This post has 8 comments.

1. Miss Julia Meecham
   14 Apr 09 8:34 am

   Would any of your readers be interested in the following:

   The Kennel Club Charitable Trust Fund Raising Promotion

   I am writing to you in order to introduce THE OFFICIAL KENNEL CLUB GENERAL & GROUP CHAMPIONSHIP SHOW COLLECTOR’S MAP which we have recently published on behalf of The Kennel Club, please see link: http://www.thekennelclub.org.uk/item/2188/pg_dtl_art_news/pg_hdr_art/pg_ftr_art

   We are offering all canine related societies, charities and foundations the opportunity to join our affiliation programme in order to assist with the marketing campaign of The Kennel Club Charitable Trust Fund Raising Promotion. Please be advised that a donation will be made to The Kennel Club Charitable Trust from every copy sold of the map and a donation of £5 will be made to your organisation from every copy sold via your introduction. I can put a copy of the publication in the post for your perusal today, if you so wish. Please contact me at julia.spotlight@btinternet.com

   We are promoting the campaign online through the not for profit website http://www.maps4pets.com This is a newly established website and we are encouraging as many charitable organisations as possible to let us know about any campaigns they may be running in the future so that we may include the information on new sections of the website.

   The direct link to the Collector’s Map is: http://www.maps4pets.com/collectors.asp

   Thank you for your time.

   Yours sincerely

   Julia Meecham
   Publishing Division
   http://www.maps4pets.com
2. Kat Wood
   15 Apr 09 6:52 am

   We pulled RC from our shelves years ago and began stocking Hill’s Rx diets only. The customer service and dvm consult services are superb. I never got anywhere with RC. Since switching to Hill’s I have not received a lot of phone calls from clients stating their pets refused to eat Hill’s.
3. maps4pets
   19 May 09 1:58 am

   Can anyone recommend a list online of all the major vets in The British Isles please?
4. tumwongsqa
   11 Jul 09 8:13 am

   GC–MS Determination of Sucralose in Splenda
   Wenlong Qiu1, Zhongfu Wang1,&, Wanli Nie2, Yuna Guo1, Linjuan Huang1,&
   1 Life Science College, Shaan Xi Provincial Key Laboratory of Biotechnology, Key Laboratory of Resource Biology and Biotechnology in Western
   China, Ministry of Education, Northwest University, Xi’an 710069, China; E-Mail: wangzhf@nwu.edu.cn; E-Mail: huanglj@nwu.edu.cn
   2 Department of Chemistry, Northwest University, Xi’an 710069, China
   Received: 25 April 2007 / Revised: 6 August 2007 / Accepted: 24 August 2007
   Online publication: 3 October 2007

   Abstract
   Sucralose (1,6-dichloro-1,6-dideoxy-b-D-fructofuranosyl-4-chloro-4-deoxy-a-D-galactopyranoside)
   is a high-intensity non-nutritive sweetener derived from sucrose. Determination of
   sucralose in food is important to ensure consistent product quality. The authors have developed
   a new method for determination of sucralose. The sucralose was converted into its trimethylsilyl
   (TMS) ether and qualitative and quantitative analysis were achieved by GC–MS and GC–FID,
   respectively, using myo-inositol ester as the internal standard. A good linear relationship
   between response and amount of sucralose TMS ether was obtained in the range 0.005–
   0.06 mg mL_1 (r = 0.9994). The detection limit was 0.25 ng.

   Keywords
   Gas chromatography–mass spectrometry
   Fragmentation patterns
   Quantification
   Sucralose
   Splenda

   Introduction
   Sucralose (1,6-dichloro-1,6-dideoxy-b-
   D-fructofuranosyl-4-chloro-4-deoxy-a-Dgalactopyranoside)
   is a high-intensity
   sweetener derived from sucrose; it is
   *600 times sweeter than sucrose. Sucralose
   is used in the manufacture of artificially
   sweetened food and beverages. It is
   important to ensure the stability of these
   products is adequate and there is no loss
   of sweetness under expected storage
   conditions.
   Analysis of sweeteners such as aspartame
   and saccharin in soft drinks has
   been achieved by reversed-phase highperformance
   liquid chromatography
   (RP-HPLC) [1, 2]. Sucralose, however,
   lacks chromophores and so other methods
   of detection must be used.
   Methods developed for analysis of
   sucralose include thin-layer chromatography
   (TLC), enzymatic assays, HPLC
   with refractive-index detection, capillary
   electrophoresis, and high-performance
   anion-exchange chromatography with
   pulsed amperometric detection (HPAE–
   PAD) [3–9]. All these methods have
   drawbacks, however, for example low
   sensitivity and time-consuming procedures.
   Gas chromatography (GC–FID)
   seems more rapid, accurate, and sensitive,
   although sucralose, a non-volatile
   substance, must be derivatized, in the
   same way as other carbohydrates
   [10–12].
   Owing to high sensitivity combined
   with the possibility of achieving efficient
   separations of complex mixtures, GC–
   FID and gas chromatography–mass
   spectrometry (GC–MS) have gained
   general acceptance for analysis of carbohydrate
   derivatives, and numerous GC
   and/or MS studies of trimethylsilyl
   (TMS) derivative of carbohydrates have
   been reported [13–15].
   External standards have often been
   used for quantitative determination of
   sucralose [3, 5, 10]; the precision is poor,
   however. In the work reported herein we
   developed an internal standard method
   and used the ratio of the analyte signal to
   that of the internal standard to compensate
   for random and systematic errors
   and to improve the precision. This
   method is useful for GC–FID instruments
   that do not have an auto-injection
   system.
   2007, 66, 935–939
   DOI: 10.1365/s10337-007-0422-4
   0009-5893/07/12 _ 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
   Full Short Communication Chromatographia 2007, 66, December (No. 11/12) 935
   Splenda is a solid artificial sweetener
   used as a sugar substitute consisting of
   dextrose, maltodextrin, and sucralose. In
   this paper we describe a new internal
   standard GC method for quantification
   of sucralose. Sucralose was derivatized to
   its TMS ether by reaction with hexamethyldisilazane
   (HMDS) and trimethylchlorosilane
   (TMS-Cl) in pyridine.
   Sucralose–TMS ether was separated from
   other compounds present in Splenda and
   quantified. We report data on linearity,
   detection limit, precision, and recovery.
   These results reveal our method is rapid,
   simple, accurate, and highly sensitive.
   Experimental
   Materials
   Chemicals and reagents were of analytical
   reagent grade. Sucralose (1,6-dichloro-
   1,6-dideoxy-b-D-fructofuranosyl-4-chloro-
   4-deoxy-a-D-galactopyranoside, chemical
   formula C12H19Cl3O8, molecular weight
   397.64, CAS no. 56038-13-2), glucose,
   maltose, myo-inositol, HMDS, and TMSCl
   were purchased from Sigma (St Louis,
   MO, USA). Dichloromethane, methanol,
   and pyridine were obtained from the
   Chemical Reagent Company of Tianjing
   (Tianjing, China). Splenda was purchased
   from McNeil-PPC (Washington, DC,
   USA). myo-Inositol ester was produced
   in our laboratory [16].
   Gas Chromatography
   GC was performed with a Shimadzu GC-
   2010 instrument equipped with a split
   injector (SPL) and a flame-ionization
   detector (FID). The capillary column was
   a 30mท 0.25 mm i.d. fused-silica column
   coated with a 0.25 lm film of the
   chemically bonded phase rtx-50. The
   injection port temperature was 270 _C;
   the column oven temperature was maintained
   at 180 _C for 2 min then increased
   at 6 _C min_1 to 250 _C which was
   maintained for 20 min. The carrier gas
   was N2 (constant flow 0.6 mL min_1)
   and the split ratio was 9:1.
   Gas Chromatography–Mass
   Spectrometry
   GC–MS was performed with a Shimadzu
   GCMS-QP2010 instrument equipped
   with an electron-impact (EI) ion
   source (electron energy 70 eV) and an
   electron multiplier detector enabling
   recording of ions from m/z 44 to m/z
   1,000. The capillary column was a
   30 m ท 0.25 mm i.d. fused-silica column
   with a 0.25 lm film of rtx-5ms; the
   temperature program was identical with
   that described above. Helium was used
   as carrier gas at a flow rate of 0.6 mL
   min_1. The temperatures of the interface
   and the ion source were 200 _C and
   250 _C, respectively.
   Silylation Reaction
   Silylation was performed by treating sucralose
   (10 mg) dissolved in pyridine
   (1 mL) with 0.4 mL HMDS and 0.2 mL
   TMS-Cl. After stirring for 5 min at room
   temperature the mixture was concentrated
   to dryness by rotary evaporation in
   vacuo at 50 _C and dichloromethane
   (5 mL) was added to the residue. The
   slurry was centrifuged and the supernatant
   was used to produce a standard
   solution (2 mg mL_1). After adding the
   internal standard the solution was analyzed
   by GC–FID and GC–MS.
   Preparation of Internal
   Standard Solution
   myo-Inositol ester was used as internal
   standard. A 1 mg mL_1 solution was
   prepared in dichloromethane.
   Sample Preparation
   Splenda (5 mg) was dissolved in pyridine
   (1 mL) and silylated as described above.
   The amount of sucralose in Splenda was
   determined from a calibration plot.
   Linearity, Limit of Detection,
   Precision, and Recovery
   The linearity of the method was investigated
   by constructing the calibration plot
   in the concentration range of interest.
   Standard sucralose–TMS ether solutions
   in CH2Cl2 at concentrations of 0.005,
   0.01, 0.02, 0.04, and 0.06 mg mL_1, each
   containing 0.2 mg mL_1 of the internal
   standard (myo-inositol ester), were chromatographed.
   The best-fit line for the
   ratio of integrated peak intensities
   against the ratio of concentrations of
   sucralose–TMS ether to internal standard
   was elucidated by linear regression analysis.
   Seven replicate measurements of the
   sucralose–TMS ether (0.06 mg mL_1)
   were performed. The precision was estimated
   by calculating the mean values,
   standard deviations (SD), and relative
   standard deviations (RSD) of the data
   obtained.
   The limit of detection (LOD) was
   estimated by continuous dilution. The
   LOD was defined as the concentration of
   Fig. 1. The EI mass spectrum of sucralose–TMS ether recorded with the GCMS-QP2010. GC–MS
   conditions: column rtx-5ms (30 m ท 0.25 mm ท 0.25 lm); injection port temperature 270 _C; flow
   rate of carrier gas (He) 0.6 mL min_1; column temperature 2 min at 180 _C, increase at 6 _C min_1
   to 250 _C, then 20 min at 250 _C
   936 Chromatographia 2007, 66, December (No. 11/12) Full Short Communication
   sample for which the signal was 3ท the
   SD of the integrated intensity above the
   mean signal from a blank sample (noise
   level).
   Recovery was examined by addition
   of 0.01, 0.02, and 0.03 mg mL_1 sucralose
   stock solution (0.5 mL) to a 1 mg
   mL_1 Splenda solution (again 0.5 mL).
   After injection, the recovery was calculated
   from the calibration plot described
   above.
   Results
   GC–MS Characterization
   of the TMS Ether of Sucralose
   Sucralose was converted to its TMS ether
   and then analyzed by GC–MS. The EI
   mass spectrum of sucralose–TMS ether is
   presented in Fig. 1. Because the 1,2-glycosidic
   linkage provides two readily
   cleaved C–O bonds, peaks of four fragment
   ions with m/z 413, 397, 359, and 343
   should be expected (Fig. 2a).
   The fragment ion at m/z 413 was not
   observed in the mass spectrum because of
   its low intensity. In general, to observe
   the molecular ion, a high sample concentration
   must be maintained in the ion
   source. A more intense peak [M–CH3]+,
   produced by abstraction of a methyl
   radical from one of the TMS groups,
   enables molecular weight determination
   if no molecular-ion peak is observed,
   however [13]. The fragment ion at m/z
   398 is formed by abstraction of a CH3
   radical from the ion at m/z 413. This ion
   (m/z 398) undergoes further fragmentation
   to give ions at m/z 308 (398–
   TMSOH) and 205 (398–TMSOH–
   CH2OTMS). The ions at m/z 310 and 383
   may arise from loss of CH2OTMS and
   two CH3 species, respectively, from the
   fragment ion at 413.
   The fragment ions at m/z 397, 359,
   and 343 originating from cleavage of the
   glycosidic oxygen are of low intensity,
   possibly as a result of the high ionization
   voltage (70 eV). The fragment ion at m/z
   397 undergoes further fragmentation to
   give ions at m/z 307 (397–TMSOH), 217
   (397–2TMSOH), 361 (397–HCl), 271
   (397–HCl–TMSOH), 181 (397–HCl–
   2TMSOH), 294 (397–CH2OTMS), 382
   (397–CH3), and 265 (397–CHOCH2OTMS).
   The other ion fragments
   derived from the fragment ion at m/z 397
   are depicted in Fig. 2b.
   The fragment ion at m/z 343 yields the
   fragment ions m/z 328 (343–CH3), 253
   (343–TMSOH), 163 (343–2TMSOH), 307
   (343–HCl), 217 (343–HCl–TMSOH), 293
   (343–CH3Cl), and 243 (343–2CH3Cl).
   The fragment ions derived from the
   fragment ion at m/z 359 are presented in
   Fig. 2b.
   The relative intensity data for some
   important fragment ions from the EI
   mass spectra are: 191 (4.15), 217 (5.48),
   243 (3.24), 271 (3.72), 307 (14.56), 308
   (20.47), 309 (10.99), 310 (10.46), 343
   (9.22), 345 (6.89), 359 (0.17), 361 (6.54),
   383 (0.65), 397 (1.16), 398 (0.6), and 412
   (0.25).
   The molecular ion of sucralose–TMS
   ether (m/z 758) and some high-information
   masses were not observed in Fig. 1,
   as described previously [17]. This may be
   explained by the instability of the TMS
   ether under the conditions used.
   Fig. 2. Proposed structures (a) and decay paths (b) of the principal fragment ions in the EI mass
   spectrum of sucralose–TMS ether
   Full Short Communication Chromatographia 2007, 66, December (No. 11/12) 937
   GC Characterization
   The TMS ether of sucralose theoretically
   yields one peak in the chromatogram,
   because sucralose is a non-reducing
   carbohydrate. The GC–FID chromatogram
   obtained from sucralose–TMS
   ether is depicted in Fig. 3; the retention
   time of the TMS ether of sucralose
   is 20.07 min. The myo-inositol ester
   elutes far away from sucralose ether
   (retention time 14.94 min). The TMS
   ethers of glucose and maltose also have
   different retention time; this makes GC–
   FID analysis of sucralose in Splenda
   feasible.
   Linearity, Limit of Detection,
   and Precision
   The linearity range for quantitative
   analysis of sucralose is from 0.005 to
   0.06 mg mL_1. The linear regression
   equation is y = 2.02923x + 0.04265
   (r = 0.9994) (where y represents the ratio
   of the integrated intensity of the sucralose–
   TMS ether peak to that of the
   myo-inositol ester peak and x is the concentration
   of the sucralose TMS ether
   relative to that of myo-inositol ester). For
   seven replicate injections the ratios of the
   integrated peak intensities were 0.650,
   0.648, 0.658, 0.656, 0.649, 0.654, and
   0.642. The mean value was 0.651 and the
   SD was 0.00407. The precision, expressed
   as the RSD, was 0.626%. Assuming a
   lowest acceptable signal-to-noise ratio of
   3, the detection limit estimated by means
   of continuous dilution was found to be
   0.25 ng.
   Determination of Sucralose
   in Splenda
   Splenda is a granular calorie-free sweetener
   that is added to food and beverages
   in a manner similar to granulated table
   sugar. The ingredients listed on the
   product manufacturer’s label include
   dextrose, maltodextrin, and sucralose. In
   this method the sucralose was separated
   from other ingredients gas-chromatographically;
   the retention time was identical
   with that of sucralose standard
   (Fig. 4). The concentration of sucralose
   in Splenda was determined by use of the
   calibration plot and found to be low—
   0.0112 mg mL_1 in a 1 mg mL_1 solution
   of Splenda.
   The recovery of sucralose was estimated
   by adding standard solution, at
   three different concentrations within the
   linearity range of the calibration plot, to
   a solution of Splenda. The results are
   listed in Table 1.
   Discussion
   Many methods have been used to detect
   sucralose [3–9]. Of these, GC, which is
   more sensitive and accurate than other
   methods, for example HPLC, has many
   advantages for detection of samples of
   low concentration. Sucralose is often
   present in very small amounts in food,
   beverages, and, especially, metabolic
   products, so GC detection could be
   advantageous.
   Saccharides are non-volatile substances
   and must be derivatized for
   determination by GC. Many derivatization
   methods are available, for example
   methylation, acetylation, and silylation.
   In contrast with methylation and acetylation,
   silylation is suitable not only for
   monosaccharides but also disaccharides;
   it was, therefore, our method of our
   choice. Formation of TMS derivatives
   by silylation of all free hydroxyl groups
   in pyridine containing HMDS and TMSCl
   occurs very rapidly at room temperature,
   so samples suitable for further
   Fig. 3. Gas chromatogram obtained from the TMS derivatives of glucose, maltose, and sucralose.
   1 = solvent, 2 = glucose, 3 = internal standard (myo-inositol ester), 4 and 5 = maltose,
   6 = sucralose
   Fig. 4. Gas chromatogram obtained after TMS derivatization of the ingredients of Splenda.
   1 = solvent, 2 = myo-inositol ester, 3 = sucralose
   938 Chromatographia 2007, 66, December (No. 11/12) Full Short Communication
   analysis can be prepared in a few minutes
   [17–20].
   Derivatization of a carbohydrate with
   TMS usually yields several peaks (formation
   of anomers) on a gas chromatogram
   unless carbohydrate standards and
   samples are thoroughly dried. Nonreducing
   saccharides (sucrose, trehalose,
   melezitose, erlose, and raffinose), however,
   produce only one peak, corresponding
   to the octakis-TMS derivative
   [21, 22]. Being derived from sucrose,
   sucralose is a non-reducing sugar, so
   persilylation of sucralose yields one peak
   only. This method does not include conversion
   of a sugar into a per-O-trimethylsilyl
   oxime (TMS–oxime) derivative;
   this not only reduces the time of the
   experimental work but also the cost.
   This newly developed method with
   use of an internal standard for quantification
   of sucralose is accurate. It eliminates
   injection error, because a relative
   value is obtained, and so is useful for
   GC–FID instruments which do not have
   an autoinjection system.
   Conclusion
   GC–MS and GC–FID, respectively, have
   been used for identification and quantification
   of sucralose in Splenda. The
   range of linearity for sucralose determination
   was from 0.005 to 0.06 mg mL_1
   (r = 0.9994) and the estimated detection
   limit was 0.25 ng. Precision, as RSD, was
   0.626%. Average recovery was 98.00%.
   The results showed that determination of
   sucralose by GC–FID with an internal
   standard is a good alternative to the
   HPLC method usually used. The method
   could be used for quality control of foods
   and beverages.
   Acknowledgments
   This research was supported by the
   Shaan Xi Provincial Key Laboratory
   Research Program of China (no.
   05JS49), the Hi-Tech Research and
   Development program of China (no.
   2006AA02Z146), and the Science and
   Technology Key Program of the Ministry
   of Education, People’s Republic of
   China (no. 206147).
   References
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   (1969) J Am Chem Soc 91:1728–1740
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   Table 1. Recovery of sucralose–TMS ether from Splenda
   Amount of standard solution
   added (mg mL_1)
   Concentration found
   in sample (mg mL_1)
   Recovery (%) Average recovery (%) RSD (%)
   1 2 1 2
   0.0300 0.0286 0.0315 95.3 105.0 98.0 3.91
   0.0200 0.0188 0.0194 94.0 97.0
   0.0100 0.00973 0.00992 97.3 99.2
   Full Short Communication Chromatographia 2007, 66, December (No. 11/12) 939
5. tumwongsqa
   11 Jul 09 8:23 am

   ORIGINAL PAPER
   Josephine McCourt Æ Joerg Stroka Æ Elke Anklam
   Experimental design-based development and single laboratory
   validation of a capillary zone electrophoresis method for the
   determination of the artificial sweetener sucralose in food matrices
   Received: 11 January 2005 / Revised: 5 April 2005 / Accepted: 13 April 2005 / Published online: 19 May 2005
   
   Springer-Verlag 2005
   Abstract A capillary zone electrophoresis (CZE) method,
   optimised chemometrically, underwent a complete
   in-house validation protocol for the qualification and
   quantification of sucralose in various foodstuffs. Separation
   from matrix components was obtained in a dinitrobenzoic
   acid (3 mM)/sodium hydroxide (20 mM)
   background electrolyte with a pH of 12.1, a potential of
   0.11 kV cm
   1 and a temperature of 22 C. Detection
   was achieved at 238 nm by indirect UV. Screening,
   optimisation and robustness testing were all carried out
   with the aid of experimental design. Using standard
   addition calibration, the CZE method has been applied
   to still, carbonated and alcoholic beverages, yoghurts
   and hard-boiled candy. The method allows the detection
   of sucralose at >30 mg kg
   1, with a linearity range of
   50–500 mg kg
   1, making it suitable for implementation
   of the recently amended ‘‘Sweeteners for use in foodstuffs’’
   Directive (European Parliament and Council
   (2003) Off J L237:3–12), which set maximum usable
   doses of sucralose for many foodstuffs, most ranging
   from 200 mg kg
   1 to 450 mg kg
   1.
   Keywords Capillary electrophoresis Æ Sucralose Æ
   Experimental design Æ Validation Æ Food
   Introduction
   Sucralose, discovered in 1976, is made from sugar
   and tastes like sugar but is an artificial sweetener
   (1,6-dichloro-1,6-dideoxy-b-d-fructofuranosyl-4-chloro-
   4-deoxy-a-d-galactopyranoside) produced by chlorinating
   sucrose. It is approximately 600 times sweeter than
   sugar and is also stable during processing and on the
   shelf. The allocation of an acceptable daily intake (ADI)
   of 0–15 mg kg
   1 body weight by the Joint FAO/WHO
   Expert Group on Food Additives (JECFA) in 1990 has
   meant that it has become a very widely used artificial
   sweetener, occurring in over 3,500 different products
   throughout the world. Due to a recent amendment [1] to
   the EU Sweeteners Directive permitting the use of sucralose
   (E955) at certain concentrations, all EU Member
   States, including the ten new EU Member States, are
   required to amend their national legislation by January
   2005 to harmonise with the Directive. The fact that from
   2005 many sucralose-containing products will be available
   in the EU means in turn that reliable methods will
   be needed for its separation from natural sugars and
   other matrix components, and for its quantification.
   Maximum usable doses have been set for sucralose, in
   this new amendment, for a broad variety of foodstuffs,
   ranging from 10 mg L
   1 in energy-reduced beer to
   2,400 mg kg
   1 in breath-freshening microsweets, with
   beverage limits set around 300 mg L
   1, yoghurt limits at
   350 mg kg
   1 and candy limits at 200 mg kg
   1 to mention
   but a few.
   The chlorination of sucrose (to produce sucralose) is
   a process whereby three hydroxyl groups of the sucrose
   molecule are substituted by three chlorine atoms (see
   Fig. 1). The very much increased sweetness of sucralose
   (as compared to sucrose) is due to the structure of the
   molecule; the two chlorine atoms present in the fructose
   portion of the molecule lead to more hydrophobic
   properties on the opposite side of the molecule.
   The common approach for sucralose determination is
   to use high performance liquid chromatography (HPLC)
   with refractive index (RI) detection [2], with sucralose
   detection limits of about 10–50 mg kg
   1 being reported
   (depending on sample type). However, since RI detectors
   are sometimes sensitive to temperature fluctuations,
   inconsistent retention times and low compatibility with
   gradient elution, other analytical possibilities have been
   looked into. Methods such as high performance thin
   layer chromatography (HPTLC) [3] with sucralose
   detection limits of 30.0 mg kg
   1 in beverages have also
   J. McCourt (&) Æ J. Stroka Æ E. Anklam
   European Commission, DG Joint Research Centre,
   Institute for Reference Materials and Measurements,
   Retieseweg 111, 2440 Geel, Belgium
   E-mail: josephine.mc-court@cec.eu.int
   Anal Bioanal Chem (2005) 382: 1269–1278
   DOI 10.1007/s00216-005-3258-5
6. tumwongsa
   11 Jul 09 8:33 am

   App1 Microbiol Biotechnol (1994) 42:173-178 © Springer-Verlag 1994
   M. P. Labare • M. Alexander
   Microbial cometabolism of sucralose, a chlorinated disaccharide,
   in environmental samples
   Received: 9 November 1993 / Received revision: 24 January 1994 / Accepted: 21 February 1994
   Abstract During the rapid mineralization in soil of sucralose
   (4-chloro-4-deoxy-o~,D-galactopyranosyl-l,6-
   dichloro-l,6-dideoxy-/3,D-fructofuranoside), a metabolic
   product was formed that appears to be the corresponding
   unsaturated aldehyde. During the slow and
   incomplete mineralization of sucralose in lake water,
   which was not increased by the addition of nitrogen
   and phosphorus, the same compound was produced.
   That product was further metabolized by microorganisms
   in lake water and soil. Mineralization was also
   slow in sewage under aerobic conditions, but organic
   products were not detected. Little or no CO2 was
   formed from the disaccharide in flooded soil or anaerobic
   sewage. Bacteria in culture did not use sucralose
   as a carbon source but did convert it to the presumed
   unsaturated aldehyde, 1,6-dichloro-l,6-dideoxy-D-fructose
   and possibly the uronic acid of sucralose. Sucralose
   carbon was not incorporated into cells of two sucralosemetabolizing
   bacteria or the microbial biomass of sewage
   or lake water. The chlorinated disaccharide was
   slowly metabolized by a galactose oxidase preparation.
   It is concluded that the chlorinated sugar is acted on
   microbiologically by cometabolism.
   Introduction
   Because certain chlorinated sugars are remarkably
   sweet, they are being considered as artificial sweeteners
   to be used for human consumption. Some of these compounds
   are not transformed readily and do not accumulate
   in the human body, and thus they will be ex-
   M. P. Labare 1 • M. Alexander ([])
   Laboratory of Soil Microbiology, Department of Soil,
   Crop and Atmospheric Sciences, Bradfield Hall,
   Cornell University, Ithaca, N.Y. 14853, USA,
   FAX: 607-255-2644
   Present address:
   XDepartment of Chemistry, United States Military Academy,
   West Point, N.Y. 10996, USA
   creted and appear in waste-waters and, for people serviced
   by septic tanks, in soils or subsoils. Although
   much attention has been given to the metabolism and
   biodegradation of halogenated hydrocarbons, both aliphatic
   and aromatic, almost no information exists on
   microbial transformations of halogenated carbohydrates.
   The chlorinated carbohydrate, sucralose (4-chloro-4-
   deoxy-o~,D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-
   /?,D-fructofuranoside: Fig. la), is mineralized in soil
   slurries (Lappin-Scott et al. 1987). A subsequent study
   demonstrated mineralization of this disaccharide in
   samples of soil, lake water, sewage and estuarine water
   CH20H COOH CHO
   OH OH OH
   A B C
   C•oH20 HO C1 0
   OH OH
   D E
   Fig. 1 The structure of (A) sucralose, (B) sucralose uronic acid,
   (C) unsaturated aldehyde of sucralose, (D) 4-chloro-4-deoxy-
   D-galactose and (E) 1,6-dichloro-l,6-dideoxy-D-fructose
7. many
   13 Jul 09 10:56 pm

   Eur Food Res Technol (2007) 226:25–31
   DOI 10.1007/s00217-006-0504-9
   ORIGINAL PAPER
   Effects of the methods of pre-treatment before freezing
   on the retention of chlorophylls in frozen leaf vegetables
   prepared for consumption
   Zofia Lisiewska · Piotr G˛ebczy´nski · Waldemar Kmiecik
   Received: 23 May 2006 / Revised: 8 September 2006 / Accepted: 17 October 2006 / Published online: 15 November 2006
   
   C Springer-Verlag 2006
   Abstract The investigation included kale, New Zealand
   spinach and spinach. The evaluation covered the raw material;
   the raw material after blanching; the raw material after
   cooking; and frozen products prepared for consumption after
   0, 4, 8 and 12 months of refrigerated storage. Both the traditional
   method of freezing (blanching before freezing) and
   the modified method of freezing (cooked before freezing)
   were used in the experiment, as well as two storage temperatures,
   T = −20 ◦C and T = −30 ◦C. The content of
   chlorophylls in fresh kalewas four times that in NewZealand
   spinach and 1.5 times that in spinach. With the exception of
   New Zealand spinach, blanching and cooking significantly
   reduced the content of chlorophylls. In kale products prepared
   for consumption, the content of chlorophylls decreased
   in each successive stage of the investigation. In products
   of New Zealand spinach and spinach, the losses were usually
   not significant. After 12 months of refrigerated storage,
   frozen kale products prepared for consumption retained 52–
   65% of total chlorophylls compared with the content in the
   raw material; products of New Zealand spinach and spinach
   retained 66–71%. In kale and New Zealand spinach, the
   content of chlorophyll a decreased more rapidly than that of
   chlorophyll b, while in spinach the converse was true. The
   kale products obtained using the modified method contained
   more chlorophylls, while in the two spinach species their
   content was lower. The lower storage temperature resulted
   in a higher retention of chlorophylls in vegetables.
   Z. Lisiewska (
   ) · P. G˛ebczy´nski · W. Kmiecik
   Department of Raw Materials and Processing of Fruit and
   Vegetables, Faculty of Food Technology,
   Agricultural University of Krakow,
   122 Balicka Street,
   30-149 Krakow, Poland
   e-mail: rrlisiew@cyf-kr.edu.pl
   Keywords Leaf vegetables . Pre-treatment . Freezing .
   Preparing for consumption . Chlorophylls
   Introduction
   Leaf vegetables, due to their abundance of nutritional components
   and wide availability, form an important part of
   the diet in many areas of the world [1–3]. These vegetables
   are usually consumed fresh or cooked, while some of
   them, including kale, spinach and New Zealand spinach, can
   also be processed. Freezing is the most popular method of
   processing vegetables. The technology of freezing includes
   several stages of pre-treatment followed by refrigerated storage;
   before consumption, the product is also subjected to
   culinary treatment. All these procedures cause decreases in
   the content of chlorophylls, which leads to deterioration of
   the colour of products [4]. Methods of preventing chlorophyll
   loss include the control of pH. According to Faboya
   [5] the optimum pH is 6; a slightly higher value of pH 7
   is given by Gunavan and Barringer [6]; while according to
   LaBorde and Elbe [7] and Tijskens et al. [8], it should be
   about 8. Gunawan and Barringer [6] and Tijskens et al. [8]
   stress that not only is the level of pH significant, but that
   the kinds of acid also have a decisive effect on this parameter.
   The researchers agree that thermal processing applied on
   the principle of “high-temperature-short-time”, which brings
   about a rapid inactivation of enzymes, is most suitable for
   preventing losses of chlorophylls [9–11]. However, Teng and
   Chen [12] suggest that not only are the time and temperature
   important, but also the environment in which the treatment
   occurs. Another important factor decisive for the retention
   of chlorophyll is the formation of metal–chlorophyll complexes;
   particularly those containing zinc, since their colour
   is similar to that of chlorophyll [13, 14].
   Springer
   26 Eur Food Res Technol (2007) 226:25–31
   In recent years, the demand for time-saving convenience
   foods has grown rapidly, particularly among younger consumers
   [15]. Frozen ready meals are the largest sector in
   terms of volume and value [16]. Frozen vegetable products
   can be of the do-it-for-me or ready-to-eat type; however,
   these require changes in the pre-treatment of the rawmaterial
   before freezing, essential to obtain appropriate consistency
   for the frozen product to be simply defrosted and heated to
   consumption temperature without cooking. Another aspect
   stressed by consumers is the appearance of the product, regarded
   as an indicator of its freshness, palatability and nutritional
   value. Crucial to the attractive appearance of fresh and
   processed leaf vegetables is the green colour, which depends
   on the amount of chlorophylls and the ratio of chlorophyll a
   to chlorophyll b [17]. As the chlorophyll content decreases,
   a deterioration in the sensory quality of vegetable products
   can be expected [4]. However, as the investigations by Jaworska
   and Kmiecik [2] on frozen spinach and New Zealand
   spinach and by Philippon et al. [18] on parsley shows, a loss
   of chlorophylls of 20% causes no, or only a slight change
   in the colour. Martins and Silva [19] also failed to find any
   deterioration in colour in frozen, unblanched French bean
   after 2 months’ frozen storage, despite losses of chlorophyll
   of up to 89%.
   The present paper describes the effects of blanching, cooking,
   freezing, frozen storage and preparation for consumption
   on the level of chlorophyll a, b and total chlorophylls in
   three species of leaf vegetable: kale, New Zealand spinach
   and spinach. The effects of the temperature of frozen storage
   (−20 ◦C and −30 ◦C) and the time of frozen storage (0, 4,
   8 and 12 months) were also evaluated.
   Materials and methods
   Materials
   The investigated material consisted of three species of leaf
   vegetables: kale (Brassica olearacea var. acephala L.—
   Winterbor cv.); New Zealand spinach (Tetragonia expansa—
   Dutch breeding, cultivar not specified); and spinach (Spinacia
   oleracea L. —Greta cv.). The content of chlorophyllswas
   investigated at the stage of raw material; blanched material;
   cooked material; and frozen vegetables obtained using the
   two different methods and prepared for consumption. Frozen
   products were stored at T = −20 ◦C and T = −30 ◦C,
   analyses being conducted directly after freezing and after 4,
   8 and 12 months of refrigerated storage.
   The raw material was harvested in the experimental field
   of the Department where the technological and analytical
   investigations were carried out. The field lies in southern
   Poland, on the western outskirts of Krakow. The soil was in
   good horticultural condition with pH in H2O 7.08 and a high
   content of potassium, phosphorus and calcium. The application
   of mineral fertilisers was determined by the fertility
   of soil and the nutritional requirements of the investigated
   species. The fertilisation of kale was: N—90 kg/ha; P2O5—
   80 kg/ha; K2O—150 kg/ha; that of the remaining species
   60, 60 and 100 kg/ha, respectively. Cultivation measures included
   mechanical weed control; sprinkler watering; and,
   where necessary, protective treatments against diseases and
   pests. The harvest of kale and spinach was carried out during
   the last 10 days of October, and of New Zealand spinach in
   mid-July.
   A sample representing the whole batch of the given
   species was taken for analysis of the raw material and for the
   preparation of frozen products. The petioles and the main
   rib were removed from kale leaves sampled for chemical
   analysis and processing. The leaf blades were than cut into
   strips 2–3 cm in width. For the preparation of frozen products
   of New Zealand spinach, whole leaves with petioles about
   15 cm in length were cut in half. Frozen products of spinach
   were prepared from whole leaves without petioles.
   Preparation of frozen products
   Two methods of processing the raw material before freezing
   were used. The first method used the traditional technology
   of blanching the raw material; after freezing and refrigerated
   storage, the frozen product was cooked in water to consumption
   consistency. In the second method, the raw material was
   cooked to consumption consistency to obtain a ready-to-eat
   product, which, after freezing and refrigerated storage, only
   had to be defrosted and heated in a microwave oven.
   In the first method, the fresh material was blanched in a
   stainless steel vessel in water, the proportion of water to the
   raw material being 5:1 and the blanching temperature T = 95–98 ◦C. The blanching time of the different species is given
   in Table 1. The applied parameters of blanching allowed a
   decrease in the activity of catalase and peroxidase to a level
   not exceeding 5% of the initial activity. After blanching, the
   material was immediately cooled in cold water and left to
   drip on sieves for 30 min.
   In the second method, the vegetables were cooked in a
   stainless steel vessel in water with 2% added salt (NaCl),
   the proportion of the weight of the raw material to brine
   being 1:1. The vegetables were placed in boiling water. The
   cooking time measured from the moment when the water
   came to the boil again is given in Table 1. After cooking to
   consumption consistency, the material was left on sieves and
   cooled in a stream of cold air.
   Blanched and cooked vegetables were packed in 500 g
   portions in polyethylene bags and frozen at T = −40 ◦C in
   a Feutron 3626-51 blast chamber. The time required for the
   inside of the product to reach the storage temperature of T
   = −20 ◦C and T = −30 ◦C was 90 min and of 120 min,
   Springer
   Eur Food Res Technol (2007) 226:25–31 27
   Table 1 Time of blanching
   and cooking before freezing and
   time of preparing for
   consumption after frozen
   storage
   Vegetable Before freezing After frozen storage
   Blanching Cooking Cookinga Defrosting and heating in microwave
   oven, goods stored at
   −20 ◦C −30 ◦C
   Kale 3 min 15min 12 min 7 min 45 s 8min 15 s
   New Zealand spinach 2 min 15 s 4 min 2 min 7 min 45 s 8 min 15 s
   Spinach 2 min 6 min 3 min 7 min 45 s 8 min 15 s
   aTime was the same for both
   storage temperature (T = −20
   ◦C and T = −30 ◦C).
   respectively. The products thus obtained were stored at T =
   −20 ◦C and T = −30 ◦C until evaluation.
   Preparation of the frozen product for evaluation
   Samples of the vegetables blanched before freezing were
   cooked in 2% brine, the proportion by weight of the brine to
   the raw material being 1:1. As was the case when cooking
   fresh vegetables, the frozen product was placed in boiling
   water. The cooking time measured from the moment when
   the water came to the boil again is given in Table 1. After
   cooking, the water was immediately drained; the product
   was cooled to T = 20 ◦C and analysed. Samples of vegetables
   cooked before freezing were defrosted and heated in a
   Panasonic NN-F621 microwave oven. In this case, a 500 g
   portion was placed in a covered heatproof vessel. The automatic
   program for heating frozen products was used with
   the maximum instantaneous power of 600 W generated by
   the oven during heating. The time required for defrosting
   and heating to consumption temperature [20] is given in Table
   1. The samples were then cooled to T = 20 ◦C and
   analysed.
   The table salt used in the experiment was always taken
   from the same batch; the pH of the tap water varied within
   7.3–7.7.
   Chemical analyses
   The content of dry matter was measured using the gravimetric
   method presented in the AOAC [21]. The content
   of chlorophylls a and b was determined using the Lichtenthaler
   and Buschmann [22, 23] method. Chlorophylls were
   extracted with pure acetone until all colour was removed
   from the sample. After suitable dilutions were obtained, colorimetric
   measurements were carried out. The measurement
   of absorbance was conducted in a Shimadzu UV-160A spectrophotometer
   at 661.6 and 644.8 nm wavelengths, i.e. at the
   absorbance maxima for chlorophylls a and b. The pigment
   concentrations were calculated on the basis of the absorbance
   coefficients at the above wavelengths.
   The content of chlorophylls in the raw material, in the
   partly processed material and in frozen products prepared
   for consumption was calculated per 100 g fresh matter. The
   level of dry matter given in Tables 2–4 allows the reader
   to calculate the content of chlorophylls on the basis of dry
   matter.
   Statistical analysis
   The differences in the content of chlorophyll a, chlorophyll
   b and total chlorophylls at the various stages of evaluation
   were established using single-factor analysis of variance
   (ANOVA) on the basis of the Snedecor F and Student’s t
   tests, the least significant differences (LSD) being calculated
   at the probability level α = 0.01. The Statistica 6.1 program
   was applied in the calculation. Chemical determinations were
   conducted in four replications.
   Discussion of results
   The species of leaf vegetables investigated varied considerably
   in their chlorophyll content (Tables 2–4). The highest
   content of these pigments was found in kale: 162.4 mg in
   100 g fresh matter; New Zealand spinach contained 25% of
   this amount, and spinach 67%. In these vegetable species, the
   chlorophyll a:b ratio was 1:0.42; 1:0.39; and 1:0.38, respectively.
   Khachik et al. [24] reported the chlorophyll a:b ratio
   as 1:0.30 in kale and 1:0.47 in spinach. Kopsell et al. [25]
   showed that the variability of the chlorophyll a:b ratio was
   23% within kale cultivars, and 45% within the years of the
   study. Suda et al. [26] stressed that the level of chlorophyll
   depended both on the species and, within the species, on the
   phase of plant growth.
   Compared with the raw material, blanching significantly
   decreased the content of chlorophylls in kale and spinach,
   while this effect was observed in all investigated vegetables
   after cooking (Tables 2–4). Prolonged thermal processing,
   i.e. cooking, increased the loss of total chlorophylls by 4–
   10% compared with the blanched material. Slightly greater
   losses were found in chlorophyll a than in chlorophyll b
   after blanching (2–3%) and cooking (3–6%). Labib et al.
   [27] claim that the blanching of spinach significantly reduced
   only the content of chlorophyll a. Bohn et al. [28]
   also reported a stronger degradation of chlorophyll a than of
   chlorophyll b in cooked spinach. The losses in total chlorophylls
   reported by Bhobe and Pai [29] and Lisiewska et al.
   [11] in blanched leaf vegetables approximated those found
   in the present work. According to Negi and Roy [30], the
   Springer
   28 Eur Food Res Technol (2007) 226:25–31
   Table 2 Content of chlorophyll in kale prepared for consumption depending on pre-treatment before freezing and temperature of frozen storage
   Stage of Material Storage temperature Dry matter Chlorophyll (mg/100 g)
   estimation ( ◦C) (g/100 g) a b a + b
   Before freezing Raw – 18.08 ± 0.18 114.5 ± 4.6 47.9 ± 1.8 162.4 ± 4.1
   Blanched – 13.06 ± 0.19 104.0 ± 3.3 44.4 ± 2.1 148.4 ± 3.7
   Cooked – 13.95 ± 0.23 97.9±4.3 43.6±1.7 141.5±2.8
   After storage (months) and preparing for consumption
   0 Blanched before freezing and cooked −20 13.48 ± 0.26 72.6 ± 3.5 35.6 ± 1.6 108.2 ± 5.1
   −30 13.63 ± 0.19 70.9 ± 3.2 35.4 ± 1.2 106.3 ± 3.2
   Cooked before freezing and prepared in −20 14.90 ± 0.25 86.1 ± 2.7 42.7 ± 1.2 128.8 ± 4.5
   microwave oven −30 15.06 ± 0.17 85.1 ± 3.3 42.6 ± 1.6 127.7 ± 2.8
   4 Blanched before freezing and cooked −20 13.54 ± 0.21 61.6 ± 3.0 33.1 ± 2.0 94.7 ± 3.1
   −30 13.65 ± 0.23 63.7 ± 1.9 34.8 ± 1.8 98.5 ± 4.8
   Cooked before freezing and prepared in −20 14.91 ± 0.18 74.1 ± 4.0 40.6 ± 1.5 114.7 ± 5.0
   microwave oven −30 14.99 ± 0.19 79.7 ± 3.2 41.8 ± 1.2 121.5 ± 4.4
   8 Blanched before freezing and cooked −20 13.52 ± 0.17 59.8 ± 2.6 30.3 ± 1.7 90.1 ± 3.2
   −30 13.64 ± 0.20 63.1 ± 3.1 33.1 ± 0.8 96.2 ± 4.4
   Cooked before freezing and prepared in −20 14.84 ± 0.23 70.9 ± 2.7 37.7 ± 1.0 108.6 ± 2.5
   microwave oven −30 14.94 ± 0.19 73.3 ± 2.1 40.0 ± 1.4 113.3 ± 3.3
   12 Blanched before freezing and cooked −20 13.54 ± 0.21 56.0 ± 2.5 28.5 ± 2.5 84.5 ± 2.9
   −30 13.60 ± 0.24 58.9 ± 1.3 30.5 ± 2.1 89.4 ± 2.6
   Cooked before freezing and prepared in −20 14.90 ± 0.17 66.0 ± 2.6 34.6 ± 1.4 100.6 ± 3.7
   microwave oven −30 14.94 ± 0.22 69.8 ± 2.3 35.7 ± 1.7 105.3 ± 4.1
   LSD α =0.01 0.335 4.39 2.43 6.11
   loss of chlorophylls amounted to 12–43% in leaf vegetables
   after thermal processing in water, and varied according
   to the species as well as the length and temperature of the
   treatment.
   In the present experiment, the fairly limited loss of chlorophylls
   after blanching and cooking might be explained by the
   pH of the water, which varied from 7.3 to 7.7 in all cases. As
   Gunawan and Barringer [6], and LaBorde and Elbe [7] claim,
   the optimum pH for inhibiting the reaction of acid converting
   chlorophyll to pheophytin by replacing the magnesium with
   hydrogen is 7–8.
   The successive stages of analysis included samples prepared
   for consumption after 0, 4, 8 and 12 months of refrigerated
   storage. Since the method of preparation was
   always the same (the samples blanched before freezing
   being cooked to consumption consistency after refrigerated
   storage, and those cooked to consumption consistency
   before freezing being defrosted and heated in a
   microwave oven), the differences in chlorophyll content
   were attributed to the effect of the prolonged refrigerated
   storage.
   Compared with the content before freezing, kale contained
   significantly less chlorophyll a and total chlorophylls
   directly after freezing; and also significantly less chlorophyll
   b in samples blanched before freezing (Table 2). The first 4
   months of storage caused a significant decrease in the content
   of chlorophyll a and total chlorophylls; the subsequent
   4 months brought about a decrease in chlorophyll b, except
   for the sample blanched before freezing and stored at T =
   −30 ◦C. After the 12-month storage period, a significant
   decrease was noted in the content of chlorophyll b and total
   chlorophylls compared with the content after 8 months, with
   the exception of the sample blanched before freezing and
   stored at T = −20 ◦C. Compared with the content found
   in the raw material, after 12 months of refrigerated storage
   kale products contained 49–61% of chlorophyll a; 59–75%
   of chlorophyll b; and 52–65% of total chlorophylls. At the
   lower storage temperature, the content of chlorophylls was
   5% higher, and with the modified method of pre-processing
   18% higher.
   Directly after freezing, as in the case of kale, samples
   of New Zealand spinach produced from blanched material
   contained significantly less chlorophyll a and total chlorophylls;
   however, no significant loss was found in samples
   cooked before freezing (Table 3). It was shown that changes
   in the level of chlorophylls between the successive stages of
   refrigerated storage were not significant in almost all cases.
   The first significant reduction due to refrigerated storage was
   found after 8 months in the content of chlorophyll a and total
   chlorophylls, with the exception of the sample blanched before
   freezing and stored at T = −30 ◦C, and in chlorophyll
   b content, but only in the sample cooked before freezing
   and stored at T = −20 ◦C. In the sample blanched before
   freezing and stored for 12 months at −30 ◦C, the content
   of chlorophylls did not significantly differ from that found
   directly after freezing. After the whole storage period, New
   Zealand spinach contained 54–75% of chlorophyll a; 65–
   82% of chlorophyll b; and 57–77% of total chlorophylls.
   Springer
   Eur Food Res Technol (2007) 226:25–31 29
   Table 3 Content of chlorophyll in New Zealand spinach prepared for consumption depending on pre-treatment before freezing and temperature
   of frozen storage
   Stage of Material Storage temperature Dry matter Chlorophyll (mg/100 g)
   estimation ( ◦C) (g/100 g) a b a + b
   Before freezing Raw – 6.19 ± 0.15 29.6 ± 1.4 11.4 ± 0.8 41.0 ± 2.0
   Blanched – 6.66 ± 0.19 27.5 ± 1.1 10.8 ± 0.6 38.3 ± 1.6
   Cooked – 9.12 ± 0.23 24.0 ± 1.1 9.9 ± 0.6 33.9 ± 1.6
   After storage (months) and preparing for consumption
   Blanched before freezing and cooked −20 8.46 ± 0.22 23.9 ± 1.3 10.0 ± 0.9 33.9 ± 2.1
   −30 8.66 ± 0.18 24.1 ± 1.2 9.9 ± 0.7 34.0 ± 1.8
   0 Cooked before freezing and prepared in −20 10.78 ± 0.21 23.5 ± 1.1 9.4 ± 0.7 32.9 ± 1.6
   microwave oven −30 10.97 ± 0.21 23.5 ± 1.5 9.3 ± 0.5 32.8 ± 2.0
   4 Blanched before freezing and cooked −20 8.42 ± 0.17 22.5 ± 1.1 9.7 ± 0.4 32.2 ± 1.5
   −30 8.50 ± 0.23 23.5 ± 1.0 9.7 ± 0.4 33.2 ± 1.3
   Cooked before freezing and prepared in −20 10.56 ± 0.21 20.4 ± 1.2 8.8 ± 0.6 29.2 ± 1.7
   microwave oven −30 10.86 ± 0.22 21.7 ± 1.2 9.0 ± 0.3 30.7 ± 1.4
   8 Blanched before freezing and cooked −20 8.42 ± 0.16 21.3 ± 1.2 9.3 ± 0.4 30.6 ± 1.3
   −30 8.46 ± 0.20 23.1 ± 1.5 9.6 ± 0.5 32.7 ± 2.0
   Cooked before freezing and prepared in −20 10.31 ± 0.16 18.5 ± 0.8 8.1 ± 0.5 26.6 ± 1.2
   microwave oven −30 10.79 ± 0.19 19.8 ± 0.9 8.7 ± 0.6 28.5 ± 1.5
   12 Blanched before freezing and cooked −20 8.44 ± 0.17 20.2 ± 1.4 8.8 ± 0.3 29.0 ± 1.6
   −30 8.43 ± 0.19 22.2 ± 1.0 9.4 ± 0.6 31.6 ± 1.6
   Cooked before freezing and prepared in −20 10.31 ± 0.21 16.1 ± 0.7 7.4 ± 0.3 23.5 ± 1.0
   microwave oven −30 10.74 ± 0.26 18.0 ± 0.9 8.5 ± 0.6 26.5 ± 1.4
   LSD α =0.01 0.376 2.18 1.07 3.03
   Frozen products stored at the lower temperature contained
   11% more chlorophylls than those stored at the higher temperature,
   while those cooked before refrigerated storage contained
   17% less than products blanched before freezing and
   cooked after refrigerated storage.
   Spinach products obtained from samples blanched before
   freezing contained significantly less chlorophylls directly
   after freezing than before, while in products from
   samples cooked before freezing the content of these pigments
   was not statistically different (Table 4). Analyses conducted
   after 4, 8 and 12 months did not show significant
   changes in the amounts of the investigated constituents between
   the successive dates of sampling. Compared with the
   content directly after freezing, the first significant reduction
   was found in the level of chlorophyll b after 8 months,
   except for spinach cooked before freezing and stored at T
   = −30 ◦C; of total chlorophylls after 8 months of refrigerated
   storage, but only in spinach blanched before freezing
   and stored at T = −20 ◦C; and of chlorophyll a after
   12 months, with the exception of spinach cooked before
   freezing and stored at T = −30 ◦C. After 12 months of
   refrigerated storage, spinach contained 68–72% of chlorophyll
   a; 62–67% of chlorophyll b; and 66–71% of total
   chlorophylls compared with the content recorded in the raw
   material. Frozen spinach products stored at the lower temperature
   contained only 5% more chlorophylls compared
   with the higher. In frozen products obtained using the modified
   method, the content of chlorophylls was 2% lower than
   that in products obtained using the traditional method of
   processing.
   Murcia et al. [17] quote the losses due to freezing, while
   in the present investigation the discussed losses resulted both
   from freezing and from preparation for consumption. With
   regard to the effect of frozen storage, Bhobe and Pai [29]
   found a 17–24% loss in chlorophyll content in five species
   of leaf vegetables during 3 months of storage at T = −18
   ◦C. However, Labib et al. [27] reported that the 3-month
   storage of spinach at −18 ◦C did not affect the level of
   chlorophylls. These authors attribute this to the lack of peroxidase
   regeneration in the stored frozen product. Other authors
   also suggest that the efficient elimination of enzymes
   results in good retention of chlorophylls during refrigerated
   storage [11, 31]. In our experiment, despite the fact that no
   regeneration of this enzyme was recorded in the investigated
   species throughout the entire storage period and preparation
   for consumption, the following losses in total chlorophylls
   were nevertheless found: 0–12% after 4 months; 4–19% after
   8 months; and 7–28% after 12 months, the losses in
   kale being greater than in the remaining species. During the
   storage of cooked leaf vegetables at −18 ◦C, Jaworska and
   Kmiecik [2] did not record losses in total chlorophylls during
   the first 6 months; however, during the following 6 months,
   the content of chlorophyll was 6–7% lower than directly after
   freezing. Differently from our experiment, the analysed
   products were not, however, prepared for consumption. As
   in the present experiment, a lower storage temperature led
   Springer
   30 Eur Food Res Technol (2007) 226:25–31
   Table 4 Content of chlorophyll in spinach prepared for consumption depending on pre-treatment before freezing and temperature of frozen
   storage
   Stage of Material Storage temperature Dry matter Chlorophyll (mg/100 g)
   estimation ( ◦C) (g/100 g) a b a + b
   Before freezing Raw – 9.13 ± 0.28 78.8 ± 3.5 30.1 ± 1.6 108.9 ± 5.0
   Blanched – 9.36 ± 0.22 68.2 ± 3.8 27.0 ± 0.9 95.2 ± 3.6
   Cooked – 11.78 ± 0.17 62.2 ± 2.0 24.7 ± 1.1 24.7 ± 1.1
   After storage (months) and preparing for consumption
   Blanched before freezing and cooked −20 10.20 ± 0.15 60.6 ± 2.9 24.7 ± 1.0 85.3 ± 3.7
   −30 10.15 ± 0.22 60.3 ± 3.2 24.9 ± 1.3 85.2 ± 4.5
   0 Cooked before freezing and prepared in −20 12.34 ± 0.20 62.6 ± 2.5 24.0 ± 0.4 86.6 ± 2.4
   microwave oven −30 12.42 ± 0.04 62.7 ± 2.7 24.1 ± 1.7 86.8 ± 4.4
   Blanched before freezing and cooked −20 10.22 ± 0.14 58.5 ± 2.4 22.5 ± 1.3 81.0 ± 3.6
   −30 10.11 ± 0.15 60.2 ± 2.2 23.1 ± 0.8 83.3 ± 2.7
   4 Cooked before freezing and prepared in −20 12.29 ± 0.19 62.4 ± 2.8 23.5 ± 1.2 85.9 ± 3.9
   microwave oven −30 12.31 ± 0.24 62.5 ± 2.2 24.0 ± 1.4 86.5 ± 3.4
   Blanched before freezing and cooked −20 10.05 ± 0.09 57.2 ± 3.5 21.1 ± 1.0 78.3 ± 4.4
   −30 10.07 ± 0.14 59.5 ± 2.9 21.9 ± 0.8 81.4 ± 2.3
   8 Cooked before freezing and prepared in −20 12.33 ± 0.20 57.6 ± 2.3 21.0 ± 1.4 78.6 ± 3.5
   microwave oven −30 12.44 ± 0.06 59.9 ± 2.2 22.2 ± 1.6 82.1 ± 3.5
   Blanched before freezing and cooked −20 10.04 ± 0.16 55.4 ± 1.9 19.1 ± 0.7 74.5 ± 2.6
   −30 10.08 ± 0.11 56.9 ± 3.0 20.3 ± 1.4 77.2 ± 4.3
   12 Cooked before freezing and prepared in −20 12.43 ± 0.21 53.2 ± 2.3 18.6 ± 1.0 71.8 ± 3.3
   microwave oven −30 12.31 ± 0.24 56.5 ± 1.6 19.8 ± 1.3 76.3 ± 2.6
   LSD α =0.01 0.339 5.08 2.27 6.79
   to better retention of chlorophylls in parsley [32]; in leaf
   blades of leaf beet [33]; in dill [11]; and in French bean
   [34].
   The sensory evaluation of frozen New Zealand spinach
   and spinach products prepared for consumption after 12
   months of refrigerated storage [35] showed that the better the
   retention of chlorophylls, the higher the evaluation of colour.
   In products of New Zealand spinach prepared using the traditional
   technology, which retained 71–77% of the initial
   chlorophyll content, and in products obtained using the modified
   technology, with a retention of 57–64% chlorophylls,
   the colour was evaluated at a level of 4.6–4.9 points and 3.9–
   4.2 points, respectively on a scale of 1–5 points. On the basis
   of earlier sensory evaluation (unpublished), this relationship
   was also found only within method used in producing frozen
   kale. In products obtained using the traditional and modified
   methods, which retained 52–55% and 62–65% chlorophylls,
   respectively, the colour was evaluated at a level of 3.7–4.9
   points and 4.3–4.8 points, respectively. During refrigerated
   storage, the levels of chlorophyll a and chlorophyll b do not
   fall at the same rate. Bohn et al. [28] and Canjura et al.
   [13] found that the level of chlorophyll a fell more rapidly
   than that of chlorophyll b in spinach. However, according to
   Teng and Chen [12], the rate of decrease of chlorophylls a
   and b in spinach depended on the technological operations
   involved: and in same cases there was a greater decrease
   in chlorophyll a, while with other operations the losses of
   chlorophyll b were greater. Premavalli et al. [3] reported that
   in six of seven species of leaf vegetables processed, chlorophyll
   a was more stable than b, the opposite result being
   recorded in the remaining species. These authors did not
   investigate species that were analysed in the present experiment.
   During refrigerated storage, the smallest changes in the
   chlorophyll a:b ratio were found in kale: from 1:0.49–0.50
   directly after freezing to 1:0.51–0.52 after 12-month storage.
   In frozen New Zealand spinach, these values increased
   from 1:0.40–0.42 to 1:0.42–0.47, respectively; in spinach
   they decreased from 1:0.38–0.42 to 1:0.34–0.35, respectively
   (Tables 2–4).
   Conclusions
   Fresh kale contained four times more chlorophylls than
   New Zealand spinach and one and a half times more than
   spinach. In blanched vegetables, with the exception of New
   Zealand spinach, and also in cooked products, the content of
   chlorophylls a and b decreased significantly. In kale products
   prepared for consumption, the chlorophyll content was
   usually significantly lower at each successive stage of the
   investigation, i.e. after 0, 4, 8 and 12 months of refrigerated
   storage; in products of New Zealand spinach and
   spinach, decreases were usually not significant. After the
   12-month period of refrigerated storage, frozen kale products
   prepared for consumption retained 52–65% of total
   chlorophylls compared with the raw material; products of
   Springer
   Eur Food Res Technol (2007) 226:25–31 31
   New Zealand spinach and spinach both retained 66–71%.
   In kale and New Zealand spinach, the content of chlorophyll
   a decreased more rapidly than that of chlorophyll b,
   but the opposite was true for spinach. Kale products obtained
   using the modified method contained more chlorophylls,
   while the two spinach species contained less. Storing
   frozen products at the lower temperature resulted in greater
   chlorophyll retention in all three species when prepared for
   consumption.
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   Springer
8. tumwongsqa
   15 Jul 09 11:35 pm

   Taste–Odor Integration in Espresso Coffee
   Abstract Espresso coffee samples were freshly prepared
   with 10% sucrose, 0.0143% sucralose (equivalent in
   sweetness to 10% sucrose), or unsweetened, each with
   and without nondairy creamer. A sensory panel rated the
   intensities of “malty,” “caramel,” “roasty,” and “coffeelike.”
   The concentrations of flavor chemicals associated
   with the latter three sensations (Furaneol, 2-ethyl-3,5-
   dimethyl [EDM] pyrazine, and 2-furfuryl thiol, respectively)
   were determined by gas chromatography, using solid-phase
   microextraction sampling of coffee headspace. Furaneol and
   furfuryl thiol were essentially unaffected by creamer addition,
   but the more nonpolar EDM pyrazine was greatly reduced.
   The malty, caramel, roasty, and coffee-like flavor intensities
   were not significantly affected by creamer addition. This
   appears to be a case of disconnect between the absence of an
   odorant and perception. Furaneol, furfuryl thiol, and EDM
   pyrazine concentrations were unaffected by adding either
   sweetener. Themalty sensationwas the samewith and without
   added sweetener. The roasty and coffee-like ratings both
   decreased to similar extents in the samples with the two added
   sweeteners. The ratings for caramel were considerably
   increased, again to a similar extent, by both sweeteners. Since
   the added sweeteners were both nonvolatile, this is clearly a
   case where taste affected odor perception.
   Keywords Chemosensory . Integration . Odor . Taste .
   Synesthesia
   Introduction
   Flavor is a multistimulus perception that involves several
   senses modulated by a number of brain states: memory,
   emotion, cognition, etc. Flavor sensations have intensities
   ranging from subliminal to unbearably strong and qualitative
   features such as the “greenness” of 2-methoxy
   pyrazines or the “sweetness” of sucrose. These qualitative
   features have been called qualia (singular quale) for over a
   century by philosophers (Lewis 1929) and more recently by
   neurobiologists studying consciousness (Koch 2004) and
   chemists studying flavor (Acree et al. 2007). The briefest
   component of a flavor experience is a sensory quale. It is a
   unitary component percept, created in the mind in response
   to stimuli acting on sensory receptors. Several elemental
   qualia are integrated into the phenomenon of flavor.
   Multiple qualia from separate modalities, e.g., taste and
   smell, can enhance or suppress each other, but the process of
   experiencing an increase in a taste quale (e.g., sweetness)
   when certain compounds are smelled (e.g., ethyl methylphenylglycidate
   “synthetic strawberry odor”) is called odor–
   taste synesthesia (Stevenson and Boakes 1998, 2004).
   During the descriptive analysis of ethyl methylphenylglycidate
   by 140 subjects (Dravnieks 1985), only 42 subjects
   used “strawberry” while 91 used “sweet” to describe its
   aroma. Whether the subjects actually experience sweet
   taste is difficult to prove and provides fodder for some
   philosophical disagreements (Dennett 1988; De Leon
   2001). In psychophysical experiments when subjects have
   been trained to associate language with standard odorants,
   a consistent increase in taste intensity simultaneous
   with the perception of specific odorants has been
   demonstrated (Stevenson et al. 1995; Stevenson and Boakes
   1998; Stevenson et al. 1999; Stevenson and Boakes 2004;
   Stevenson et al. 2005). This work supports the proposition
   Chem. Percept. (2008) 1:147–152
   DOI 10.1007/s12078-008-9018-0
   A. Chiralertpong : T. E. Acree : J. Barnard : K. J. Siebert (*)
   Department of Food Science and Technology, Cornell University,
   Geneva, NY 14456-0462, USA
   e-mail: kjs3@cornell.edu
   that synesthesia is an integral part of the processing of
   sensory information rather than a rare, abnormal, or even
   pathological brain state (Prescott 2004).
   In the experiments reported here, the effects of the taste
   quale “sweetness” on the intensity of several odor qualia
   were investigated in a real world system, espresso coffee.
   Using a globally available espresso machine and coffee
   beans, an experimental system easily reproducible in other
   laboratories was developed. Descriptive analysis was used
   to assess odor qualia in sweetened and unsweetened
   espresso coffee. Two different sweeteners, the high-potency
   sweetener sucralose and the common sweetener sucrose,
   were used to distinguish the difference between the effects
   of the sweeteners on volatility and the more central effect of
   quale–quale interactions. Since many coffee drinkers use
   creamer, it was also included in the study. Chemical
   analysis was used to test for changes in volatility of key
   odorants.
   Materials and Methods
   Chemicals 2-Furfuryl thiol, 2-ethyl-3,5-dimethylpyrazine,
   Furaneol® (2,5-dimethyl-4-hydroxy-3(2H)-furanone), 3-
   methylbutanal, guaiacol, β-damascenone, and 2,3-butanedione
   were purchased from Sigma-Aldrich (St. Louis, MO,
   USA). Sucralose was a gift from Tate & Lyle® (Decatur,
   IL, USA).
   Sample Preparation Dannon® Spring Water (Danone
   Waters of North America, California, USA) and Medium
   Roasted Arabica Coffee Beans (Illy® Caffè North America,
   Scottsdale, AZ, USA) were used to prepare espresso coffee
   with a Saeco® Royal automatic espresso machine (Saeco®
   International Group, Italy). The grind was set to level “4,”
   which corresponds to medium ground coffee, and 7.0 g of
   beans was used for an approximately 47-mL cup. Each
   aliquot from the espresso machine was split into two 23.5-
   mL portions, and one portion constituted one sample.
   Nestlé CoffeeMate® (Nestlé Beverage, California, USA),
   2.45 g, 25% fat content, was used for the creamer-added
   samples, resulting in a final volume of 24.5 mL. For the
   sweetened samples, the sweetener used was either sucrose
   (Domino® granulated sugar, Domino Foods, New York,
   USA) or sucralose.
   Sensory Evaluation Sensory tests were conducted in isolation
   booths under moderate fluorescent lighting in a wellventilated
   room designed for sensory testing. Subjects were
   from the Food Science and Technology Department at the
   New York Agricultural Experiment Station, Geneva, NY,
   USA. Unsalted crackers and rinse water were provided for
   the judges during the evaluation period.
   Sensory Test for Iso-sweetness To find the sucralose
   concentration that provided sweetness intensity comparable
   to that of 10% sucrose (iso-sweet), a sensory test format
   was adapted from the difference-from-control (Meilgaard et
   al. 1999) and the relative-to-reference tests (Stoer and
   Lawless 1993). The sensory panel had 27 members, 17 men
   and 10 women; all liked coffee and drank it occasionally,
   and their average age was 37. They reported they were
   generally healthy, had normal senses of taste and smell, and
   no history of medical problems with coffee, sugar, or any
   other sweetener. Three levels of sucralose (0.0143%,
   0.0196%, and 0.025% [w/v]) in espresso coffee were
   compared with a 10% (w/v) sucrose control. Five espresso
   coffee samples were served in 4-oz paper hot cups. The first
   sample was a labeled 10% sucrose control. The other four
   consisted of the three sucralose-added samples plus one
   blind control; these were labeled with random three-digit
   codes and were presented in random order across sessions
   and subjects. Subjects were asked to taste the control
   sample first and then to evaluate the size and direction of
   the difference between each sample and the control, which
   was assigned a value of “5” on a nine-point scale. Since
   most, if not all, psychometric tests suffer scaling bias, the
   use of a blind control removed the scale from the
   determination of iso-sweetness. The test was carried out
   three times, each on a different day.
   Sensory Descriptive Analysis Eleven subjects, six men and
   five women, with an average age of 33, participated in this
   study. Subjects reported that they had no dentures, diabetes,
   oral or gum disease, hypoglycemia, food allergies, or
   hypertension and that they took no medications that
   affected their senses of taste or smell. Training sessions
   were conducted with reference odorants (see “Training”)
   and preliminary sensory descriptive analysis (SDA) tests
   adapted from quantitative descriptive analysis (Lawless and
   Heymann 1998) were carried out prior to the coffee
   evaluations to evaluate individual performance and general
   trends. Two subjects were removed from the panel because
   their responses were too noisy, and two new subjects were
   trained for the SDA session, providing the minimum 10–12
   judges recommended for quantitative descriptive analysis
   (Lawless and Heymann 1998).
   Training Subjects were introduced to four aroma compounds
   orthonasally that were the focus of this study: 2-
   furfuryl thiol (coffee-like), 2-ethyl-3,5-dimethylpyrazine
   (roasty), Furaneol® (caramel-like), and 3-methylbutanal
   (malty). Experiencing single odorants should produce the
   same quale whether ortho- or retronasally administered,
   although this would not be true of mixtures. These samples
   were prepared by dipping filter paper strips into solutions of
   the compounds and then placing a strip in a screw cap vial.
   148 Chem. Percept. (2008) 1:147–152
   Subjects were each provided with a set of labeled aroma
   standards for individual familiarization and self-training.
   After 3 days, subjects were presented with seven samples
   labeled with three-digit codes in random order. Four of
   these contained the target compounds, while the other three
   were other compounds that have been identified in coffee:
   guaiacol (medicinal), β-damascenone (fruity), and 2,3-
   butanedione (buttery). Subjects were asked to match each
   target aroma descriptor with one of the seven samples, or
   mark “other” if they did not think the sample matched any
   target descriptor. Each subject was subjected to at least
   three matching tests, depending on how well he or she did
   on the previous test. If the subject could not match the four
   target aromas correctly on three consecutive matching tests,
   he or she was required to return for another test until at least
   three consecutive correct records were produced. Subjects
   were required to wait at least 2 h before attempting another
   matching test. In later testing, the reference standard for the
   “malty” character was changed from 3-methylbutanal to
   actual ground malt. The self-training and matching test
   were repeated with the new “malty” reference before
   continuing.
   SDA of Espresso Treatments Six coffee samples with
   different combinations of additives were presented to each
   subject (see Table 1). To avoid bias from sample appearance,
   the samples were divided into two groups, the black
   coffee group and the creamer-added coffee group. The
   black coffee group contained the three samples that did not
   have added coffee creamer. The study was carried out in
   triplicate over six sessions, with three sessions for each
   sample group. Each sample was prepared immediately prior
   to serving by dispensing from the espresso machine into a
   3-oz white ceramic coffee cup labeled with a three-digit
   code and containing the appropriate additives (water blank
   or additive[s] in water). The cup was immediately covered
   with a watch glass, which remained in place until a subject
   removed it to taste the sample. The samples were served
   one cup at a time in random order across sessions and
   subjects. Subjects were asked to drink the coffee and then
   rate each sample for the perceived intensity of each of the
   four target aromas on 15-cm line scales anchored with “not
   detectible” on the left and “extremely strong” on the right.
   Then, the subjects returned the cup with any remaining
   coffee and, to reduce lingering taste and odor, ate some
   crackers and rinsed with water before the next sample was
   served. Electronic ballots were used to collect the data. The
   ballots were created using the program FileMaker Pro 7®
   (FileMaker, California, USA). Subjects were asked to wait
   at least 2 h before participating in the next tasting session to
   avoid fatigue.
   Sample Headspace Analysis
   Sample Preparation The six coffee samples were prepared
   in the same way as for the descriptive analysis (see Table 1).
   Analysis of 2-Furfurylthiol and 2-Ethyl-3,5-Dimethylpyrazine
   A 6-mL portion of each coffee sample was placed in a 15-
   mL vial and was immediately sealed with a crimped-on,
   Teflon-faced silicone rubber serum cap (Supelco, Bellefonte,
   Pennsylvania, USA). Each vial was equilibrated at
   50 °C for 30.0 min in the temperature-controlled autosampler
   carousel of a model CP-8200 SPME mode autosampler
   (Varian, Palo Alto, CA, USA). A 50/30 μm divinylbenzene/
   carboxen on polydimethyl siloxane fiber (Supelco) was
   then exposed in the vial headspace for 1.0 min.
   Analysis of Furaneol® The same procedure as for 2-
   furfurylthiol and 2-ethyl-3,5-dimethylpyrazine was used
   except that the SPME fiber coating was polyacrylate
   (Supelco) and the extraction time in the headspace was
   30.0 min instead of 1.0 min.
   Gas Chromatography Parameters After exposure in the
   vial headspace, the fiber was retracted into its housing and
   Table 1 Six coffee samples and their coffee additive combinations
   Group Sample code Additives added for each cup
   2.45 g creamer 2.45 g sucrose 1.00 mL sucralose solutiona 1.00 mL spring waterb
   Black samples B x
   BSl x
   BS x x
   Creamer-added samples C x x
   CSl x x
   CS x x x
   a The sucralose solution concentration was 0.35%(w/v).
   b Spring water was added to adjust the final cup volume to 24.5 mL.
   Chem. Percept. (2008) 1:147–152 149
   was then inserted into the gas chromatograph (GC) inlet
   port, which was held at 250 °C. The fiber was exposed
   5.0 min for desorption. The purge valve was opened during
   the last 2 min of desorption. The column oven initial
   temperature was 35 °C; this was maintained for 5.0 min and
   then programmed to 250 °C at the rate of 6 °C/min. The
   carrier gas was helium. The GC was a Hewlett Packard
   model 5890 series II instrument equipped with a flame
   ionization detector (FID). The column was 30-m, 0.25-mm
   inner diameter fused silica, coated with a 0.5-μm film of
   methylsilicone (DB-5; Agilent). All samples were analyzed
   in triplicate.
   Identification Chromatographic peaks were identified by
   comparison with the retention of authentic standards and
   confirmed with an Agilent GC equipped with an Agilent
   5973 Quadrupole mass-selective detector.
   Quantification External standards prepared in Dannon
   spring water containing 0.2% (v/v) ethanol and adjusted to
   pH 5.4 (the pH of the Espresso coffee) were used to
   calibrate the GC-FID procedure. The concentration ranges
   used for each compound spanned the range found in the
   coffee samples. Peak areas were integrated using the
   Agilent ChemStation program (Rev A.09.03). Results were
   averages of at least three replicate determinations of each
   standard concentration used. If the coefficient of variation
   (CV) was greater than 15%, additional replicates were
   analyzed until the CVs were less than or equal to 15%.
   Results and Discussion
   Difference Test
   A sensory difference test was carried out to find the
   sucralose concentration that provided sweetness intensity
   comparable to that of 10% sucrose in the espresso coffee.
   The results are shown in Table 2. The lowest sucralose
   concentration used (0.0143%) produced a sweetness intensity
   closest to and not significantly different from 10%
   sucrose. This is a 700-fold concentration ratio for equally
   sweet sucrose and sucralose.
   The Effect of Whitener on Coffee Aroma
   Figure 1 shows the liquid-phase concentrations of key
   coffee odorants equivalent to those measured in the
   headspace above black and whitened coffee. As expected,
   the nonpolar pyrazine was much less volatile in whitened
   than in black coffee, while the extremely polar compounds
   were unaffected by the lipophilic whitener. Changes in the
   relative volatility of odorants when lipids are added to an
   aqueous matrix occur because lipophilic odorants such as 2-
   ethyl-3,5-dimethylpyrazine (log P=1.96, where P is the
   octanol–water partition coefficient) are extremely soluble
   and less volatile in the lipid phase, while less lipophilic
   compounds like 2-furfurylthiol (log P=0.9) or the extremely
   nonlipophilic compound Furaneol® (log P=−1.69)
   become more volatile in the presence of lipids. This
   differential effect on volatility can distort odor perception
   if the magnitude is significant. However, as shown in
   Fig. 2, none of the qualia were affected by the addition of
   whitener (p=0.05). The “malty” and “caramel” qualia were
   the weakest while the “roasty” and “coffee” notes were the
   most intense. Greater changes upon the addition of creamer
   have been reported in brewed coffee (Bücking and
   Steinhart 2002; Calvino et al. 1990). Perhaps the higher
   lipid content of espresso minimizes the effects of added
   lipid. These results are like those of Maeztu et al. (2001),
   which found no correlation between the “malty” character
   and the concentrations of Strecker aldehydes that are
   thought to be malty aroma contributors. In this study, the
   whitener had a significant effect on relative odorant
   volatility but no effect on perception.
   Table 2 Mean scores for sweetness intensity of the blind control and
   coffee with several levels of added sucralose
   Sample Sweetness score, mean±SD
   Blind control (10% [w/v] sucrose) 4.62±1.80 a
   0.0143% (w/v) sucralose 4.76±2.09 a
   0.0195% (w/v) sucralose 6.30±1.80 b
   0.0250% (w/v) sucralose 7.10±1.66 c
   All pairwise comparisons were made by Tukey’s test at the 95%
   confidence level. Means with the same letter were not significantly
   different.
   Furaneol EDM-Pyrazine Furfuryl-SH
   0
   500
   1000
   1500
   ng/mL (aqueous equilivant)
   Black Creamer
   }
   Fig. 1 The concentrations in nanograms per milliliter liquid-phase
   equivalent of the key coffee odorants in the headspace above black
   and whitened coffee (mean±standard errors)
   150 Chem. Percept. (2008) 1:147–152
   The Effects of Sweeteners on Coffee Aroma
   Figure 3 shows the concentrations in nanograms per
   milliliter (liquid phase equivalent) of the key coffee odorants
   in the headspace above unsweetened, sweetened with
   sucrose, and sweetened with sucralose coffees (mean±
   standard errors). It is striking how completely unaffected
   the concentrations of the most potent odorants in coffee
   headspace were by any of the treatments. Although there
   may be a slight reduction in the apparent volatility of 2-
   ethyl-3,5-dimethylpyrazine in the coffee sweetened with
   sucrose, there was no significant difference (at the 95%
   level) between the sucrose and sucralose samples. Ebeler
   (2001) stated that compound volatility would only be
   affected when sucrose concentration is above 20% (w/w).
   This implies that at the olfactory receptor, the concentrations
   of the key odorants released from espresso coffee
   are unaffected by the presence of different sweeteners, even
   though sucrose is 700 times more concentrated than
   sucralose. A previous report predicted that the retention of
   hydrophobic compounds should be greater due to an
   increase in hydrophobicity of the matrix after sucrose is
   added (de Roos and Wolswinkel 1993). The current study
   does not support this idea even for Furaneol® (log P=
   −1.69), one of the most hydrophilic compounds found in
   foods. Therefore, any changes in odor due to the presence
   of sweeteners must be the result of top-down processing
   (the modulation of responses initiated at the periphery by
   signals from more central areas of the brain, Wilson and
   Stevenson 2006) and not due to modulation of the chemical
   processes at the periphery.
   Figure 4 shows the effects of sweetener on the four
   coffee attributes or qualia. It is assumed that, as a result of
   training, these qualia change only in intensity and not in
   their nature, but we have no way to verify this experimentally.
   There were no significant changes in the “malty”
   qualia caused by either sweetener, while significant
   increases (>100%) in the “caramel” note were caused by
   the presence of either sweetener. The magnitude of the
   effect was the same for both sucrose and sucralose even
   though their concentrations were 700-fold different. That
   these two sweeteners had the same sweetness intensity
   indicates that the enhanced caramel intensity was caused by
   a top-down process from a place in the brain remote from
   the periphery. In contrast, the “coffee-like” and “roasty”
   notes were significantly (p=0.05) reduced by the presence
   of both sweeteners to similar extents. This again supports
   the notion that the effects are central and not peripheral. It
   is possible that some of these shifts in relative perceptual
   intensity are the result of “dumping,” the process in which
   an attribute appears to change in nature due to ballot
   restrictions (Lawless and Heymann 1998). Dumping has
   only been observed with naïve consumer panels (Clark and
   Lawless 1994); untrained subjects tend to colocalize taste
   and smell and lack the ability to parse their perceptions.
   Fig. 2 Intensity scores for each sensory attribute (mean±standard
   errors)
   Fig. 3 The concentrations of the key odorants (mean±standard errors)
   above the coffee treatments as described in Fig. 1
   Fig. 4 Intensity scores for each sensory attribute (means±standard
   errors)
   Chem. Percept. (2008) 1:147–152 151
   This effect has been shown to diminish with training
   (Bingham et al. 1990).
   Prescott reported that flavor perception is highly dependent
   on both past experience with specific odor–taste
   combinations (the origin of congruence) and on cognitive
   factors that influence whether or not the flavor elements are
   combined. In their studies, subjects reported that they
   tended to associate “roasty” character with barbeque sauce,
   which is often sweet. Thus, it is possible that the “roasty”
   character may be associated with “sweetness” for some
   people, and the relevant example is the usual co-occurrence
   of the two characters in barbeque sauce. Once more, this
   might be a result of the interaction between smell and taste
   (Blake 2004). This phenomenon is not uncommon, as prior
   studies have shown that pairings of certain tastes and
   certain odors can cause mutual enhancement or suppression
   in perceptions of the sensory qualities (Hornung and Enns
   1994) (Keast et al. 2004). It is possible that the subjects
   associated “coffee-like” odor with bitterness and/or astringency
   and thus felt the suppression of “coffee-like” odor
   once sweetness, which suppresses perception of other
   tastes, was detected.
   According to Frank and Byram (1988), odors take on
   taste qualities through frequent co-occurrence with particular
   tastes. Since the co-occurrence of sweet taste and
   “caramel” odor is common in many foods and beverages, it
   is possible that the sweet taste increased the intensity of the
   “caramel” quale. It has been reported that sweet taste can be
   evoked when caramel odor is present; such behavior is an
   example of odor–taste synesthesia (Stevenson et al. 1995).
   The results reported here show that the converse is also
   true, that taste can increase the intensity of odor. This is an
   example of taste–odor synesthesia in a real food system,
   espresso coffee.
   Acknowledgment This material is based upon work supported by
   the Cooperative State Research, Education and Extension Service, US
   Department of Agriculture, under project NYG 623-496 and NYG
   623-482.
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