Experimental Design and Methodologies in the Research on Olfactory Perceptual Interactions

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Icewine Samples

Three ranks experimental icewines, based on the level of pressing, Black label, Blue label and Yellow label, were made from Vidal grapes harvested in 2010 from ChangYu Winery in Huanren-on-the-Huanlong Lake, Liaoning province (North-east China). The Black label icewine received the lightest pressing, followed by the Blue label, with the Yellow label reserved for wines from the highest press fraction. Grapes were harvested, destemmed, crushed and pressed at –8ºC to –9ºC, and then, the grape juice was transferred to a stainless-steel container and mixed after adding 60–80 mg/L SO2 and 30 mg/L of pectinase HC (Lallemand, France). Alcoholic fermentation was carried out at 10–12°C for 40–60 d with 200 mg/L of dried active yeast K1 (LALVIN, Canada). Malolactic fermentation was not induced. Stabilization, fining and filtration were involved before bottling, and were commercialized after 12-month aging time. All samples were stored horizontally at 18ºC in the dark prior to analysis. Three bottles were provided for each sample and were analyzed in duplicate.

Aroma Extraction Methods

Solid-phase extraction (SPE) method was used to extract volatile compounds. The column (LiChrolut EN, Merck; 0.5 g of phase) was first rinsed with 6 mL of dichloromethane, then 6 Rules and Mechanisms of Perceptual Interactions in Odor Mixtures: Application to Icewine Aroma mL of methanol and 6 mL of a water-ethanol mixture (11%, ethanol by volume). 50 mL of sample was passed through the column at a flow rate of 1 mL/min. Sugars, pigment and other low-molecular-weight polar compounds were eliminated with 20 mL of ultrapure water. Finally, the sorbent was eluted with 10 mL of dichloromethane. Using nitrogen stream, the organic phase was concentrated to a final volume of 250 μL for GC-O and GC-MS analysis.

Gas Chromatography-Olfactometry and Gas Chromatography Mass Spectrometric Analysis

The instruments used were an Agilent 6890 gas chromatograph equipped with an Agilent 5975 mass-selective detector (MSD) and a sniffing port (ODP 2, Gerstel, Germany). The analytical columns were a DB-FFAP column (60 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent, Torrance, CA) and HP-5MS column (30 m × 0.25 mm i.d., 0.25 μm film thicknesses, Agilent, Torrance, CA). The front inlet was programmed in splitless mode for SPE (1 uL injected), and the oven temperature was initially held at 50°C for 2 min, then raised to 230°C at 6°C/min and held for 15 min. The carrier gas was helium at constant flow rate of 2 mL/min. The effluent supplemented with Helium was split to the olfactory port installed back of the GC detector. The sniffing time was 45 min for each analysis and the capillary, which was connected with the sniffing port, was kept at 250°C. The data acquisition [electron impact (EI) at 70 eV] was in scan mode, 35–500 Da for compound identification.
GC-O analysis was conducted by a panel of four well-trained assessors (two females and two males) from Laboratory of Brewing Microbiology and Applied Enzymology at Jiangnan University. The assessors first analyzed the extracts on both DB-FFAP column and HP-5MS column and record the retention time and descriptors of the odor peak for each compounds. After discussing, checking the aroma descriptor with the chemical standards and remembering the aroma characteristic, aroma extract dilution analysis (AEDA) was used for searching important odorants.

Aroma Extract Dilution Analysis

For AEDA, the concentrated fraction was diluted stepwise (1:3) with dichloromethane. Each dilution was submitted to GC-O analysis under the same GC conditions described above until no odorant could be detected. The flavor dilution (FD) factor of each compound represented the maximum dilution in which the odorant could be perceived. Analysis was repeated in duplicate by each assessor. Only the odorants detected among more than two assessors were recorded.

Aroma Identification and Quantitation

Aroma compounds identification was achieved by comparison of their odors, NIST 05 a.L database (Agilent Technologies Inc., Santa Clara, CA, USA), and their retention indices (RI) on both columns with those of pure standards. RI of the odorants were calculated from the retention times of n-alkanes (C5–C30), according to a modified Kovats method (Cates & Meloan, 1963).
Three methods were involved in aroma quantitation (Table 5). Standard curve concentrations and compounds were quantified on a DB-FFAP column, based on the ratio of the peak area of the compound relative to the peak area of the internal standard to determine the concentration of the analytes. Standard curve concentrations and compounds were quantified in icewine model solution. The formula was referred from icewine model solution (Bowen & Reynolds, 2012) and prepared based on the true concentration in Chinese icewine (12.2 g/L total acid, tartaric acid was used; 159.0 g/L residual sugar, fructose was used; and 11.0% ethanol by volume, with a pH of 3.4).
Methional, guaiacol, furaneol, homofuraneol and γ-decalactone were enriched by SPE methods and quantified by GC-MS. L-menthol (314 mg/L) was used as internal standards. Selective ion monitoring (SIM) mass spectrometry was used to quantitate some aroma compounds, m/z 104 for methional, m/z 128 for furaneol and m/z 142 for homofuraneol. The ion monitored of L-menthol in the SIM run was m/z 138.
Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry. A 50/30 μm DVB/CAR/PDMS fiber (Supelco, Inc.,Bellefonte, PA) was used for aroma extraction. Except for methional, guaiacol, furaneol, homofuraneol and γ-decalactone, other compounds were enriched by headspace solid-phase microextraction (SPME) method and quantified by GC-MS. L-menthol (314 mg/L) and octyl propionate (181 mg/L) were used as internal standards. 8 mL of sample was placed into a 20 mL glass vial with a silicon septum, then added to 10 μL internal standard and saturated with 3 g sodium chloride, and was equilibrated at 60°C for 15 min and extracted for 30 min under stirring at the same temperature. After extraction, the fiber was inserted into the injection port.
Headspace Solid-Phase Microextraction−Gas Chromatography−Mass Spectrometry after Derivatization. 2, 3-butanedione and 1-octen-3-one were quantified after derivatization with PFBHA. First, 8 mL of sample was placed into a 20 mL glass vial and saturated with 3 g sodium chloride, then added to 10 μL of p-fluorobenzaldehyde (1.24 mg/L), which was an internal standard. Finally, 120 μL of PFBHA (50 g/L in water) was added. Then, it was equilibrated at 65°C for 10 min and extracted for 45 min under stirring at the same temperature, then transferred the fiber to the injector for desorption at 250°C for 300 s.
The front inlet was programmed in splitless mode, and the oven temperature was initially held at 50°C for 2 min, then raised to 100°C at a rate of 6°C/min and held for 0.1 min, then to 160°C at a rate of 2°C/min and held for 0.1 min, and finally at 5°C/min to 230°C and held for 10 min. The carrier gas was helium at constant flow rate of 1 mL/min.
The mass spectrometer was operated in electron ionization mode at 70 eV with SIM.
The ion monitored for p-fluorobenzaldehyde after derivatization was m/z 319. Monitored ions of 2, 3-butanedione and 1-octen-3-one after derivatization were 279 and 140 respectively.
Rules and Mechanisms of Perceptual Interactions in Odor Mixtures: Application to Icewine Aroma
Aroma compounds were recombined in odorless icewine and compared with the corresponding real wine. Odorless icewine was prepared as follow. The icewine was extracted by the SPE method until the remaining liquid was odorless, and was freeze-dried to obtain the lyophilisate matrix. Before the recombination, the lyophilisate matrix was dissolved by aqueous solutions containing 10% of alcohol, and was adjust to the icewine concentration level which included 12.2 g/L total acid, 159.0 g/L residual sugar, and 11.0% ethanol by volume, with a pH of 3.4. The aroma compounds with FD≥ 9 (Table 6) of icewine were added into the odorless icewine according to their occurring concentrations (Table 7).
Twelve assessors (seven females and five males, 24 years old on average) were involved in descriptive analysis. They were recruited from Jiangnan University and trained according to the standard (ISO-8586, 2012). ‘Le nez du vin’ (Jean Lenoir, Provence, France) was used as aroma standard to help assessors to describe the odor qualities of 54 odorants. After one year trainings and tests, they showed good performance in flavor memory and discrimination, and also showed good ability in consistency, stability and repeatability for giving scores.
After assessors meeting to discuss the lexicon terms and reach consensus, the final lexicon was generated. The six major descriptions were honey, caramel, apricot, rose, tropical fruit and raisin. Then, assessors were given the icewine reconstitution samples and real icewine samples one by one in a randomly order with three-digit-coded. Assessors needed to score the intensity of each attribute on a seven-point scale from 1 (extremely weak) to 7 (extremely strong). During the session, the assessors evaluated these samples with a 5-min break after each sample.

Table of contents :

Chapter I Review of Literature on Odor Perception
1.1 Olfactory System
1.1.1 Olfactory Epithelium
1.1.2 Olfactory Bulb
1.1.3 Olfactory Cortex
1.2 Dimensions of Odors
1.2.1 Odor Intensity
1.2.2 Odor Pleasantness
1.2.3 Odor Quality
1.3 Odor Mixtures
1.3.1 Perception of Odor Mixtures
1.3.2 Interactions Occurred at Different Stages of the Olfactory System
1.4 Research Progress Related to Olfactory Perceptual Interactions
1.4.1 Perceptual Interactions between Odorants Observed in Foods and Beverages
1.4.2 Methodologies Involved in Research Related to Odor Mixture Interactions
Chapter II Review of Literature on Aroma of Icewine
2.1 Introduction
2.2 Uniqueness of Icewine and Its Aromas
2.3 Origin of Icewine Aromas
2.4 Factors Affecting the Primary Aromas of Icewine
2.4.1 Grape-growing Environment
2.4.2 Grape Varieties
2.4.3 Dehydration and Freeze-thaw Cycles in Icewine
2.4.4 Harvest
2.4.5 Other Vineyard Management
2.4.6 Healthy Grape-growing Condition
2.5 Factors Affecting the Secondary Aromas of Icewine
2.5.1 Pressing
2.5.2 Yeast and Fermentations
2.6 Factors Affecting the Tertiary Aromas of Icewine
2.7 Conclusions and Perspectives
Chapter III Characterization of the Key Aroma Compounds in Chinese Vidal Icewine 
3.1 Introduction
3.2 Materials and Methods
3.2.1 Chemicals
3.2.2 Icewine Samples
3.2.3 Aroma Extraction Methods
3.2.4 Gas Chromatography-Olfactometry and Gas Chromatography Mass Spectrometric Analysis
3.2.5 Aroma Extract Dilution Analysis
3.2.6 Aroma Identification and Quantitation
3.2.7 Aroma Recombination of Icewine by Descriptive Analysis
3.2.8 Aroma Omission Test by Discrimination Analysis
3.3 Results and Discussions
3.3.1 Odor-active Compounds Identification of Icewine
3.3.2 Quantitative Analysis in Yellow Label Icewine
3.3.3 Aroma Recombination and Omission Experiments
3.4 Conclusions
Chapter IV The Contribution of Aroma Compounds in Icewine Considering Odor Mixture-Induced Interactions
4.1 Introduction
4.2 Materials and Methods
4.2.1 Samples
4.2.2 Chemicals
4.2.3 Aroma Extraction Methods
4.2.4 Gas Chromatography−Olfactometry (GC-O) and Olfactoscan Analysis Conditions
4.2.5 Subjects
4.2.6 Gas Chromatography-Olfactometry and Olfactoscan Analysis
4.2.7 Data Process for Detection Frequency (DF) Method
4.2.8 Identification of the Impact Compounds
4.2.9 Data Analysis
4.3 Results and Discussions
4.3.1 Odor Zone Defined in GC-O and Olfactoscan Analysis by the Detection Frequency (DF) Method
4.3.2 Peak Identification and Odor-active Compound Contribution in GC-O and Olfactoscan Analysis
4.3.3 Mixture-induced Effect of Icewine Background Odor on the Detection and Identification of Odor-active Compounds
4.3.4 General Discussion
4.4 Conclusions
Chapter V Factors Influencing the Perception of Odor Mixtures in Icewine
5.1 Introduction
5.2 Materials and Methods
5.2.1 Chemicals and Samples
5.2.2 Stimuli and Delivery Apparatus
5.2.3 Subjects
5.2.4 Experimental Procedure
5.2.5 Rate-All-That-Apply (RATA) Procedure
5.2.6 Data Processing
5.3 Results and Discussions
5.3.1 Sensory Data Quality Assessment
5.3.2 Influence of the Addition of Single Odorants on the Perception of Icewine Odor
5.3.3 Influence of the Combined Addition of Odorants on the Perception of Icewine Odor
5.3.4 Odor Perception of Binary Odor Mixtures Composed of the 11 Key Odorants
5.3.5 General Discussion
5.4 Conclusions
Chapter VI General Law of Olfactory Interaction in Binary Odor Mixtures
6.1 Introduction
6.2 Materials and Methods
6.2.1 Subjects
6.2.2 Stimuli
6.2.3 Sample Preparation
6.2.4 General Procedures
6.2.5 Data Processing
6.3 Results and Discussions
6.3.1 Panel Performance and Repeatability
6.3.2 Intensity and Pleasantness of the 72 Odorants (dataset II)
6.3.3 Perception of Components’ Odor within Mixtures (dataset I)
6.3.4 Overall Intensity of Binary Mixtures (dataset I)
6.3.5 Binary OdorPleasantness Perception (dataset II)
6.3.6 Pleasantness Prediction (dataset II)
6.3.7 General Discussion
6.4 Conclusions
Chapter VII General Discussion and Perspective
7.1 Odor-active Compounds in Icewine and Their Contribution to Icewine Aroma
7.1.1 Icewine Odor-active Compounds Identification
7.1.2 The Contribution of Single Odorants to Icewine Aroma
7.1.3 The Contribution of Different Odorant Combinations to Icewine Aroma
7.2 The Perception of Odor Mixtures
7.2.1 Elemental Coding or Configural Coding
7.2.2 Separation Processing or Completion Processing
7.2.3 Olfactory Perceptual Interactions
7.3 Experimental Design and Methodologies in the Research on Olfactory Perceptual Interactions
7.4 General Perspectives

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