METHOD OF DETERMINING INTERACTIONS BETWEEN SENSORY STIMULI AND APPARATUS FOR USE IN SUCH METHOD

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of determining interactions between sensory stimuli, especially interactions that affect the perception of these stimuli by animals, such as humans. The present method may suitably be applied to detect unknown interactions between different stimuli. The method may, for instance, be used to identify molecules that are capable of masking the impact of molecules causing off- flavours. Likewise, the method may also be used to identify molecules capable of enhancing the perceived intensity of particular flavour molecules.

BACKGROUND OF THE INVENTION

The scientific understanding of interactions between sensory stimuli is very fragmented and largely empirical. It is well-established that, for instance, flavour molecules can strongly affect the olfactory, gustative and/or somato -sensory perception of other flavour molecules. These interactions can, for instance, result in a reduction or increase of the perceived intensity of a particular flavour molecule. It has also been observed many times, however, that flavour molecules affect the perceived flavour character/profile of another flavour molecule rather than its perceived intensity. It is widely accepted that besides olfactory, gustative, and/or somato-sensory

(OGS) stimuli also other stimuli can influence the perceived impact of primary OGS stimuli and vice versa. Examples of such non-OGS sensory stimuli include visual stimuli, acoustic stimuli, tactile stimuli etc.

In order to determine interactions between olfactory/gustative stimuli and other sensory stimuli, complex and laborious studies need to be performed. An important bottleneck in these studies is the availability of OGS stimuli. Although at present thousands of flavour molecules can be purchased as pure compounds, this arsenal of chemical compounds represents only a fraction of the flavour imparting molecules

provided by nature and found, for instance, in spices, herbs, fruit, vegetables, processed food, flowers, etc. Hence, it would be advantageous if a method existed that can suitably be employed to determine interactions between a first OGS stimulus that is provided by an unidentified flavour molecule that is contained in a complex mixture with other flavour molecules (e.g. a flavour extract) and a second well-defined sensory stimulus.

In addition, it would be highly desirable to have available a practical screening method in which a complex mixture of flavour molecules (e.g. a natural extract) can be screened for molecules whose OGS stimulus exhibits an interaction with a particular second sensory stimulus.

SUMMARY OF THE INVENTION

The inventors have developed a screening method that delivers the aforementioned benefits. The present method makes it possible to determine interactions between a first olfactory, gustative and/or somato-sensory (OGS) stimulus provided by a flavour molecule that is contained in a flavour mixture (e.g. a natural extract) and a second sensory stimulus. The method of the present invention employs a sample containing a plurality of flavour molecules, which sample is subjected to a molecular separation to obtain a fraction containing a subset of the mixture of flavour molecules contained in the original sample, which fraction is delivered to an organism's olfactory, gustative and/or somato-sensory (OGS) receptors whilst simultaneously providing a second sensory stimulus to one or more receptors of the same organism.

The present method offers the important additional advantage that it can suitably be used as an effective screening method in which a complex mixture of flavour molecules (e.g. a natural extract) is screened for molecules whose OGS stimuli exhibit an interaction with a particular second sensory stimulus. Thus, the present method can be used as an effective tool for identifying flavour molecules that, for instance, are capable of suppressing off- flavours (e.g. oxidation notes, bitter or sour taste) or that are capable of enhancing aroma and taste (OGS) (e.g. salt, sweet, umami, savoury aroma, CO ₂ taste). Also the present method can be used to investigate the

effect of visual, auditive or other sensory stimuli on the perception of certain flavour molecules and vice versa.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the present invention relates to an integrated method of determining interactions between sensory stimuli, said method comprising:

• providing a first olfactory, gustative and/or somato-sensory (OGS) stimulus by: - subjecting a sample containing a plurality of flavour molecules to a molecular separation to obtain a fraction containing a subset of the flavour molecules contained in the original sample; and

- delivering said fraction to an organism's olfactory, gustative and/or somato- sensory (OGS) receptors; • simultaneously providing a second sensory stimulus to one or more sensory receptors of the same organism; and

• recording the combined impact of the first and second stimulus on said organism.

The term "integrated" as used herein refers to a method in which the action of generating a fraction containing a subset of flavour molecules coincides at least partly with the recording of the combined impact of the first and second stimulus. More particularly, in the present integrated method the combined impact of the first and second stimulus is being recorded whilst the sample that is used to generate the first stimulus is still being subjected to the molecular separation. Since the present method is operated in an integrated fashion the separated fraction can be used to provide the first stimulus without any need for intermediate sample preparation or sample storage.

It should be understood that in case the organism employed in the present method is an animal, the present method is not a method for the treatment of the animal body by surgery or therapy or a diagnostic method practised on the animal body.

According to a preferred embodiment the present method is executed with the help of an integrated apparatus that comprises (i) means for molecular separation of the sample into two or more fractions, (ii) means for delivering at least one of said two or more fractions to an organism's OGS receptors and (iii) means for providing a second sensory stimulus, notably an OGS stimulus.

The term "flavour" as used herein refers to the combination of taste and odour, influenced by sensations of pain, heat and cold and tactile sensations (definition of flavour according to the British Standards Institute). The latter three sensations are also referred to as somato sensory sensations. Thus, the term flavour in the context of the present invention encompasses odour, taste and somato sensory sensations. The term "odour" refers to the part of the flavour that is perceived through receptors in the olfactory epithelium/mucosa (including the trigeminal nerve) or any other receptors located in the nasal cavity. The term "taste" refers to the part of the flavour that is perceived through the taste buds and any other receptors that are located in the oral cavity, the pharynx and/or the upper part of the esophagus. Somato-sensory sensations may be perceived through receptors located in the nasal or oral cavity, the pharynx and/or the upper part of the esophagus.

The OGS impact of a flavour molecule may be generated directly by the flavour molecule itself or it may manifest itself through the perceivable effect said flavour molecule has on the flavour impact of another flavour molecule. It is noted that the term "flavour molecule" as used herein also encompasses fragrance molecules, i.e. odourants that are used in non-food applications, such as perfumes, detergents and personal hygiene products.

According to a particularly preferred embodiment of the present method, the second stimulus is selected from the group consisting of an olfactory stimulus, a gustative stimulus, a trigeminal stimulus, an in-mouth texture stimulus, a temperature stimulus, a visual stimulus, an auditive stimulus or a combination of two or more of these stimuli. Even more preferably, both the first and second stimuli are OGS stimuli.

The term "organism" as used herein encompasses animals, animal tissues, receptors and in vitro systems that are capable of generating discriminating responses to different OGS stimuli. Preferably, the organism in the present method is an animal, more preferably a mammal and most preferably a human.

The present method can suitably be used to, for instance, detect flavour molecules that are capable of suppressing the flavour impact of an off- flavour substance. In order to detect flavour molecules having this particular masking ability, in the present method the off-flavour substance is used to provide the second sensory (olfactory) stimulus. By applying the off- flavour substance in a concentration that is above its flavour threshold level, it is ensured that flavour molecules having a significant masking effect can be

detected because the flavour impact of the off- flavour substance is not detected or its intensity is substantially reduced when the masking flavour molecule provides the first stimulus and the off- flavour molecule the second stimulus. Naturally, a similar protocol may be used to detect flavour molecules that are capable of enhancing the flavour impact of another flavour substance, e.g. sodium chloride, CO ₂ , sugar etc.

If the present method is used to determine the effect of the first stimulus on, for instance, the perceived flavour profile of a second olfactory stimulus, it is important to have a reference profile reflecting the effect of said second olfactory stimulus per se. Thus, according to a preferred embodiment, the present method additionally comprises a comparison of the record of the combined impact of the first and second stimulus with a reference record, said reference record being obtained by:

• providing to the same organism the same second OGS stimulus without simultaneously providing the first sensory stimulus; and

• recording the impact of the second OGS stimulus on the organism. Particularly reliable results can be obtained from the comparison of the record of the combined impact of the first and second stimulus and the reference record if both records are obtained from the organism within a relatively short timeframe, e.g. within 1 hour. Preferably said records are collected within a time frame of less than 15 minutes, more preferably of less than 10 minutes. The results obtained by the present method are particularly useful if both the first and second stimulus are provided repeatedly to the same organism. Typically, in the present method, both the first and second stimulus are provided to the same organism at least three, more preferably at least five times and the combined impact of these stimuli is recorded as many time, i.e. preferably at least three times and more preferably at least five times. Typically, the aforementioned repetition of providing the first and second stimuli is conducted within a time frame of less than 40 hours, more preferably within a time frame of 12 hours, most preferably within a time frame of 2 hours.

According to another preferred embodiment, different organisms are used in the present method whilst applying the same first and second stimulus for each organism, and the results obtained for the different organisms are averaged or otherwise aggregated statistically. Thus, for instance, a panel of individuals may be used in the present method and a reliable result can be obtained by averaging or aggregating the combined impact that was recorded for each individual.

In case the same stimulus is to be provided repeatedly, it is important to ensure that said stimulus is generated in a reproducible manner. In case the second stimulus is an OGS stimulus, this may advantageously be achieved by using an olfactometer or a gustometer. The present method may suitably employ any molecular separation technique that is capable of partitioning a sample into a plurality of samples containing different subsets of the flavour molecules present in the original sample. Examples of suitable molecular separation techniques include chromatography, distillation, membrane filtration and combinations of these techniques. Most preferably the molecular separation technique employed in the present method is a chromatographic separation technique, e.g. gas chromatography or liquid chromatography.

In case of gas chromatography, for instance, a cryo-focussing modulator may advantageously be used to obtain separated fractions containing flavour molecules. Cryo-focussing offers the advantage that it facilitates synchronisation of the first and second stimulus.

In the present method the first OGS stimulus may be provided by directly providing the fraction obtained from the molecular separation to the organism. Thus, for instance, the eluent obtained from gas chromatography or liquid chromatography can be transferred immediately to the organism, e.g. by introducing said eluent into the nasal or oral cavity.

The combined impact of the first and second stimulus on the organism may be recorded, for instance, by means of self-reporting and/or by measuring fluctuations in the animal's brain activity or biological functions. In case the organism is a human, the combined impact is advantageously recorded by means of self-reporting. In case a sequence of first OGS stimuli is provided to the organism, it is advisable to ensure that there is sufficient time between these stimuli to ensure that there is no "carry-over" effect. Typically, this can be achieved by interspacing the first OGS stimuli with a period of at least 0.1 second, e.g. 0.1-300 seconds, during which no first OGS stimulus is provided. The duration of the first OGS stimulus is typically within the range of 0.1 -60 seconds.

In the present method advantageously at least two different fractions of the original sample are delivered to the organism's sensory OGS receptors. According to a

particularly preferred embodiment of the present method, said method is conducted in recursive fashion as defined below: a. provide a sequence of first OGS stimuli by: o subjecting a sample containing a plurality of flavour molecules to a molecular separation to obtain x fractions containing a subset of the flavour molecules contained in the original sample; o delivering each of said x fractions to an organism's OGS receptors; b. simultaneously provide a synchronised sequence of identical secondary stimuli to one or more receptors of the same organism; c. record the combined impact of each of concurrent first and second stimulus; d. select a fraction that has the desired impact on the organism and repeat a. to c. by treating this fraction as a sample. Naturally, when the separated fraction is subjected to yet another molecular separation other conditions or even another separation technique may be employed than were employed in the molecular separation of the original sample.

Using this methodology, only a limited number of cycles are required to identify a very small subset of flavour molecules or even a single flavour molecule that exhibit(s) a particular interaction with the secondary stimulus. Typically, the number of cycles required to achieve the desired result is in the range of 2-15, especially in the range of 3-6. In the aforementioned protocol, x advantageously is an integer in the range of 2-20, especially 2-10.

Another aspect of the invention relates to an apparatus for determining sensory interactions between flavour molecules in an animal, said apparatus comprising:

• a molecular separation device selected from the group consisting of gas chromatographs and liquid chromatographs comprising conduit means for delivering a first gaseous or liquid flow containing a molecularly separated fraction;

• an olfactometer or a gustometer comprising conduit means for delivering a second gaseous or liquid flow containing at least one pre-selected flavour molecule; wherein the exits of the conduit means of the olfactometer or the gustometer and the conduit means of the separation device are in close proximity (e.g. less than 5 cm) and aligned or wherein the conduit means of the olfactometer and the conduit means of the separation device are joined to produce a combined flow comprising the first and the

second flow, the apparatus being provided with an exit through which the combined flow can leave said apparatus.

According to a particularly preferred embodiment of the present apparatus, the molecular separation device comprises a means for selectively controlling the introduction of the molecularly separated fraction to the first flow and the olfactomer or gustometer comprises means for selectively controlling the introduction of the at least one selected flavour molecule to the second flow. Even more preferably, the apparatus comprises means for selectively controlling the introduction of the at least one selected flavour molecule and/or for selectively controlling the introduction of the molecularly separated fraction dependent upon programmed input. It should be understood that the molecularly separated fraction can also be used as such as the first flow, in which case introduction of said fraction to the first flow can be controlled, for instance, by switching between a flow consisting of said first fraction and another baseline flow. According to another advantageous embodiment of the present apparatus, the molecular separation device comprises means for collecting, holding and releasing the flavour molecules contained in the first gaseous or liquid flow, wherein said means is arranged to release the collected fraction at timed intervals. This particular set-up of the present apparatus facilitates simultaneous delivery, at timed intervals, of a stimulus from the olfactomer/gustometer and a stimulus from the molecular separation device. The invention is further illustrated by means of the following examples.

EXAMPLES

An apparatus comprising a gas chromatograph with a sniffing port and an olfactometer was used to investigate the effect of odourants contained in a flavour mixture on the off- flavour impact of methional (a typical off- flavour note in alcohol- free beer).

Trained panellists (N=8, 2 repetitions, age 28-52, 1 male) were exposed to an odour sequence (orthonasal delivery) containing methional pulses (saturated headspace, 3-9 ppm in propylene glycol), delivered by a computer controlled air dilution olfactometer. These pulses were mixed with other odourants delivered by the aforementioned chromatograph system. The concentrations of the latter odourants had

been pre-set to a level generating the same perceived intensity as a 6 ppm methional pulse (following evaluation by 3 panelists). For that purpose, the odourants listed in Table 1 were all dissolved in methanol in concentrations also specified in Table 1. The sample was subjected to GC separation applying a liquid injection of 3 μl and a split 1:7.

Table 1

During the evaluations, pulse delivery from the olfactometer was continuous with an inter stimulus interval of 20 sec. to avoid carry-over effects. The latter was achieved by means of a modulator that allows capturing and triggered release from the GC. Synchronisation of the mixed pulses was achieved by ensuring that the methional pulses were delivered at the same time that each of the above odourants eluted from the gas chromatograph column through the sniffing port. Panellists indicated by hand sign when they perceived methional. Correct identifications were pooled per odourant. The collected data were analysed statistically according to paired comparison tests. The results obtained from the screening study are summarised in Table 2.

Table 2

From the above results it can be concluded that in case of octanal the number of correct identifications decreased strongly with decreasing methional concentration, whereas no such effect was observed for the other odourants. These results suggest that odour-odour interactions between octanal and methional cause a perceptual masking of methional.

Furthermore, these results show that olfactometry, chromatography and sensory analysis may advantageously be combined in an in vivo method that enables rapid screening of e.g. odour-odour interaction. A similar set-up using a taste-delivery system (e.g. a gustometer) may be used to study cross-model aroma-taste interactions to enhance (sweetness, saltiness) or mask (bitterness, sourness) taste qualities.

Example 2

In order to verify whether indeed odour-odour interactions between octanal and methional can cause a perceptual masking of methional in foodstuffs and beverage, the effect of octanal on methional perception was tested in orange juice.

In orange juice, methional was introduced at a level of 12 ppb, thereby causin a musty, potato-like off-note. A total of five different orange juice samples were evaluated:

1. oranges juice (reference), 2. orange juice with 12.5 ppb methional,

3. orange juice + 1.25 ppm octanal,

4. orange juice + 12.5 ppb methional + 1.25 ppm octanal,

5. orange juice + 12.5 ppb methional + 2.25 ppm octanal.

The sensorial characteristics of the samples were evaluated by 10 trained panellists using Quantitative Descriptive Analysis (17 sensory descriptors). The results are depicted in Table 3.

* significant at 5 % ** significant at 1 % *** significant at 0,1 %

In Table 3 the letters A, B and C and the pairs formed by these letters are used to show which samples scored significantly different for the indicated sensory attribute at the indicated significance level (right hand column). A sample classified as "A" is significantly different from samples classified as "B","C", "BC" and vice versa. Likewise, a sample classified as "AB" is significantly different from samples classified as "C".

Clearly, these results show that the addition of octanal to orange juice containing methional greatly improved the flavour quality. The typical orange note was restored and methional associated attributes such as "potato, musty, decayed orange, hydrolysate" were suppressed. At higher levels (2.25 ppm), octanal not only masked methional perception, but also contributed a chemical, grapefruit-type note.

Overall, these results demonstrate, that the masking interactions of octanal on methional perception, as previously identified by the present screening technique, are also observed in a real food system (orange juice). Hence, it is clear from this data that the screening technique of the present invention provides meaningful data about interactions between olfactory stimuli.

Example 3

The procedure described in Example 1 was repeated, except that this time the olfactometer produced citronellal-pulses instead of methional pulses. Furthermore, in this particular study the citronellal concentration was kept constant (200 ppm in propylene glycol) but the injection volume of the compounds of Table 1 was varied (2- 3 μl; split 1:7). The results of this study are shown in Table 4.

Table 4

The results show that octanal suppressed citronellal perception in a statistically significant, concentration-dependent fashion. A similar effect was not observed for the other odourants, These findings suggest that, for the odour pair octanal-citronellal, a similar competition mechanism for binding sites at the olfactory level exists in humans as has been described for rats (Kay et al., 2003, Shirokova et al., 2005, Schmiedeberg et al, 2007).

Odourant specific competition for receptors is a proposed mechanism for aroma masking (Kay et al., 2003; Oka et al, 2004). These masking interactions can be identified in a practical fashion with the screening methodology according to the present invention. Also this data shows that olfactometry, chromatography and sensory analysis may advantageously be combined in the present screening method for rapid screening of e.g. odour-odour interaction. A similar set-up using a taste-delivery system (e.g. a gustometer) may be used to study cross-model aroma-taste interactions to enhance (sweetness, saltiness) or mask (bitterness, sourness) taste qualities.

Literature:

Kay LM, Lowry, CA, Jacobs HA (2003). Receptor contributions to configural and elemental odor mixture perception. Behavioral Neuroscience, 2003, 117: 1108-1114. Oka Y, Nakamura A, Watanabe H, Touhara K (2004). An odorant derivative as an antagonist for an olfactory receptor. Chemical Senses, 29: 815-822.

Schmiedeberg K, Shirokova E, Weber HP, Schilling B, Meyerhof W, Rrautwurst D (2007). Structural determinants of odorant recognition by the human olfactory receptors ORlAl and ORlAs. Journal of Structural Biology. 159: 400-412. Shirokova E, Schmiedeberg K, Bedner P, Niessen H, Willecke K, Raguse JD, , Meyerhof W,

Rrautwurst D (2005). Identification of specific ligands for orphan receptors. The Journal of Biological Chemistry. 280: 11807-11515.