technique to the analysis of authentic food, beverage and flavour samples. These included espresso-roast ground coffee, fruit juice beverage and butter flavour in vegetable oil. They concluded that SPME is a useful tool for flavour analysis and can be considered as complimentary to commonly used methods such as solvent extraction, SDE, conventional SPE and purge-and-trap sampling.

Unlike these techniques, in which the quantitative recovery of analytes is necessary for quantitation, SPME sampling is a single-batch process, so that quan- titative adsorption is often very difficult, if not impos- sible. Any changes in experimental conditions that affect the adsorption distribution will be reflected in the sen- sitivity and reproducibility of the analytical method. An external calibration method for SPME generally is not suitable for quantitation, because a synthetic matrix can hardly match that of an authentic sample. Although the matrix effect could be significantly reduced by diluting samples and saturating with salt, it is not always applicable.

Recently, however, Zhang & Pawliszyn (1995) have developed an extraction system where the sample matrix

is heated while the fibre coating is simultaneously cooled with liquid CO*. This not only facilitates the mass transfer and release of analytes into the headspace, but also creates a temperature gap between the cold fibre coating and the hot headspace which significantly increases the partition coefficient of the analytes. With this method, quantitative extraction was achieved for toluene, ethylbenzene and xylene isomers from gas, water, or soil in less than 5 min.

For SPME/GC analysis, cryogenic focusing is often used to improve peak resolution. Yang & Peppard (1994) found that the internal diameter (i.d.) of the GC injection liner can influence the peak width, especially for early eluting compounds. Instead of a conventional split/splitless injector with a 3.5 mm i.d. liner, a pro- grammable temperature vaporising injector with a 1 mm i.d. liner was used and the GC resolution improved markedly. These workers also studied salt effect, the influence of sample volume and compared liquid and headspace sampling. SPME sensitivity chan- ged significantly with increasing salt concentration and gave rise to different types of behaviour with different compounds. The amount adsorbed on the SPME fibre is dependent not only on the initial sample concentration but also on the sample volume. With increasing sample volume/headspace volume, the extent of SPME adsorp- tion increased rapidly initially (up to 1 ml) and then remained relatively constant (l-3 ml). If the property change of the adsorption surface in gas and liquid pha- ses can be ignored, the adsorbed amount of an analyte on the SPME fibre is thermodynamically independent of the sampling method, because at equilibrium the che- mical potential of a compound in the three phases is the same. However, liquid and headspace samplings do dif- fer in kinetics. Differences in the rates of evaporation, dissolution and diffusion in gas and liquid phases, and the concentration difference between liquid phase and its headspace can make SPME liquid and headspace samplings very different. In general, the adsorption rate is higher when the concentration of analyte is higher. If an analyte exists predominantly in the liquid phase, a SPME liquid sampling method is more sensitive for a given sampling time and vice versa. As expected, it was found that the extent of SPME adsorption decreased with increasing headspace volume, the amount of liquid phase being kept constant.

Louch et al. (1992) have studied the dynamics of organic compound extraction from water using liquid- coated fused-silica fibres. Mathematical descriptions of the adsorption and desorption processes were developed and compared with experimental results. One model assumes a perfectly agitated solution which resulted in extraction times dependent only on diffusion of analyte in the coating. The second model considered extraction from a static solution, In this case, extraction times were determined by diffusion of analyte in water. Results indicate that when standard stirring equipment is used

274 R. J. Stevenson, X. D. Chen, 0. E. Mills

as a means of agitation, the dynamics of the extraction process is controlled by diffusion of analyte through the thin static aqueous layer located around the fibre. The mass-transfer limitation caused by the diffusion of ana- lyte in the aqueous phase may be overcome by sampling volatile analytes from the headspace above the aqueous sample since diffusion coefficients of molecules in the gas phase are four orders of magnitude higher than in water. Headspace extraction can be improved by ensur- ing rapid mass-transfer between the aqueous and vapour phases. The use of a spray system may help in this regard. Sonication (using a laboratory glassware cleaner) improved mass transport compared with the unstirred case but is still not as efficient as stirring at the maximum rate. The sensitivity of the method is depen- dent on the coating volume and the coating/water dis- tribution constant, the linear range is over several orders of magnitude and the relative precision is a few percent and is dependent on the thickness of the coating.

Membrane-based systems (Van De Merbel et al., 1993; Boyd-Boland et al., 1994) have also been used for sample preparation prior to chromatographic analysis. Recently, an analytical method combining the hollow fibre membrane, cryofocusing and thermal desorption technologies has been developed (Pratt & Pawliszyn, 1992a,b; Yang & Pawliszyn, 1993; Yang et al., 1994a,b). It is claimed that this method of membrane extraction with a sorbent interface is simple, effective, solvent-free and easy to automate.

Xu & Mitra (1994) have developed a method for continuous monitoring of volatile organic compounds in water, by passing the aqueous sample through a hol- low fibre membrane. The volatile organics selectively migrate across the membrane into an inert gas stream, followed by trapping and concentration on a microtrap and then desorption by an electrically generated tem- perature pulse.

GC analysis

Currently; when sample extracts have been cleaned up and concentrated, they are almost exclusively analysed in gas chromatographs (Eiceman et al., 1992; McNair, 1993) using open-tubular (capillary; Ettre, 1992) col- umns with chemically linked separation phases. GC is most suitable for flavour studies since it has excellent separating powers and extreme sensitivity. These quali- ties are essential for analysis of complex flavour extracts containing hundreds of volatiles. Capillary columns provide about 3000 theoretical plates per metre whereas a good packed column will provide about 2500. How- ever, a packed column is limited to about 7 m in length while a capillary column may be up to 100 m long. Thus, capillary columns give vastly improved resolution within the same time frame.

Whole column cryogenics, cryogenic traps or on-col- umn injections are often used in conjunction with

capillary columns to further enhance resolution, espe- cially for the lower boiling point volatiles.

A number of GC cryo-trap devices are available commercially. Manura (1994) has shown one such device, cooled down to -70°C with liquid COz, to be useful for the analysis of both volatiles and semi-vola- tiles via either headspace GC introduction techniques or purge-and-trap thermal desorption techniques: the ana- lytes are trapped in a narrow band at the front of the column and subsequently thermally desorbed.

It is generally accepted that the injection process is the weakest point in chromatographic analysis, and there are a number of papers in the literature concerned with injection into open tubular columns (Jennings & Mehran, 1986; Hinshaw, 1989a; Jennings, 1990; Kane, 1993; Poy, 1993; Grob, 1994; Grob & Frohlich, 1994). In a recent review dealing with injection techniques, Grob (1994) states that although capillary GC is more than 30 years old, injection methods have not achieved the degree of reliability needed for routine analysis. He considers that injection has never been optimised with sufficient professionalism, and that without more work in this field, GC is in danger of stagnation and perhaps ultimately of degeneration! Although quantitative results of acceptable accuracy and precision depend mostly on the skill of the operator, the introduction of autosamplers can avoid some human errors. The auto- sampler, however, may not be suitable for all con- ceivable samples. When a liquid sample is injected into a GC vaporising chamber with a microsyringe, the main source of error in this process is the syringe needle. To avoid the possibility of deviation, the literature suggests a number of syringe-handling methods (Poy, 1993): fill- ing needle method, cold needle method, hot needle method, slow injection method, wet needle method, solvent flush method and air plug method.

In trace analysis by capillary GC, it is often desirable to use larger than normal injection volumes to obtain sufficient sensitivity. Yan & Nikelly (1994) have shown that larger injection volumes with little or no loss in column efficiency are permissible if a short pre-column is connected to the front of the analytical column. The pre-column should be coated with a thick film of a liquid phase of polarity dissimilar to that of the column.

The sample-introduction modes most widely used in open-tubular columns are split, splitless and on-column. McCalley (1989) has analysed a mixture of various volatile fatty acids by capillary (polar thick-filmed) GC using on-column injections of aqueous solutions. The programmed temperature vaporiser has also received more attention recently (Jennings & Mehran, 1986).

Capillary columns come in standard commercial lengths of 15, 30, 60 and 105 m and some companies also produce 25 and 50 m lengths. Longer columns generate more plates, but resolution is proportional to the square root of column length, and analysis time is directly proportional to column length for the fixed

Analyses and binding studies of flavour volatiles 215

purge gas flowrate. Commercial i.d.s include 100, 250, 320 and 530 urn. Both 250 and 320 urn i.d. columns are efficient and fast. The 250 urn i.d. column will provide slightly better resolution, but the 320 urn i.d. column, with approximately four-fold greater capacity, will provide slightly better sensitivity by permitting larger sample size.

dynamic headspace GC. In a cheese sample and a test mixture of 37 volatile flavour components, a 30 m x 320 pm i.d. column coated with a 4 urn layer of 100% polydimethylsiloxane was found to be most suitable.

The most important part of the column is the sta- tionary phase (Guthrie & Harland, 1994), a polymeric film, which is coated on the inner wall. Differences in the chemical and physical properties of the injected mixture’s components and their interactions with the stationary phase are the basis for the separation process. How long the analytes are retained in the column (retention time) is a measure of the analyte-stationary phase interaction. A wide variety of stationary phases are commercially available providing specific combina- tions of interaction for different classes of compounds. The general chemical principle that ‘like dissolves like’ can be applied to the stationary phase. Thus, a non- polar column is best for analysis of non-polar com- pounds and vice versa. Interactions between non-polar compounds and non-polar stationary phases are dis- persive so separation is based on the boiling points of the molecules. Dipole and/or acid-base interactions play a part in interactions between polar compounds and polar phases. The nature of the sample component and phase affect sample capacity: nonpolar phases have higher capacities for nonpolar analytes while polar phases have higher capacities for polar analytes. Tem- perature programming can increase the sample capacity of a column, although resolution is sacrificed. Thinner films (0.10-0.25 urn) are more efficient (except for very volatile compounds) and faster, but thicker films (l- 5 urn) have greater capacity and are more inert (due to better coverage of the fused-silica surface) and better for very volatile compounds.

After choosing an efficient column, the most impor- tant operating parameter is column temperature. For isothermal operation, a lower column temperature means better resolution but a longer analysis time. Modern GC systems come with both temperature and pressure programming capabilities. Temperature pro- gramming can be thought of as a series of isothermal steps in which the sample is injected at a low column temperature and remains frozen on the column inlet until an ideal range of column temperature (40-SOC below the elution temperature of the peak) is reached and then the components begin to move down the col- umn. The initial temperature should be low enough to cause all the injected sample to be frozen on the column inlet which means that the programming rate deter- mines the elution temperature. Thus, cryogenics may be required to freeze volatile mixtures. The final tempera- ture should be hot enough to elute all the important analytes during the programmed temperature gradient, but high enough to elute all compounds. The program- ming rate (typically 2-lO”C/min) determines both ana- lysis time and resolution: a higher programming rate (similar to a higher column temperature) results in fas- ter analyses and a loss in resolution. If cryogenic cooling is not possible, holding the temperature until important peaks are eluted may provide the necessary resolution.

Detectors

Grob Jr. & Schilling (1983) developed a solvent effect known as ‘phase soaking’ that occurs in the coated col- umn beyond the flooded inlet section and may influence the shape and the retention of early eluting peaks. Sol- vent is retained by the stationary phase and increases the retention capacity of the system. ‘Retention factors’ for some solutes were determined and they varied between 1 and 10; that is, between no influence of the added solvent vapour and a more than lo-fold slower migration in the soaked than in the pure stationary phase.

A number of detectors (Hinshaw, 1990a; Eiceman et al., 1992; McNair, 1993) are available to give evidence of the substances separated in the gas chromatograph. Flame ionisation detectors (FIDs; Hinshaw, 1990a) respond to all organic compounds except formic acid and formaldehyde. They have great sensitivity, excellent dynamic linearity (10’) and provide good quantitative results. FIDs are simple to operate and robust: they are insensitive to minor changes in flow rate, column tem- perature or trace amounts of oxygen and water. A dis- advantage is that they require three separate gas supplies: helium, nitrogen or argon as carrier gas; hydrogen and air as combustion gases.

The most common source of deteriorating perfor- mance of capillary columns used for the analysis of natural products is the sample itself. Rood (1990) has outlined symptoms of contamination by sample residues, diagnosis of sample-induced fouling and treatment of the problems.

Thermal conductivity detectors are universal detec- tors producing a signal for all molecules. They are easy to use, only one gas (helium or argon) supply is required, they possess no fire hazard, they respond to inorganic compounds and they are non-destructive. Unfortunately, they have limited sensitivity (> 10 ppm analyte), limited dynamic linearity (104) and are not recommended for < 320 urn i.d. capillary compounds.

Imhof 8z Bosset (1994a) have studied the performance of a number of capillary columns for the analysis of

Electron capture detectors (halogenated compounds), flame photometric detectors (for either sulfur- or

volatile flavour compounds in dairy products by phosphorus-compounds) and alkali flame ionisation

R. J. Stevenson, X. D. Chen, 0. E. Mills

detectors (nitrogen- and phosphorus-compounds) are selective detectors that determine only certain mole- cules. These detectors can be difficult to use, easily overloaded, easily contaminated and may require frequent calibration.

Although GC is basically a separation technique, it can be used in many ways for qualitative identifications. By themselves, none of the GC methods give unambig- uous identification of an unknown compound, but combinations play an important role in flavour analysis. Under constant GC conditions, the retention time of an analyte remains constant. Retention times are char- acteristic of analytes but many compounds have the same retention time and, therefore, unambiguous iden- tification is virtually impossible. Use of different sta- tionary phases may solve the problem, but relative retention times are often calculated as they are more reliable than absolute retention times. Relative retention times are obtained by relating the retention time of the unknown compound to that of a standard compound or a series of compounds. The Kovats retention index sys- tem (Kovats, 1958), using n-alkanes as calibration stan- dards, is the most favoured and widely adopted. By definition, the n-alkanes have an index equal to the number of carbon atoms multiplied by 100. Ettre (1973) has given a comprehensive discussion of this method. The original Kovats indices were developed by using isothermal conditions. Polar calibration standards such as l-alcohols (Grobler, 1972; Hawkes, 1973), l-alka- nones, propyl ethers and fatty acid methyl esters (Woodford & Van Gent, 1960; Ackman, 1972) have also been recommended.

These systems are not suitable for prediction of retention index from structure and, conversely, for retrieval of structural information from retention data. Correlations of retention, structure and physicochemi- cal properties have been intensively studied, and Peng (1994) has outlined these in a recent review. Peng has shown that ,the chromatographic identity of a com- pound can be determined by four parameters: Z (Kovats retention index), A (regression coefficient), Z (number of atoms in the molecule) and GRF (group retention factor, which is the intercept when Z of the members of a homologous series is plotted against 2). A linear corre- lation was found to hold for homologous series of acids, alcohols, esters, amines, aromatic hydrocarbons etc., on both non-polar and polar columns (Peng et al., 1988, 1991). Thus:

Z = AZ + (GRF),

is the basis for predicting retention index from structure and, conversely, for retrieving structural information of mono-functional compounds from retention data. Farkas et al. (1994) have attempted to create a highly repro- ducible GC retention indices library as an extension of the TN0 database of volatile compounds in food. They used a procedure for the adjustment of operational

conditions on two apolar and one polar capillary columns, enabling high reproducibility of standard relative retention indices.

McReynolds constants (McReynolds, 1966) were developed by using temperature programming and, thus, they are more suitable for complex-mixture matrices such as foodstuffs. Jennings & Shibamoto (1980) have listed McReynolds constants for some common flavour and fragrance compounds. A similar system based on the retention time of the unknown analyte relative to a series of ethyl esters of aliphatic acids has been used by Mussinan (1993).

Recently, two theoretical procedures (Al-Bajjari et al., 1994~) were described for the calculation of peak widths in programmed temperature gas chromatographs from corresponding isothermal widths. One of these methods was adapted to the analogous calculation of peak asymmetries (Al-Bajjari et al., 19943). Comparison with experimental data was satisfactory. Composite predic- tions of retention times, widths and asymmetries were then used to predict (a) the forms of complete chroma- tograms comprising close peaks, (b) resolution char- acteristics of such chromatograms and (c) optimum programmed temperature conditions.

Meyer (1995u,b) has investigated quantitation by peak area or height for small peaks which are accom- panied by a large neighbour of 100 or 1000 times greater size. By varying peak order, relative peak width, tailing and resolution, it was found that the size of small peaks can be determined accurately if they precede the large one and if their height is used for quantitation. Under these conditions, even poor resolution and tailing can be tolerated. If the small peak is eluted after the large one, the only recommendation is to achieve true baseline resolution if possible. However, if the peaks are truly Gaussian, the elution order is of minor importance.

Recently, Bicchi et al. (1994) have used a combination of retention indices and specific multidetection respon- ses to identify the constituents of a complex mixture.

MS coupled with GC is a major method used to identify volatile flavour compounds (Cronin & Caplan, 1987). Complex mixtures result in complex spectra which make interpretation extremely difficult. Hence, coupling of a GC (the separation method) to an MS is a logical step and permits a mass spectrum on each com- ponent of a mixture to be obtained as that component elutes from the GC.

The advent of the quadrupole mass spectrometer (Finnigan, 1994) helped to establish MS as an impor- tant technique for organic analysis. Many believe that MS, including hyphenated techniques such as GC-MS will become the largest segment of the analytical instru- ment market by the year 2000. A recent review (Wach, 1994) compares the features of nine currently available benchtop GCMS instruments.

The mass spectrum alone of a flavour compound may be sufficient for identification, especially if the GC

Analyses and binding studies offlavour volatiles 277

retention index is known. If the mass spectrum of the analyte does not match any of the library spectra, or isomers are indistinguishable, infrared (IR) and/or nuclear magnetic resonance spectra may be required. However, both these techniques require much larger quantities of sample than does MS. Casabianca et al. (1995) have outlined the application of hyphenated techniques to the chromatographic authentication of flavours in foods and perfumes.

If possible, the attribution of an unknown GC-MS chromatographic peak to a specific substance should be confirmed by comparison with a standard compound analysed under the same experimental conditions. Imhof & Bosset (1994a) applied a standard addition method with purge-and-trap GC-MS to quantitatively analyse volatile flavour compounds in pasteurised milk and fermented milk products. A total of 33 volatile organic compounds detected in the headspace of milk or fermented dairy products were added in various con- centrations to pasteurised milk as a reference and chro- matographically analysed.

The range of chemicals supplied by various compa- nies is very wide but can never be completely comprehensive. Sturaro et al. (1994) have proposed two simple ways by which a reference compound may be obtained. Firstly, it may occur as a synthetic byproduct in a commercial sample. Alternatively, the compound may be synthesised by a simple procedure, preferably by a single step one.

Recently, MS-MS (Startin, 1987) has been applied in various ways to the analysis of foods and flavours. Samples have been analysed using a solids probe with- out any extraction, derivatisation, distillation or other sample preparation. Two mass spectrometers are used in tandem and consist of three quadrupole mass analy- sers. A limitation is that it cannot be routinely used to analyse all compounds of a complex mixture.

Correlation to human perception

For the evaluation of consumer preference, there is a natural dominance of sensory procedures over instru- mental techniques. However, Dirinck & De Winne (1994) have described several cases where measurement of flavour characters in food can best be approached by both sensory and instrumental techniques. They con- cluded that, to obtain good correlation with sensory results, instrumental flavour characterisation should give major attention to the isolation procedures and to the selection of the relevant components to be quantified.

Grigoryeva et al. (1994) have correlated the sensory and GC characteristics of Dutch cheese. GC and pro- file-rank methods were used for the quantitative and qualitative evaluation of the odour and taste of Dutch cheese samples of different quality, time of ripening and storage. It was possible to classify samples into ‘good’, ‘moderate’ and ‘unsatisfactory’ without using a

standard. The correlation between sensory and GC characteristics was used to optimise the conditions of cheese production.

King et al. (1994) have applied flavour concentration adjustments for correlation between GC-headspace measurements and sensory evaluations. Two methods for obtaining correction factors between a model fruity flavoured aqueous solution and milk were studied. The first method compared GC-headspace measurements of each compound in water as opposed to milk suspen- sions. In the second method, an intensity function was obtained by sensory analysis for each compound in water and milk. From extrapolation or interpolation of the functions, it was possible to obtain a concentration of the compound in milk giving the same intensity as the aqueous concentration specified by the original flavour formula.

In an interesting application of MS, Soeting & Hei- dema (1988) have developed a MS method for measur- ing flavour concentration/time profiles at the nose. Although there were very large variations in the release curves among different subjects, major effects of the nature of the foodstuff on the release of volatiles could never the less be observed.

Although GC-MS has identified a large number of compounds in foodstuffs, a test of tlavour activity is missing from most experiments. Piggott (1990, 1994) has outlined the problems relating sensory and chemical data to the understanding of flavour. He concluded that, although a range of methods including the calcu- lation of odour units, fractionation of chromatographic effluent, ‘nasal appraisal’ of chromatographic effluent and a range of multivariate statistical procedures have been used to link composition data to flavour data, defects in understanding of the mechanism of operation of the chemical senses have limited the success achieved.

A number of monographs (Engen, 1982; Lawless & Klein, 1991; Overbosch et al., 1991) have been written on the subject of chemosensory behaviour and the tests that are used to measure it. Irwin et al. (1992, 1993) have reviewed and applied the Receiver Operating Characteristic in the study of taste perception. Williams et al. (1988) and Williams (1994) have discussed sensory and chemical/physical information in the context of food acceptance. They deal with the stimulus response relationship, review the chemical and physical data potentially related to perception, types of sensory methods available for characterising products and the various approaches that have historically been applied to make links between the sensory and chemical/physi- cal information. Brand & Bryant (1994) have discussed the importance of sensory receptor systems (taste, olfaction and chemesthesis) in relation to the sensation of food flavour. Booth (1994) has discussed flavour quality as cognitive psychology.

One detector is especially important in aroma research-the human nose-and preferably that of a

278 R. J. Stevenson, X. D. Chen, 0. E. Mills

trained flavour analyst. To be able to judge the sensory relevance of separate substances, the eluate in the GC column is divided in an output splitter and part of it (N 90%) drawn into a sniffer mask, after being mixed with moist air to prevent drying out of the mucous membranes. The technique is known as gas chromato- graphy-olfactometry (GC-O; Acree & Barnard, 1994). The remainder of the eluate is passed to another detec- tor to obtain a chromatogram that can be correlated with the sensory impressions recorded, thus creating an aromagram-a chromatogram in which all odours per- ceived are indicated on a time basis.

Karpe et al. (1995) have described a multi-coupling analytical system consisting of a thermal desorber installed with a gas chromatograph, a FID, a mass spectrometer and a sniffer port. The sniffer was con- nected at the output of the GC column leading to the FID. The retention times obtained by the FID and the sniffer port were well correlated, allowing the detection of odorous compounds, which were then identified by MS. This device is, therefore, able to identify and quantify volatile organic compounds and odorous compounds in only one analytical run.

Driscoll et al. (1985) have studied the sensory quality of non-fat dry milk after long-term storage, while Hough et al. (1992) have investigated the sensory thresholds of flavour defects in reconstituted WMP.

Plumas et al. (1993) have used differential GC-0 to measure overall food odour intensity of conched choc- olate versus extruded chocolate. The differential olfacto- meter based on reciprocal inhibition is not perturbed by qualitative differences between an odour to be measured and a standard.

The reproducibility of sensory impressions depends on both the experience of the analysts and whether or not they are on form that day. Some training is certainly needed to smell and describe compounds in the effluent of a GC column, and to separate ‘real’ odours from the distraction of background smells in a consistent man- ner. Early elutants may appear every 5-10 s, so fast ‘translations’ of sensory impressions into descriptions have to be made. Sniffing periods of longer than 30 min usually result in olfactory fatigue.

The sensitivity of the human nose and that of a GC detector may bear no relation to one another, so when one is sniffing it is preferable not to observe the chro- matogram at the same time. Intense peaks may be odourless, while odour impressions may appear at ‘empty’ areas in the chromatogram. Synergistic effects may be important when considering overlapping peaks. This implies that a completely odourless pure substance in the overall sample matrix may be capable of making a significant contribution to the aroma of the product under investigation. However, there is controversy over this issue and Acree (1993) has stated that responses to mixtures of stimuli are characterised by inhibition and suppression and not by synergy. Murphy et al. (1977)

studied the perceived intensity of odour-taste mixtures relative to unmixed components. The outcome approxi- mated simple additivity.

Flavour units (Acree et al., 1984) and isointensity scores (D’Andrea, 1975), which are somewhat empiri- cal, have been used as bioassays in the study of flavour compounds.

It is claimed that both Combined Hedonic Response Measurements (CharmAnalysis, the software of which is now commercially available; Acree et al., 1984; Cunningham et al., 1986; Marin et al., 1988; Acree & Barnard, 1994, Guichard et al., 1995) and Aroma Extraction Dilution Analysis (AEDA; Schieberle & Grosch, 1987; Ulrich & Grosch, 1987; Schieberle & Grosch, 1988; Grosch, 1993; Guth & Grosch, 1993) provide reproducibility in determining relative flavour units of unknown components in foods (i.e. quantify the importance of individual aroma constituents in a fla- vour isolate) by sniffing the GC effluent at increasing dilution. As the flavour isolate is diluted, less and less aroma constituents would be present in the GC column effluent at sufficient concentrations to give an aroma sensation. Both methods create an aromagram which presents a plot of the last dilution of the isolate (a number) where the aroma was last smelt versus GC retention time (Kovats index). Aroma constituents with the highest dilution value should be the most important to the aroma of the foodstuff. CharmAnalysis measures the dilution value over the entire time the compounds elute, whereas AEDA determines the maximum dilution value detected-AEDA measures peak height; Charm- Analysis yields peak heights and areas. It has been pointed out (Anon., 1994; Da Silva et al., 1994) that both procedures may contain a number of defects.

Moio et al. (19936) have used CharmAnalysis to ascertain the main compounds responsible for the aroma of bovine, ovine, caprine and water buffalo milk. Vacuum extraction and extract dilution sniffing analysis using CharmAnalysis were applied by Moio et al. (1994) to investigate the odour impact compounds of raw, pasteurised and UHT bovine milk.

Osme is a time-intensity approach for evaluating the significance of odour compounds in GC effluent and provides an interpretable FID-style aromagram called an Osmegram. Da Silva et al. (1994) assessed Osme’s capability and reliability using four trained panellists who recorded directly the intensity, duration and qual- ity of each sample odorant in the GC effluent. The panellists were in good overall agreement in rating each compound’s odour potency and quality and Osme was considered to be fairly quantitative when compared with traditional olfactometry techniques.

Starting from an established correlation between spe- cific molecular vibration patterns and the olfactory responses of organisms, Wright (1977) has devised a method by which equivalently specific patterns of neural excitation may be identified electrophysiologically. A

Analyses and binding studies ofJavour volatiies 279

molecular mechanism for the interaction is proposed which also takes into account such related matters as olfactory thresholds and the possibility of both ‘specia- list’ and ‘generalist’ receptors.

Amoore (1967) has experimented with specific anos- mia indicating that sensations of smell might be deter- mined by reactions of primary odours, analogous to the primary colours in theories of colour vision.

Because it is an interaction between consumer and product (Fishken, 1990; Goldman, 1994; Dijksterhuis, 1995), flavour cannot be measured directly by instru- ments. However, an electronic nose (Pelosi, 1989; MacKay-Sim, 1991; Newman, 1991; Coghlan, 1994; Benady et al., 1995) has been developed that could be used to ‘sniff out’ tainted food, perfumes and drugs. It is a simplified version of the human nose, but whereas the human nose has about 10000 sensors, one electronic device has only 12. The sensors are made of polypyrrole polymers that conduct electricity and adsorb volatile ingredients in aromas, vapours and gases. The 12 sen- sors are made from different polymers and each of them adsorbs different combinations of ingredients in the sample, producing visual fingerprints. Coupled with neural network technology, the electronic nose should prove a useful and complementary tool to MS and trained sniffing panels.

Binding studies and future research

Although much technological progress has been made in developing spray-dried WPC that contains -80% protein, residual lactose, lipids, phospholipids, lipopro- teins and Cut may undergo chemical reactions such as oxidation and Maillard browning, producing stale and aged off-flavours. Improved flavour and functionality may be achieved by reducing these residues with new technologies such as chemical pretreatment (Kim et al., 1989; Karleskind et al., 1995; Laye et al., 19956) physi- cal pretreatment (Rinn et al., 1990), microfiltration (Rinn et al., 1990; Gesan et al., 1995; Karleskind et al., 1995; Laye et al., 19956) and ultrafiltrationdiafiltration (UF-DF; Rinn et al., 1990; Laye et al., 19953). Storage under nitrogen, the use of Cu+-complexing reagents and antioxidants may also assist in flavour improve- ment. For instance, Graf (1994) has shown that the addition of ascorbic acid (0.05-0.400/,) and trace amounts of copper gluconate (10-65 ppm) to salsa or guacamole effectively removed oxygen dissolved in the food within l-5 min and depleted the headspace oxygen within a few days. Unlike other cations treated, copper- catalysed ascorbate-mediated reduction of oxygen to water without concomitant generation of hydroxyl radicals or other activated oxygen species. Thus cop- per(H) ascorbate protected foods against lipid perox- idation, discolouration and other oxidative damage. It also inhibited bacterial growth and, thereby, increased the microbiological stability of high-moisture foods.

Preliminary work with a falling-film evaporator has shown that relatively short product residence time required to concentrate the UF-DF retentate fraction, compared with a conventional vacuum evaporator, results in significantly less browning and off-flavour (Morr & Richter, 1988).

Lee & Morr (1994) found the order of decreasing lipid oxidation rates under accelerated storage (6 days at 60°C in the dark) for dried dairy products was WPI and PC < WMP < WP, NFMP and WPC. Under the accelerated storage conditions, the WPC GC-MS pro- file exhibited higher concentrations of hexanal, hepta- nal, octanal and nonanal than all the other dried products. WPC also demonstrated the poorest flavour stability. The reason for the high susceptibility of WPC to lipid oxidation was not concluded. However, WPC contained 7% milkfat and the powder was quite fluffy, thus allowing a more intimate contact of substrate with the residual oxygen from the headspace atmosphere. It was also likely that WPC had a smaller particle size distribution than the other dried products. Information on the fatty acid composition of the residual lipids in WPC is lacking, but it is possible that these lipids may contain a higher percentage of fatty acids that are more susceptible to oxidation than the other dried products.

The purpose of spray-drying (Refstrup, 1995) is to obtain products of good quality and of low moisture content. The behaviour of thermolabile constituents is important for obtaining this quality. Some thermolabile compounds (e.g. amino acids in drying of foods) should escape destruction as much as possible, while for others (e.g. bacteria in drying of foods) destruction is desirable. Daemen and coworkers (Daemen, 1981; Daemen & Van Der Stege, 1982; Daemen et al., 1983; Daemen, 1984) have investigated the destruction of some enzymes and bacteria during the spray-drying of milk and whey under various conditions. The outlet air temperature had a large effect, but the effect of the inlet air tem- perature was only slight. It was expected that a larger particle size would correspond to a greater destruction, but the inclusion of air in the particles masked this effect. The destruction of bacteria was not only ascribed to a thermal effect, but also to a non-thermal drying effect.

As previously stated, more than 6000 volatiles have been identified in foodstuffs. It is considered that the greatest hindrance of relating physicochemical data to sensory data in flavour research is the lack of sufficient understanding of the chemical senses. Piggott (1994) has suggested three important areas of research in this regard:

1. Favour release (Mills & Solms, 1984; Overbosch et al., 1991; Salvador et al., 1994), the effect of food structure and what happens when food is eaten.

2. Chemoreceptor mechanisms (Booth, 1994; Brand & Bryant, 1994; Nagodawithana, 1994) and the effect of molecules on the receptors.

280 R. J. Stevenson, X. D. Chen, 0. E. Mills

3. Neural networks as a solution to some of the dif- ficult mathematical modelling problems (Powers, 1988; Schmid et al., 1990; Vellejo-Cordoba & Nakai, 1993; Dijksterhuis, 1994; Dijksterhuis et al., 1994; Elmore et al., 1994; Vellejo-Cordoba & Nakai, 1994a,b).

Because overall flavour, as perceived by the con- sumer, is important in determining the acceptability of foods, interactions of flavours with proteins such as WPC (Kinsella & Whitehead, 1989) is of practical interest. At this stage, the components responsible for off-flavour development in WPC have not been quanti- tatively identified, but may include some volatile fatty acids, carbonyls from oxidised lipids (e.g. phospholipids or lactic acid/esters) and amino acid byproducts includ- ing nitrogen- and sulfur-containing compounds. These compounds are possibly ‘bound’ to whey proteins. Thus, binding of both off-flavours and flavours (King & Solms, 1979, 1982; O’Neill & Kinsella, 1987, 1988; Kim & Min, 1989; Kinsella, 1989; Haring, 1990; Solms & Guggenbuehl, 1990; Overbosch et al., 1991; Plug & Haring, 1994) to foodstuffs should be important phen- omena, as attempts are made to fabricate and flavour new foods with proteins. Consequences of flavour binding to food industry are two-fold: (a) off-flavour development and (b) loss of desirable flavour in for- mulated foods. Mills & Solms (1984) studied the bind- ing effects of hexanal, heptanal, nonanal, hexanone, octanone and nonanone to WPC in an attempt to obtain better understanding of off-flavour problems. They found both reversible and irreversible binding under various conditions. A taste sensation will result when competition for flavour compounds between food proteins and proteins of the sensory receptors is won by the latter. They concluded that, since little is known about this area at present, the best course of action is to reduce the level of bound off-flavour compounds as much as possible. Although it may be functionally acceptable, the release of reversibly bound off-flavours from the proteins will certainly restrict its widespread use in foods. It was shown that for the binding of some classes of compounds to WPC, this may be controlled by careful choice of pH and temperature during pro- cessing and by reduction of residual lipid.

Hansen & Heinis (1992) have studied the effect of aqueous WPC and sodium caseinate on benzaldehyde, d-limonene and citral flavour intensity by quantitative descriptive analysis deviation from reference using a 12- member trained panel. It was found that intensity of benzaldehyde and d-limonene flavour decreased in the presence of both WPC and sodium caseinate, but citral did not show a decrease in flavour perception. These workers concluded that, when such dairy proteins are used in formulated foods, they may interact with flavour volatiles and, thus, reduce flavour intensity which may be unacceptable to consumers.

Landy et al. (1995) have determined the retention of diacetyl, ethyl acetate, ethyl butanoate and ethyl hex- anoate in a solution of sodium caseinate from their volatility measured by headspace analysis or exponen- tial dilution.

A number of research groups have demonstrated that constituent whey proteins (B-lactoglobulin, a-lactalbu- min and bovine serum albumin) bind various types of compounds. For example, B-lactoglobulin binds varying amounts of butane, pentane and iodobutane (Wishnia & Pinder, 1964, 1966), alkanes (Mohammadzadeh et al., 1967) and alkanes and benzene (Mohammadzadeh et al., 1969a,b); a-lactalbumin binds varying amounts of aldehydes and methyl ketones (Franzen & Kinsella, 1975); and bovine serum albumin binds long-chain fatty acids (Spector, 1975; Morrisett et al,, 1975), hydro- carbons (Wishnia & Pinder, 1966; Mohammadzadeh et al., 1967, 1969u,b) and aldehydes and ketones (Beyeler & Solms, 1974; Franzen & Kinsella, 1975; Damodaran & Kinsella, 1980). Jasinski & Kilara (1985) used equili- brium dialysis to determine whether WPC or its con- stituent proteins bind 2-nonanone and nonanal. They found that the largest number of binding sites with the strongest binding affinity was observed with WPC. Bovine serum albumin bound 2-nonanone and nonanal to a slightly lesser extent, while S-lactoglobulin and cl-lactalbumin did not bind them. However, O’Neill & Kinsella (1987), using the same method, found that 2-heptanone, 2-octanone and 2-nonanone readily bound to B-lactoglobulin, most probably by hydrophobic interactions. These workers (O’Neill & Kinsella, 1988) also noted that the flavour binding behaviour of B-lac- toglobulin was significantly altered by thermal or che- mical modification‘ Thus, upon heat-treatment at 75°C for 10 and 20 min, the binding affinity for 2-nonanone was reduced and the number of sites for binding was increased. King & Solms (1979) have found reversible adsorption of [i4C]benzyl alcohol on denatured bovine serum albumin and that adsorption is directly propor- tional to the amount of protein and decreases linearly with increasing flavour concentration. Adsorption was reduced by 50% when the protein was suspended in media containing both dissolved protein and lipid material.

Kinsella (1989) has monitored the reversible adsorp- tion of flavours, including entrapment, using dynamic microgravimetry in a vacuum system which provides control of the vapour pressure of various flavour com- pounds. Adsorption isotherms were constructed from the equilibrium mass gains of the food, that is, powder at several equilibrated pressures of the flavour com- pound. Desorption isotherms were obtained by evacu- ating the chamber to specific decreasing pressures and continually determining the mass of the powder. B-Lactoglobulin showed a much greater adsorption of 2pentanone than casein at a relative pressure of 0.7

(P/Pv).

Analyses and binding studies ofJavour volatiles 281

Le Thanh et al. (1992) have studied the interactions between volatile and non-volatile compounds in the presence of water. They used headspace analysis and sorption coupled with GC to conclude that hydrogen bonds exist between carbohydrates, water and aroma constituents, and also that hydrophobic interactions occur in the presence of casein.

Maier (1970, 1972a,b, 1973, 1974, 1975) has used ‘desiccator’ experiments to study the binding of volatile flavour compounds to dry (or almost dry) foodstuffs or pure nutrients. He found that, for five dried instant foods, binding of volatiles approximated to that of the main ingredients. A near-rectilinear increase in molarity of sorbed component with the square root of time, with ethanol and such proteins as casein, zein, gelatin and ovalbumin was observed. Sorbed amounts of hexane, ethyl acetate, acetone and ethanol, to gelatin, ovalbu- min, casein and zein, increased in that order, in which also the hydrophobicity of the proteins increased. The irreversibly sorbed amounts increased, however, in the opposite order. Calculations of the hydrophobicity and polarity, respectively, of the individual proteins, show that, at least with ethanol, and, to a lesser extent, with acetone and ethyl acetate, there exists a relation to the quantities sorbed. Thus, it seems that larger amounts of volatiles are bound by hydrophobic interactions and are weakly bound while smaller amounts are relatively strongly bound by polar forces.

It seems that the ability to either mask off-flavours or simulate the desired food flavour is influenced by the flavour binding capacity of proteins. Binding of flavours by proteins results in the suppression of their primary flavouring impact. Therefore, techniques for controlling adsorption of specific flavours should be investigated. The excessive binding of flavours during processing and storage, and/or the preferential release (or retention) of some components of a flavour blend during mastication present challenges to researchers. Flavour binding and release are, therefore, extremely important because the perceived flavour is the ultimate criterion in determining food acceptability. Concentration can pray a major part in the difference between desirable and undesirable flavour impact. Thus, knowledge of flavour binding behaviour of food components is critical in determining food acceptability.

Hills & Harrison (1995) have proposed a new theory for flavour release from solid or semi-solid foods. They used two-layer stagnant film theory to calculate the mass transfer coeficient for the flavour across the food- saliva interface. By using colours which dye a sweet as a substitute for flavour, it was found that theoretical pre- dictions compared well with experiments, both for sim- ple solution of the dye into stirred water and for the more realistic case of mass loss on sucking in the mouth.

ZS-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important ffavour compound that has been isolated from fruits such as pineapples, strawberries, grapes and

raspberries. Glycosidically bound DMHF has been also found in some of these fruit. Krammer et al. (1994) have isolated DMHF B-D-glucopyranoside from fresh toma- toes and also performed enzyme-mediated hydrolysis to liberate DMHF from its bound form. Although further quantitative studies are necessary to learn about the influence of DMHF B-D-glucopyranoside on the volatile flavour of tomatoes, it is known that enzymes (in the mouth, for instance) are required to liberate certain tlavour-impact compounds from their bound form (Williams, 1993).

Young & Paterson (1995) have studied bound vola- tiles in kiwifruit. Volatiles were released by enzymic hydrolysis with B-glucosidase and several new flavour components in kiwifruit have been identified from non- volatile precursors. These compounds are probably glycosidically bound and sensory data indicates that they may be important aroma constituents.

Microencapsulation (Balassa & Fanger, 1971; Dzie- zak, 1988; Aguilera & Stanley, 1993; Arshady, 1993; Reineccius, 1994) is a packaging technology by which small droplets of liquid or solid particles are packed into continuous individual shells or ‘walls’. Scanning elec- tron microscopy (SEM) has been applied to the mor- phological study of various microcapsule systems by Rosenberg et al. (1985). The shells can protect the encapsulated materials from factors which may cause deterioration (e.g. oxygen, moisture and light). They can also be designed to enable controlled release of the encapsulated materials under desired conditions. Spray-drying is a well-established technique in the food industry and is the most commonly used technique to microencapsulate food ingredients. Rosenberg and coworkers (Moreau & Rosenberg, 1993; Rosenberg & Lee, 1993; Rosenberg & Young, 1993; Young et al., 1993a,b; Sheu & Rosenberg, 1995) have used SEM to study the microencapsulating properties of whey pro- teins. The retention of volatiles and the protection of these compounds in a microencapsulated product are related to the porosity and degree of integrity of the microcapsules. Successful spray-drying microencapsu- lation of volatiles depends on achieving high retention of volatiles during processing and storage. Rosenberg et al. (1990) have used SEM to study factors affecting retention of aroma (volatiles) in spray-drying micro- encapsulation. They used model systems in which gum arabic was the wall material and various esters were the core (volatile) materials. The outer and inner structure of the spray-dried microcapsule and the structural change induced by exposure to various relative humid- ity (RH) levels were observed. The retention depends on capsule composition and drying conditions and, as long as no structural damage is indue& high solids con- centration and drying temperatures enhance volatiles retention. It was shown that the volatiles are organised within the capsule in small droplets embedded in the ‘wall’, and that they are protected and retained (up to

282 R. J. Stevenson, X. D. Chen, 0. E. Mills

90%) as long as the capsule structure is intact. Aroma losses occur during the early stages of the drying process by stripping of volatile droplets, and the losses are enhanced by internal mixing in the drying capsule. Once the structure is destroyed (e.g. increasing RH to 97%), total loss of the volatiles was observed. Thus, providing no chemical reactions are taking place during proces- sing, proper manipulation of the drying conditions when applied to dairy protein products such as WPC, could lead to a dried product that retains large amounts of volatiles or, on the other hand, complete loss of volatiles.

CONCLUSIONS

Although the older methods of isolation of mixtures of volatiles such as headspace and distillation/liquid-liquid extraction will continue to play a dominant role, the newer techniques such as SPME and membrane-based sampling should become increasingly important, espe- cially since solvents can be avoided. The benchtop GC- MS has revolutionised volatile separation/identification. MS-MS, GC-IR and GC-Raman spectroscopy etc., will also become important. Understanding of the mechanisms of flavour binding and release and those of human receptors are not advanced. Much work needs to be done in this area.

ACKNOWLEDGEMENTS

The research fellowship for the first author was kindly provided by the New Zealand Dairy Board and the New Zealand Dairy Research Institute. Their generous sup- port for the research group is greatly appreciated.

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