HEADSPACE VOLATILES AND PHYSICAL CHARACTERISTCS OF VACUUM-MICROWAVE.

AIR AND FREEZE-DRIED OREGANO (lippia berlandieri Schauer) ABSTRACT: Mexican oregano (lippia berlandieri schauer) was dried using a freeze dier, conventional hot air drier, and a recently developed vacuum-microwave drier. The effect of the drying method on the relative content of major flavor volatiles, rehydration rate, color, and structural integrity of the herb was evaluated. Dynamic headspace analysis of volatiles present in fresh or dried oregano revealed that - myrcene, - terpinene, - terpinene, p-cymene, and thymol were the major volatile compounds of the plant. The level of thymol in vacuum-microwave- dried oregano was found to be comparable to that of fresh or freeze-dried samples (1.3 times the thymol concentration of air-dried). Air-dried samples of oregano were darker, were less green, and exhibited lower rehydration rates than those prepared by vacuum-microwave or freeze-drying. Structures of vacuum microwave and freeze-dried oregano appeared to be quite similar as observed on electron micrographs. Key words: oregano, volatiles, vacuum-microwave, drying INTRODUCTION A survey of the literature REVEALED the numerous plants in various plant families are known colloquially as oregano, oreganum, or some similar name. This herb is a perennial plant which is characterized by its pungent spicy odor. The herb is marketed in the fresh or dried state. The air dried plant and its extract are used in flavoring vegetables, meats, sauces, and other food products. Generally, air drying of aromatic herbs, including oregano, is a effective method of preservation that inhibits the growth of microorganism and delays the onset of some biochemical reaction in the final product. Hot air drying, however, can cause thermal damage and can severely modify the physical and chemical characteristic of the marketed product. Although freeze drying can be used to avoid damage caused by heat ,producing a product with a superior physical and chemical qualities ,it is considered a costly and time consuming process. Vacuum -microwave drying offers an alternative way to improve the quality offers dehydrated products .the low temperature and fast mass transfer conferred by vacuum (Huxsoll and Morgan 1968),combined with rapid energy transfer of microwave heating ,generates very rapid low-temperature drying ,the absence of air during drying may inhibit it oxidation. Therefore ,physical properties such as structure color and sensory qualities of products can be better preserved .To date ,vacuum microwave drying has been successfully use and the dehydration of animal materials such as shrimp (Lin and others 1998b),and krill(Durance 1997) ,as well as plant materials such as potatoes chips (Durance and Liu 1997),carrots(Lin and others 1998a),cranberries (yongsawatdiguul and GunaseKaran 1996a,b)and sweet basil (Yousif and others 1999) ,regardless of their nature vacuummicrowave-dried foods showed better retention of key constituents and better sensory properties than air-dried equivalents. This study was undertaken to investigate the potential use of vacuum microwave methods for drying oregano, and to compare the effect of various drying methods on the physical and chemical qualities of this aromatic herb. MATERIALS AND METHODS PLANT SOURCE Mexican oregano, lippia berlandieri was purchased from a local wholesale market in surrey, B.C, but it was originally produced in and imported by air from united state. The herb was generally available with the stems and leaves attached, in all experiments; the whole plant was used, excluding the roots. The initial moisture of the plant material was 82.4% on a wet weight basis. DRYING A sample (600g) of fresh oregano was placed in the drying drum ( constructed from high- density polyethylene) of a 4 KW maximum power microwave vacuum chamber (26 in by 20 in) SS 304 stainless steel ,Enwove corp,Vancouver,B.C.,CAN) The drum was rotated at a rate 11 rotations per min. After a vacuum of 27 in of Hg was achieved, the magnetron was powered at 3.2 kw for 12 min, followed by 1 KW for 6 min, and then 0.5 KW for 5 min, microwave power was measured by the IMPI 2-L test (Buffler 1993), Under these conditions, the temperature of the material was 45C as measure d at the end of the drying period using an infrared

thermometer (Model 39650-04 Cole Parmer Instruments Co. Chicago Ill., U.S.A.).Ambient airflow rate through the chamber was 3 L /MIN. The final moisture content and water activity of the plant material were 13.2% and 0.55, respectively. Fresh oregano from the same batch was air- dried using a commercial dryer as follws:1 kg of fresh basil leaves was loaded on versa-belt dryer (wal-dor industries Ltd., new hamburg,Ont.).the dryer temperature was set at 48C which an air flow rate of 3 2.3m /and relative humidity of 25 % .After 11.5 h in the dryer, moisture content and water activity of the plant material were 13.2 % and 0.53,respectively. A 3 sample of fresh oregano was freeze-dried under vacuum (1.6 mm Hg).chamber and condenser temperatures were 20 C and 55 C .respectively. The dried material had a moisture content and water activity of 9% and 0.54 %, respectively.
rd

WATER ACTIVITY (aW) AND MOISTURE CONTENT DETERMINATION Water activity (aW) of all dried samples was determined using the Aqualab (model CX.2 Decagon Devices, Inc., Pullman Wash., U.S.A.) moisture content of dry samples was determinate (in triplicate) using a laboratory oven at 103C samples were dried to a constant weight. HEADSCAPE VOLATILE COMPOUNDS ANALYSIS Volatile compounds of oregano were extracted and collected by a dynamic headspace technique, separated on a varian 3700 gas chromatograph (Varian Associates Inc., Palo Alto Calif., U.S.A.) and identified by gas chromatography mass spectrometry (GC-MS). Six samples of fresh or dried oregano were weighed in clean Zip lock bags so that the content of each bag delivered a final concentration of 0.6% (w/v, based on moisture content studies)when suspended in the preheated (60C) distilled water contained is a 1 purge and trap apparatus (Wheaton , Millville,N.J.,U.S.A ) . The temperature of the apparatus was held at 60 C throughout the experiment by circulating water from a water bath. Fresh samples were blend (using a household blender) in 100 ml of preheated (60 C) distilled water until completely homogenized (30 sec.) and then placed in a purge and trap apparatus that contained 400ml. of preheated water (60C). Dried samples of the herb were crushed while inside the sealed plastic bag and the flakes were immediately added to 500ml of 60 C water in the purge and trap vessels. An internal standard tetradecane (Aldrich Chemical, Millwaukee, Wis, U.S.A.) dissolved (1:100) in diethyl ether (BDH chemicals, Toronto, Ont.) was added (500 uL) to each of the vessel that were attached to a horizontal shaking platform. The headspace of the shaken herb-containing vessels was purged with purified N2 (Linde specialty gas, Vancouver, B.C.) AT 50 mL /min for 2 h and passed through an absorbent trap containing tenax GC (60-80 MESH Alltech Co. Deerfield.Ill.,U.S.A.) about 100 mg of tenax GC was packed into each glass tube (18 cm,6mm,o.d.,4 mm i.d.,)which was secured at both ends with glass wool deactivated with SY-LONO-CT (Supelco Inc., Toronto, Ont.). The tenax GC was conditioned prior to first use as recommended by the manufacturer. Subsequent use of the traps was preceded by stripping the absorbent with diethyl ether and drying at 60C with N 2 flowing at 30 ml/min for 30 min. Diethyl ether (2ml) was used to elute the volatile compounds from the Tenax GC, and the extract was concentrated to approximately 300 uL by directing a gentle stream of N2 onto the surface. A sample (uL) of the concentrated extract was injected into the GC that was equipped with a flame ionization detector (FID) and a polyethylene glycol (PEG) capillary Supelcowax-10 column (30 m, 0.25 mm i.d., 0.25 um film thickness, Supelco Inc., Toronto, Ont.). The column temperature was held at 35C for 5 min, programmed at 4c per min to 200C and held at 200c for 5 min. The injector port and detector were set at 220c and 250C, respectively. The flow rates for helium (carrier gas) and hydrogen gas were set at 30 ml/min and for air at 300mL /min. Splitless injection was employed. Data was collected and processed with the JCL 600 Chromatography Data System for PC (Jones Chromatography, Lakewood, Colo., U.S.A.). The relative amount of major volatile compounds was determined by dividing the area of a compound by peak area of the internal standard and multiplying by 100.

For identification of the volatile compounds, separation was performed using a longer (60 m) Supelcowax-10 fused silica capillary column that was housed in a Hewlett-Packard 5890-5970 GC-MSD system (Hewlett-Packard, Avondale, P., and U.S.A.). Oven temperature was initially held at 35C, then increased by 3C/min to 200C with a final hold time of 10 min. The carrier gas was helium and the column head pressure was maintained at 30 psi. The MSD operating conditions were scan range 25 to 200 amu, threshold 400, sample rate 2.7 scan/s, and EM voltage 1800.Mass spectral identification was obtained with a HP G1034C MS Chem Station containing a HP G1035A Wiley (138.1) PBM library. COLOR All dried samples and a commercially available air- dried sample were analyzed colorimetrically. Five grams of each treatment samples were ground (in triplicate) in a household coffee grinder for 10 s to produce a powder of a uniform color. The samples were then transferred to a 10 cm Petri dish, and subsequently read by a Hunter LabScan II Spectrocolorimeter (Hunter Lab, Reston, Va., U.S.A.). The instrument, equipped with a D65 illuminant and 2 observer optical position, was standardized using a black plate and a standard white plate (No. LS-13685, X=79.8. Y=84.67, Z=91.23). The results were expressed as Hunter Lab L (whiteness/darkness), a (red/green), and b (yellow /blue) values. REHYDRATION The rehydration potential of dried oregano leaves was evaluated by immersing pre-weighed samples, held by plastic netting in small glass containers in water at (1) 30 C and (2) 100C. The samples (in triplicate for each time interval and treatment temperature) were drained under vacuum (in a Buchner funnel, for 30 sec.) and re-weighed at 0, 10, 20, 30, 60, 90, and 120 min for samples rehydrated at 30C; and at 0, 0.5, 4.0, 6.0, 8.0, and 10.0 min for samples rehydrated at 100C. Then weight of water absorbed (g) divided by the dry sample weight (g) was expressed as the rehydration ratio. The slope of the rehydration ratio against rehydration time was defined as the rehydration rate. SCANNING ELECTRON MICROSCOPY (SEM) Dried whole leaves of similar size and moisture content were chosen at random from the various drying methods for scanning electron microscopic examination. The leaves were fragmented, under a dissection microscope, into smaller pieces. Leaf fragments were then attached to SEM stubs and subsequently coated with gold (about 25 nm) using the Nanotech SEMPREP II Sputter Gold Coater, and finally stored under desiccation until examined by the scanning electron microscope (Stereos can 250, Cambridge instruments Ltd., Cambridge, U.K). Polaroid pictures were taken and processed as specified by the manufacturer. STATISTICAL ANALYSIS Statistical analysis software (Instant for Macintosh, version 2.01) was used to evaluate the significance of the difference between the various treatment groups. Student t-test was used to compare the mean values of the various treatments. Mean values were considered significantly different when p-0.05. RESULTS AND DISCUSSION Aroma loss is one of the important changes that occur during drying of herbs. Aroma is an important quality factor that influences consumer acceptability of herbs. In oregano, flavor is mainly determined by one or both of its two character-impact compounds, thymol and /or carvacrol. The literature shows a wide variation in the essential oil composition and concentration of oregano. The concentration of thymol and carvacrol shows an interesting pattern depending on the region where oregano is produced. Oregano used in this research was classified as belonging to the family verbenaceae, a cultivar indigenous to Mexico and the southern United States. Mexican oregano is characterized by its high content of thymol, present as the main constituent 40% to 60% of total volatiles, and carvacrol at 5 to 25% (Lawrence 1984). Monoterpene hydrocarbons such as -myrcene, -terpinene, y-terpinene, and p-cymene are also present in Mexican oregano oils at concentrations between 20% and 40%, with g-terpinene and p-cymene being the most prevalent.

The aroma of a food product is determined by detection of volatile compounds which escape from the food matrix and are present in the headspace above the food material. Volatile can be released to the headspace at varying concentrations depending upon their solubility in the matrix, absolute amount, and volatility. Therefore, trapping analysis of the headspace volatiles, as used in this study, can be more closely related to the sensory aroma profile of the product than solvent extraction of total volatiles. More than twenty four volatiles collected by the headspace method were detected in oregano by capillary GC. Compounds that were identified are listed in table 1. A typical chromatogram of headspace flavor volatiles of fresh oregano is shown in fig. 1. In this study, the peak areas (relative abundance) of most of the identified compounds, including carvacrol, were small. Therefore for further discussion only the quantitatively major compounds found in the sample analyzed were considered, including the main aroma volatiles, for example, - myrcene, -terpinene, - terpinene, P-Cymene, and thymol. Fig.1- typical chromatogram of volatile compounds extracted from fresh oregano L. berlandieri leaves by a purge and trap technique. (Peak 16=C14, tetradecane internal standard, numbers correspond to compounds identified by GC/MS in table 1).

Table 1-headspace volatile compounds from oregano, L. berlandieri extracted and col-lected by headspace procedure analyzed and indentified by GC/MS
a

Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

compound -pinene -thujene p-Xylene m-Xylene o-Xylene 3-heptanone - Myrecene -terpinene Limonene -Phellandrene .terpinene 3-Octanone p-Cymene -Terpinolene 3-Octanole Tetradecane(internal standard) 1-Octen 3-ol Terpinen-1-ol linalool 2,3-butanediol -coryphyllene(+terpinen-4-ol) -terpiniol borneol thymol carvacrol

The peak correspond to the numbers in fig.1.

numbers

Fig. 2-effect of drying method on relative abundance of -myrcene, -terpinene, -terpinene, p-cymene, and thymol of oregano, L. berlandieri leaves. F = fresh. AD = air-dried. VND = vacuum-microwave-dried. FD=freeze-dried. For each compound, different letters above the bars indicate a significant difference (p<0.5). Figure 2 shows the effect of the drying method on the abundance of the five volatile compounds .the level of monoterpenes, myrcene, and -terpinene, in the air or vacuum-microwave-dried samples was not significantly different from levels found in the fresh samples. The significant increase in concentration of -myrcene upon freeze drying is noteworthy. A combination of enhanced loss of this monoterpene during air or vacuum-microwave drying as compared to freeze drying, and/or greater hydrolysis of nonvolatile conjugates of the freeze-dried samples during the volatiles extraction in the purge and trap apparatus may be responsible for this elevation in concentration. This phenomenon is known to occur among various volatile compounds of aromatic herbs (Braja and others 1989; stahl-biskup 1987) although the mechanism responsible for these observations is not understood. The amount of -terpinene showed a significant decrease from original levels present in the fresh samples when the plant was air-or vacuummicrowave-dried, but not freeze-dried. P-cymene concentration in fresh oregano was significantly reduced upon drying by all of the three methods. Thymol, a key character-impact compound, was also affected by air-drying. Statistical analysis showed a significant reduction in thymol concentration in air-dried samples, while its concentration remained unaltered in vacuum-microwave or freezedried samples. Although formal organoleptic testing of the samples dried by the various methods was not performed, it was evident that the intensity of odor (presumably due to thymol) in vacuum microwave and freeze dried samples was stronger in comparison whit air dried samples. The effect of a particular drying proto-col on the release or retention of aroma compounds is not predictable ,and varies with each herb .for example,Venskutonis (1997) found the five flavor volatiles discussed above (fig.2) were dramatically increase when thyme, thymus vulgaris, Was air-dried at 60C but did not increase when the herb was dried at 30C or freeze-dried .similarly , myrcene,-terpinene ,-terpinene,p-cymene, and thymol in sage, salvia officinalis ,showed a significant reduction in concentration when the plant was dried at 60C compared to 30C. The combination of heat at atmospheric oxygen in air drying facilitates enzymatic activity of poliphenol oxidase (PPO) which results in the browning effect that characterizes many air-dried food materials (Howard and others 1996).during the shorter period of drying in the vacuum-microwave and the resulting reduced exposure to oxygen less browning .

Fig.3-rehydration curves of freeze dried (FD),vacuum microwave dried (VMD),and air dried (AD) oregano,L.berlandieri leaves, at (a) 30C or (b) 100 C .at each time interval, different letters above/below y-axis values indicate a significant difference (p> 0.05) between treatments. Table 2 hunter lab color values of dried oregano, L. berlandieri Color parameter L a b
a a

FD

VMD

AD

commercial

51.11 0.91

44.94 1.99b -4.35 0.57b 15.21 0.81a

34.340.72c -1.04 0.13c 10.43 0.51b

29.570.96d 2.46 0.34d 9.000.19c

-6.79 0.42

15.370.66a

for each parameter, values (mean standard deviation) followed by different letters are significantly different at p<0.05 from each other FD = freeze- dried; AD = air-dried; VMD = vacuum microwave dried. Was absorbed, as indicate by the significantly higher L values of vacuum-microwave dried samples (table 2). Dehydration processes affect the quality attribute of color to a varying degree. While the effect of the drying protocol on the concentration of chlorophyll was not directly assessed in this study ,color analysis showed that vacuum microwave- dried samples were significantly greener in color (large negative a values)that either air dried oregano or a commercially air dried sam ples of the herb. A similar effect on color during vacuum-microwave and air drying was also evident in sweet basil (Yousif and others 1999) and carrot chips (Lin and others 1998a) these authors observed that air dried samples had darker hues (smaller L value s)than vacuummicrowave dried samples. Rehydration of plant materials is affected by their polarity /hydrophobicity and equally by other physical structures such as the presence or absence of international open space. The rehydration curves of dried oregano at 30C and 100C are shown in fig.3a st and b, respectively. Statistical analysis of the data was limited only to the 1 two readings of the rehydration assay (10 and 20 min at 30 C, 0, 5 and 1 min at 100 C)because of the observation that most rehydration in this study occurred during the first 20 min of the process, and also because of the fluctuation of the readings past that point most likely as a result of the heterogeneous nature (that is ,size,thickness,age)of oregano leaves at low temperatures (30 C )vacuum microwave dried oregano exhibited

significantly higher rehydration ratios as compared to air dried samples (fig.3a). At higher temperatures, less time was required for reconstitution of all samples, and similarly vacuum-microwave-dried samples exhibited significantly higher rehydration ratios than air dried ones (fig.3b).similarly, in and others81998a,b)observed better rehydration of vacuum microwave-dried carrot slice and shrimp ,further ,at high temperatures higher rehydration rates occurred at the beginning of rehydration ,this was especially noted in the vacuum microwave and freeze dried samples. In oregano, the epidermis layers are covered by hairs or trichomes. in addition to this surface structure ,oregano plants are known to synthesize and store their essential oils in glandular type of trichomes.These capitates glands have an unicellular stalk and a unicellular globular to ovoid head with the cuticle raised to form a blander like covering ; they occur in depression in the epidermis .The epidermal cells surrounding the stalk radiate to give a very characteristic appearance to the glands visible on the surface. The 1,2,3 effect of each drying method on the leaf structures was observed under the scanning electron microscope (fig.4a ) .After air drying ,oregano leaves exhibited significant changes in their botanical structures such as the severe shrinkage of the cuticle and its 1 associated structures and the underlying epidermal layers (fig 4a ). Internally, the cells of both the palisade and spongy mesophyll 1 were extremely affected and appeared collapsed (fig.4b ). The effect of drying on the structures of the cuticle in vacuum 2 microwave -dried samples was moderate as judged from the extent of shrinkage to the layers(fig. 4a ) and cells of the epidermal and mesophyllic layers of vacuum -microwave or freeze dried samples had and almost identical stretched or buffed appearance 2 3 (fig. 4b and b ).this is consistent with the rehydration data that shows enhanced rehydration rates when oregano was dried by vacuum-microwave or freeze dryer, compared to hot air .This ability to rehydrate at a higher rate is likely attributed to the presence of open structures within the leaf. It was previously reported that the structure of carrot (Lin and others 1998a)and potato (Durance and Liu 1996) slices could be puffed or expanded by vacuum microwave drying because the chamber pressure was kept low during vacuum microwave drying ,and the internal pressure within products was elevated by water vapor .this pressure differential generates an outward force, causing the material to expand beyond its original dimensions ,resulting in a puffing effect, alternatively the low temperature that is maintained during vacuum-microwave drying may minimize the collapsing forces of heat during drying. Under both circumstances the original open structure is retained.

Fig.4-oregano, L. berlandieri leaf. Scanning electron micrographs of: (A) epidermis x 100 and B cross sectional view x 480 .1 =airdried.2=vacuum-microwave dried .3 = freeze dried .cut. =cuticle.c.tr. = covering trichome.c.g.=capitates gland.s=stoma.s.m=spongy mesophyll .p.m=palisade mesophyll. The quality of dried oregano depends on the drying method. Vacuum-microwave or freeze drying were found to be better for drying the oregano plant materials used in this study without decreasing the concentration of its character impact compound, thymol. Unlike freeze drying ,however, vacuum-microwave drying of oregano is a rapid process (0.4 compared to 11,5 h for air drying ) that can produce a dry plant material with similar qualities(in terms color ,rehydration ,and structure ) to that or freeze-dried samples.