The authors would like to acknowledge the support of the Australian National Health and Medical Research Council and Deakin University. The work performed at the Deakin University Sensory laboratory was supported by the National Health and Medical Research Council Grant (1043780)(RSJK) and Horticulture Australia Limited (BS12006)(RSJK).

The following techniques have been approved for use by the Deakin University Human Research Ethics Committee.
1. Demographics and Anthropometry
<ol>
	<li>Record demographic information from participants, including date of birth and gender.</li>
	<li>At baseline (and other study points if temporal study design) take height and weight measurements. Ensure that participants have taken off shoes, heavy jackets or other clothing items, and have removed any heavy items from their pockets.
	<ol>
		<li>Measure participant&#39;s height using a stadiometer. Record measurements to the nearest cm.</li>
		<li>Weigh participants using dedicated scales. Record weight to the nearest g.</li>
		<li>Calculate BMI using the equation:&#160;weight (kg)/height2&#160;(m2). From this, participants are categorized according to standard BMI definition values; healthy 18.5-25 kg/m2, overweight 25-30 kg/m2 or obese &#62;30 kg/m2 27.</li>
	</ol>
	</li>
</ol>
2. Producing Samples for Oral Fatty Acid Threshold Assessment
<ol>
	<li>Use non-fat UHT milk as the base for fatty acid taste threshold assessment. Product can be purchased and stored in bulk if necessary, and will keep unopened for up to 6&#160;months or until the product has reached its expiry. Prepare 2&#160;types of vehicles: vehicle with added fatty acid and a control vehicle. The volume of solutions prepared for testing will depend on participant number. The following protocol provides typical amounts&#160;for&#160;2&#160;participants.</li>
	<li>Prepare a base milk solution to use for both the control and fatty acid vehicle by placing 5% w:v food grade gum arabic (e.g., 100 g per 2 L of milk) into a 3&#160;L glass beaker. If required, hydrate gum prior to use (this will vary depending on gum manufacturer).</li>
	<li>Add 0.01% w:v EDTA to the gum to prevent oxidation (e.g., 200 mg per 2 L of milk).</li>
	<li>Allocate approximately 1 L of non-fat milk per participant (e.g., for 2&#160;participants, use 2 L milk) and pour into beaker.</li>
	<li>Using a laboratory grade mixer with emulsor screen, homogenize the solution at 12,000 rpm for 2 min. Set solution aside.</li>
	<li>Prepare the fatty acid solutions using food grade C18:1. Oxidation can be assessed through gas chromatography if necessary.</li>
	<li>Prepare a series of 13 variants of the fatty acid vehicle (UHT non-fat milk) with increasing concentrations of C18:1 (0.02, 0.06, 1, 1.4, 2, 2.8, 3.8, 5, 6.4, 8, 9.8, 12, and 20 mM/L). To do this, label 250-ml&#160;glass beakers with each concentration.</li>
	<li>Add 5% liquid paraffin to each beaker (e.g., 5 ml&#160;paraffin per 100 ml&#160;of milk solution).</li>
	<li>Based on C18:1 concentration, add the appropriate amount of C18:1 to each beaker (see Table 1).</li>
</ol>
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C18:1 concentration (mM)</td>
			<td>&#956;l/100 ml</td>
		</tr>
		<tr>
			<td>0.02</td>
			<td>0.56</td>
		</tr>
		<tr>
			<td>0.06</td>
			<td>1.9</td>
		</tr>
		<tr>
			<td>1</td>
			<td>31.5</td>
		</tr>
		<tr>
			<td>1.4</td>
			<td>44.1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>63.1</td>
		</tr>
		<tr>
			<td>2.8</td>
			<td>88.4</td>
		</tr>
		<tr>
			<td>3.8</td>
			<td>119.9</td>
		</tr>
		<tr>
			<td>5</td>
			<td>157.8</td>
		</tr>
		<tr>
			<td>6.4</td>
			<td>202</td>
		</tr>
		<tr>
			<td>8</td>
			<td>250</td>
		</tr>
		<tr>
			<td>9.8</td>
			<td>309</td>
		</tr>
		<tr>
			<td>12</td>
			<td>380</td>
		</tr>
		<tr>
			<td>20</td>
			<td>631.2</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Example C18:1 concentration per 100 ml&#160;solution. Increasing concentrations (&#181;l/L) of C18:1 are used to prepare the series of 13 emulsions for oral fatty acid threshold testing.
<ol start="10">
	<li>Following use, fill the C18:1 container with N2 to minimize oxidation and store below 4 &#176;C.</li>
	<li>Add the base milk solution to each fatty acid beaker&#160;to a total volume of 100 ml.&#160;Set aside.</li>
	<li>Using the remaining base solution, prepare the control vehicle. In a 2&#160;L glass beaker, add 5% of the remaining volume in liquid paraffin (e.g., 35 ml&#160;liquid paraffin in a final volume of 750 ml) together with the remaining base solution and homogenize for 30 sec per 100 ml&#160;of liquid.</li>
	<li>Homogenize the control vehicle for 30 sec per 100 ml. This step is conducted prior to the fatty acid solutions to prevent contamination with C18:1.</li>
	<li>Homogenize each fatty acid vehicle, beginning with the lowest concentration for 30 sec per 100 ml.</li>
	<li>Sanitize the homogenizer both prior to and following testing.</li>
	<li>As the homogenization process can raise the temperature of the solutions, check temperature of control and C18:1 samples with a thermometer. Serve all samples at RT (20 &#176;C).</li>
	<li>Milk samples must be freshly prepared on the same day as testing. Taste each solution prior to testing to assess freshness and suitability. 
	Note: Depending on volume required, solution preparation will take a minimum of 60 min.</li>
</ol>
3. Oral Fatty Acid Threshold Testing
<ol>
	<li>Ensure that participants have refrained from eating or drinking (including coffee, gum, mouthwash, etc.) for at least 1&#160;hr prior to testing.</li>
	<li>Minimize non-taste cues by conducting testing under red lighting with participants wearing nose clips.</li>
	<li>Use the ascending forced choice triangle procedure to determine oral fatty acid thresholds. Label 30-ml&#160;plastic portion cups with a three-digit identification number. Provide each participant with a set of three 20-ml&#160;solutions in random order; two control vehicles and one fatty acid vehicle with the lowest concentration of C18:1 (0.02 mM).</li>
	<li>To determine a participant&#39;s oral fatty acid threshold, instruct the participant to taste each solution from left to right and expectorate into a sink. Ask participants to not swallow the samples.</li>
	<li>Ask the participant to identify which of the 3&#160;samples is &#39;odd&#39; or &#39;different&#39; and if they are unsure, they must guess (forced choice).</li>
	<li>Have participants rinse their mouth with deionized water after each set of samples.</li>
	<li>If correctly identified, provide the participant with a second set of 3&#160;solutions (2&#160;control and 1&#160;fatty acid solution in random order) with the same fatty acid concentration. If incorrectly identified, provide the participant with a second set of 3&#160;solutions, but with the next highest concentration of C18:1 (0.06 mM).</li>
	<li>Continue this procedure until the participant is able to correctly identify the &#39;odd&#39; sample 3x in a row at the same concentration. The concentration at which they are able to correctly identify the &#39;odd&#39; sample is recorded as the participants C18:1 detection threshold. See Figure 1 for a graphical representation of this process.</li>
	<li>Based on detection threshold, characterize participants as hypersensitive, or hyposensitive to C18:1. In line with previous literature, hypersensitive individuals can detect C18:1 at concentrations &#60;3.8 mM, while hyposensitive individuals require concentrations &#62;3.8 mM. 
	Note: Depending on the number of incorrect answers, the testing procedure can take between 10-30 min to complete.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig1highres.jpg" src="/files/ftp_upload/51236/51236fig1.jpg" /> 
Figure 1.&#160;Ascending forced choice triangle procedure used for determining fatty acid detection thresholds. Participants are provided with three solutions (two control solutions and one C18:1 solution at a given concentration) and asked to identify the &#39;odd sample&#39;. If correct, participants are given a second set of samples with the same C18:1 concentration. If incorrect, the participant is provided with another set of samples with a higher concentration of C18:1. This procedure continues until three &#39;odd&#39; solutions are correctly identified at a given concentration. This point is deemed the individuals &#39;fatty acid detection threshold&#39;.
4. Fat Ranking Task
This task involves participants tasting four samples of instant custard, each with different fat contents (0, 2, 6, and 10%) and ranking them in order of perceived ascending fat concentration.
<ol>
	<li>Prepare 1&#160;batch of custard using non-fat instant vanilla custard powder according to packet instructions. Mix 2 tablespoons custard powder, 1 tablespoon&#160;sugar, and 2 cups&#160;non-fat milk in a microwave safe bowl. If the suggested product is unavailable, this may be substituted with a similar non-fat instant product (e.g., cook and serve custard).</li>
	<li>Using high power, heat the mixture using a 1,400 W microwave in 30 sec intervals for a total of approximately 5 min, or until thick. This may vary depending on the brand of custard and the wattage of the microwave used. Allow custard to cool.</li>
	<li>Label four 500-ml&#160;kitchen bowls (or similar) with fat percentages.</li>
	<li>Divide the custard into 4&#160;separate 100-g batches.</li>
	<li>Add 0, 2, 6, and 10% vegetable oil to each bowl to achieve the desired fat content (e.g., in a 100 g batch, add 0 ml, 2 ml, 6 ml, and 10 ml vegetable oil to achieve respective fat percentages) and combine. Stir each sample well to ensure all ingredients are completely amalgamated.</li>
	<li>Label four 30-ml plastic portion cups with randomized three-digit numbers. Fill the portion cups with 20 g of each custard (1&#160;type of custard per cup).</li>
	<li>Refrigerate samples prior to testing and serve cold (4 &#176;C).</li>
	<li>Carry testing out under red lights to minimize visual cues.</li>
	<li>Have participants taste, swallow and rank the 4&#160;custards from perceived lowest to highest fat content and receive a score out of 5&#160;depending on their responses.</li>
	<li>Scoring for this task is shown in Table 2. 
	Note: Approximate preparation time for the custard samples is 30 min. The fat ranking task should take no longer than 10 min to complete.</li>
</ol>
<table align="left" border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ranking order</td>
			<td>Score</td>
		</tr>
		<tr>
			<td>0, 2, 6, 10</td>
			<td>5</td>
		</tr>
		<tr>
			<td>2, 0, 6, 10</td>
			<td>4</td>
		</tr>
		<tr>
			<td>0, 2, 10, 6</td>
			<td>3</td>
		</tr>
		<tr>
			<td>0, 6, 2, 10</td>
			<td>2</td>
		</tr>
		<tr>
			<td>1, 6, 10, 2</td>
			<td>1</td>
		</tr>
		<tr>
			<td>6, 0, 2, 10</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2, 10, 6, 0</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2, 6, 0, 10</td>
			<td>0</td>
		</tr>
		<tr>
			<td>6, 2, 10, 0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>0, 10, 2, 6</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Fat ranking task scoring. Participants are given 4&#160;samples of custard with 0, 2, 6, or 10% fat added. Participants are asked to rank samples from lowest to highest fat content and score 0 to 5 points (5 being the maximum).
5. Fatty Food Liking
<ol>
	<li>Prepare small samples (5-20 g) of both regular and low-fat options of commercially available foods. Foods include regular and low-fat versions of: cream cheese (served on a cracker), chocolate mousse, cheese, dry biscuits, peanut butter dip served on a piece of carrot, mayonnaise, salad dressing (served on a slice of cucumber), and yogurt.</li>
	<li>Label each sample with a random three-digit number for identification.</li>
	<li>Present samples in a random order to prevent order effects.</li>
	<li>Instruct participants to taste each sample individually. Food items are ingested, but participants can eat as much or as little of each sample as they desire.</li>
	<li>Have participants rate how much they like or dislike each sample. Measure liking using a 100-mm hedonic generalized magnitude scale (gLMS; see Figure 2) ranging from strongest imaginable dislike to strongest imaginable like. Record liking by placing a vertical line at the point which represents the participants like or dislike of the food.</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig2highres.jpg" src="/files/ftp_upload/51236/51236fig2.jpg" /> 
Figure 2.&#160;Hedonic gLMS. The hedonic gLMS30,31 used to assess liking of both regular and low fat commercially available foods. Participants taste and rate each sample and place a vertical line at the point which best represents their like, or dislike of the sample.
<ol start="6">
	<li>Non-taste inputs are not minimized for this task, so carry this task out under normal light and do not have participants wear nose clips.</li>
</ol>
6. Tongue Photography
<ol>
	<li>Set up a camera and tripod for photography. Regular indoor lighting is sufficient.</li>
	<li>Set the camera to macro mode (or similar) for close up photography.</li>
	<li>Use a hole punch to create a 6-mm diameter circle on a 1.5 cm x 1.5 cm (or similar) square of filter paper. Label the paper with the participant&#39;s identification number.</li>
	<li>In a 50-ml beaker, combine blue food coloring with deionized water at a 1:20 ratio. A small amount is required per participant.</li>
	<li>Pour 30 ml food grade ethanol into a 50-ml&#160;beaker for tweezer sterilization.</li>
	<li>Using masking tape, mark a 20 cm x 30 cm rectangle on the side of the testing table (this should be regular desk height), as shown in Figure 3a.</li>
</ol>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig3highres.jpg" src="/files/ftp_upload/51236/51236fig3.jpg" /> 
Figure 3.&#160;A) Tongue photography setup. Demonstration of the table setup required prior to tongue photography. B) Tongue photography. Demonstration of the tongue photography method
<ol start="7">
	<li>Have participants place their elbows on the marked corners of the rectangle, rest their chin in their palms and to comfortably protrude their tongue, using the lips to steady this position (Figure 3b). The participant must remain in this position for the duration of the testing.</li>
	<li>Using a rectangular (1.5 cm x 3 cm) strip of filter paper, briefly dry the bottom portion of the tongue.</li>
	<li>Dip a cotton bud into the food coloring/water solution and transfer a small amount of dye onto the anterior dorsal surface of tongue, immediately right of the midline point and close to the tip (see Figure 4). Dry the tongue for a second time with filter paper.</li>
	<li>Dry ethanol sterilized tweezers with paper towel and using tweezers, place the pre-labeled 1.5 cm2 filter paper onto the participant&#39;s tongue, with the 6-mm hole over the blue food coloring (see Figure 4).</li>
	<li>Using flash, take three-digital photographs of the participant&#39;s tongue. For confidentiality, ensure only the participant&#39;s mouth and tongue are visible.</li>
	<li>Remove the 1.5 cm2 filter paper from the participant&#39;s tongue with tweezers that have again been sterilized in food grade ethanol. Upload photographs to a photo editing software and with the zoom function, count all visible fungiform papillae.</li>
	<li>Differentiate fungiform papillae from other papillae as larger mushroom shaped, elevated structures. They do not take on the dye solution as strongly, and as such appear much lighter in color. 
	Note: Tongue photography should take no longer than 10 min to complete.</li>
</ol>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig4highres.jpg" src="/files/ftp_upload/51236/51236fig4.jpg" /> 
Figure 4.&#160;Quantifying fungiform papillae density. Location of the 6-mm area for fungiform papillae assessment. Using photography editing software, numerical figures indicate each fungiform papilla.

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L., Bagla, R., Morgenson, M., Mirza, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NaCl+thresholds:+relationship+to+anterior+tongue+locus,+area+of+stimulation,+and+number+of+fungiform+papillae.">NaCl thresholds: relationship to anterior tongue locus, area of stimulation, and number of fungiform papillae.</a> Physiol Behav. 72, 373-378 (2001).</li><li>Hayes, J. E., Duffy, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+sugar-fat+mixtures:+sweetness+and+creaminess+vary+with+phenotypic+markers+of+oral+sensation.">Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation.</a> Chem Senses. 32, 225-236 (2007).</li><li>Tepper, B. J., Nurse, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+perception+is+related+to+PROP+taster+status.">Fat perception is related to PROP taster status.</a> Physiol Behav. 61, 949-954 (1997).</li><li>Tepper, B. J., Nurse, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PROP+taster+status+is+related+to+fat+perception+and+preference.">PROP taster status is related to fat perception and preference.</a> Ann N Y Acad Sci. 855, 802-804 (1998).</li><li>Shahbake, M., Hutchinson, I., Laing, D. G., Jinks, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantitative+assessment+of+fungiform+papillae+density+in+the+human+tongue.">Rapid quantitative assessment of fungiform papillae density in the human tongue.</a> Brain Res. 1052, 196-201 (2005).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Database+on+Body+Mass+Index.+BMI+classification.">Global Database on Body Mass Index. BMI classification.</a> World Health Organisation. , (2006).</li><li>Astrup, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+as+an+adaptation+to+a+high+fat+diet:+Evidence+from+a+cross+sectional+study.">Obesity as an adaptation to a high fat diet: Evidence from a cross sectional study.</a> Am J Clin Nutr. 59, 350-355 (1994).</li><li>Mattes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+preference+and+adherence+to+a+reduced-fat+diet.">Fat preference and adherence to a reduced-fat diet.</a> Am J Clin Nutr. 57, 373-381 (1993).</li><li>Duffy, V. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+preference+questionnaire+as+a+screening+tool+for+assessing+dietary+risk+of+cardiovascular+disease+within+health+risk+appraisals.">Food preference questionnaire as a screening tool for assessing dietary risk of cardiovascular disease within health risk appraisals.</a> J Am Diet Assoc. 107, 237-245 (2007).</li><li>Duffy, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+food/beverage+liking:+A+tool+for+epidemiological+studies+to+connect+chemosensation+with+health+outcomes.">Surveying food/beverage liking: A tool for epidemiological studies to connect chemosensation with health outcomes.</a> Ann NY Acad Sci. 1170, 558-568 (2009).</li><li>Running, C. A., Mattes, R. D., Tucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+taste+in+humans:+Sources+of+within-+and+between-subject+variability.">Fat taste in humans: Sources of within- and between-subject variability.</a> Prog Lipid Res. 52, 438-445 (2013).</li><li>Ralston, A. W., Hoerr, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+solubilities+of+the+normal+saturated+fatty+acids.">The solubilities of the normal saturated fatty acids.</a> J Org Chem. 7, 546-555 (1942).</li><li>Parodi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+fat+in+human+nutrition.">Milk fat in human nutrition.</a> Australian Journal of Dairy Technology. 59, 3-59 (2004).</li><li>Pepino, M. Y., Love-Gregory, L., Klein, S., Abumrad, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fatty+acid+translocase+gene+CD36+and+lingual+lipase+influence+oral+sensitivity+to+fat+in+obese+subjects.">The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects.</a> J Lipid Res. 53, 561-566 (2012).</li><li>Lawless, H. T., Popper, R., Kroll, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+labeled+magnitude+(LAM)+scale,+an+11-point+category+scale+and+the+traditional+9-point+hedonic+scale.">A comparison of the labeled magnitude (LAM) scale, an 11-point category scale and the traditional 9-point hedonic scale.</a> Food Qual Prefer. 21, 4-12 (2010).</li><li>Bartoshuk, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedonic+gLMS:+a+new+scale+that+permits+valid+hedonic+comparisons.">Hedonic gLMS: a new scale that permits valid hedonic comparisons.</a> , (2010).</li></ol>

The authors have nothing to disclose.

The techniques described for determination of oral fatty acid thresholds, fatty food liking, and tongue papillae density have been validated and used in a number of published works in recent years and we suggest oral fatty acid threshold assessment, the fat ranking task and fatty food liking be performed in duplicate at each relevant time point in a study. There has been some discussion regarding the optimum method for assessing detection thresholds32. In particular, the composition of solutions used varies between laboratories, as does the methodology itself. Specifically, the fatty acid used in this protocol, C18:1, we believe is a generally representative and easy to use fatty acid, as opposed to other fatty acids, including linoleic acid (C18:2) and lauric acid (C12:0), which have been used previously9. C18:1 is commonly found in the food supply and unlike C12:0 is liquid at RT, and is more resistant to oxidation than C18:29. C18:1 has also been shown to provide reliable data across multiple testing sessions, and is highly correlated with C18:2 and C12:010. Furthermore, C18:1 has been examined extensively throughout relevant literature, and is thus more helpful for comparisons.
A major point of difference between the protocol outlined within the present paper and other procedures used in other laboratories are the vehicles used for presenting fatty acid stimuli and the systematic approach by which detection thresholds are determined. Two major fatty acid vehicles used within the literature are non-fat milk10,17 and water emulsions6. While both have demonstrated efficacy for fatty acid threshold determination, participants may be more likely to identify the taste of fat within milk, that is, it is unusual to taste fatty acids in water, which may result in lower levels of external validity for studies utilizing a water base. Non-fat milk provides a vehicle for fatty acid chemoreception, without compromising validity. Although these two methods are yet to be directly compared in the literature, it is known that fatty acids are poorly soluble in water33. As a result of fatty acid solubility in milk-based solutions, this emulsion could both be kept longer and be more homogenous than water-based solutions, though this is yet to be confirmed. When implementing this methodology, it is important to note free fatty acids may be naturally present in milk34 and consequently, the product should be used well within its expiry to prevent the increase of free fatty acids (which develop with age) and potential interference with taste threshold performance. Successful preparation of the solutions depends on numerous factors. Firstly, the order in which the &#39;ingredients&#39; are added is imperative. Vehicle preparation steps should carefully follow those outlined earlier to ensure proper vehicle composition and a stable emulsion. Secondly, temperature must be controlled for. Each sample must be presented to participants at RT to ensure participants do not detect the &#39;odd sample&#39; due to factors other than &#39;taste&#39;. Finally, all samples must be correctly homogenized for the suggested period of time. While the emulsion of fatty acids and no-fat milk is more effective than if water were to be used, there is still a chance of emulsion separation within the sample.
The specific testing method used in oral fatty acid threshold determination must also be considered. Two sensory-based methods have been commonly described in the literature; one being the ascending forced choice triangle procedure and the alternate, the staircase method35. The ascending forced choice triangle methodology is an established method for taste threshold determination and can be considered useful for several reasons, including the fact that, unlike the staircase method, the ascending method begins with the lowest concentration of C18:1 (0.02 mM) and increases until the participant is able to detect the presence of fatty acid in solution9. Conversely, the staircase method involves increasing or decreasing the fatty acid concentration from a predetermined midpoint11. However, starting a threshold determination at a point above threshold may cause a desensitization of response impairing ones tasting ability. Further, the ascending method has a lower probability of random chance influencing results (3.7%) compared with the staircase method (11.1%)11. As such, we suggest the ascending forced choice triangle method, combined with non-fat milk as a vehicle for taste testing appears to be an effective means of accurately determining oral thresholds.
Food acceptance or liking testing is one of the more straightforward assessments performed within sensory research and as such there are few problems that tend to arise. However, the type of liking scale used is an important focus. In this case, a hedonic gLMS is the most effective, as it has good discriminatory power and is easy for participants to use36. The end points of the hedonic gLMS are labeled with the descriptors &#39;strongest imaginable dislike&#39; and &#39;strongest imaginable like&#39; and participants evaluate liking against all hedonic experiences, not solely foods30,31. This is effective in controlling for ceiling effects produced by standard 9-point scales, as all experiences are considered and compared. Further, the hedonic gLMS is more able to demonstrate greater individual variance, as the scale is broader36. Food acceptance testing itself may be limited by the foods presented, in that we only present two options per food type. Further research may include several more brands or types of each food, each with differing fat contents, or perhaps specifically made products where fat content can be controlled and is the only variable. It is important to note that the interpretation of all data must be done with caution. While a potential link between liking, preference and intake is plausible and intriguing, results are generated within a laboratory environment and there may be limits to the applicability of these findings to real-world situations.
Assessing papillae density through tongue photography is a more difficult process, with specific steps that must be taken in order to produce appropriate and applicable results. In particular, it is important to identify the correct papillae type. Three types of taste papillae are visible on the human tongue; fungiform, foliate and circumvallate4. Fungiform papillae can however be easily distinguished as mushroom-shaped structures26, and are generally the papillae that are recorded during sensitivity assessments. Fungiform papillae tend to range in concentration from 5-60 per 6-mm area37 (depending on sensitivity), though there have been studies indicating that some individuals may have upwards of 230 papillae the same area21. The type of camera used is fundamental to obtaining appropriate results and may account for this variability. Prior to the use of digital photography in this area, videomicroscopy was the gold standard for identifying and recording papillae density. However, it has been determined that the same level of identification is possible using an appropriate digital camera26. Further, digital photography takes only several minutes, where videomicroscopy may take up to an hour26. Not only this, but digital photography has the potential to be far less costly, and more portable, which may be helpful for use with various participant groups26. Finally, while we aim to measure fungiform papillae density for associations with oral fatty acid detection, we also suggest taste thresholds for the five prototypical tastes also be performed in parallel. Given previous linkage with papillae density and taste function, this could serve as an additional &#39;checking measure&#39; which may add integrity to the data, especially given this is a new area of research.
The area of oral chemoreception research, particularly regarding fatty acids, is an emerging one, and as such it is important for all research to be performed to a high standard, preferably with the use of consistent protocols to allow for direct comparisons.

Excessive dietary fat consumption is a potential contributor to weight gain1-3 and obesity has become a modern day global epidemic. Research suggests higher levels of fat intake, particularly as part of an ad libitum diet, may be associated with a higher BMI2,3, however the factors influencing dietary fat consumption and preference are far from clear. Searching for the mechanisms which underlie fat consumption is therefore an obvious goal and of particular interest is an oral mechanism responsible for fat detection, commonly termed &#39;fatty acid taste&#39;2.
From an evolutionary standpoint, the taste system presumably served as a gatekeeper of the digestive system, guiding the consumption of energy dense nutrients and expulsion of potentially toxic compounds4. The sense of taste is elicited through specialized taste receptor cells which are distributed within three types of tongue papillae; fungiform, foliate, and circumvallate papillae, which can each contain up to several hundred taste buds5. In addition to the widely accepted five prototypical tastes (sweet, salty, sour, bitter, and umami), it is not entirely surprising that there has been suggestion of an oral mechanism for detecting fat, or more likely their breakdown products fatty acids6.
Previous research has consistently shown fatty acids can be detected in the oral cavity over a range of concentrations7-11, despite the fact that it is not a &#39;taste&#39; in the traditional sense, as it has no single discernible perceptual quality associated with it (i.e. sweet)12. Work from our laboratory has highlighted functional implications of impaired fatty acid chemoreception, namely on body weight and dietary fat consumption. Those who are less able to detect fatty acids (hyposensitive) appear to have a higher body mass index (BMI) and consume more energy9, while a relationship between oral fatty acid sensitivity and dietary fat consumption has also been observed; that is, fatty acid hyposensitive individuals have been shown to consume more animal fats, including, meat, high fat dairy, and fatty spreads&#160;all of which are been implicated as contributors to weight gain13. Additionally, individuals who are more sensitive to fatty acids appear to be better equipped at differentiating between samples with varying fat contents9. While other research groups have failed to find similar associations10,14,15, this growing area of research remains intriguing.
These individual differences in fatty acid chemoreception appear to be somewhat modulated by environmental factors, including diet. Habitual fat intake has been associated with impaired oral fatty acid chemoreception and consequently, a heightened preference for, and increased consumption of dietary fat16. In addition to gustatory adaptation, the gastrointestinal tract (GI) also appears responsive to such changes in fat intake17 and impaired GI fatty acid sensitivity may be implicated in the inability to generate appropriate satiety signaling responses which discourage excess energy consumption18.
In addition to environmental factors, fatty acid chemoreception may also be dictated by genetic or physiological differences between individuals, including the concentration of fungiform papillae density (and presumably taste receptors) on an individual&#39;s tongue19. Higher density of fungiform tongue papillae has been linked to heightened oral sensitivity for numerous orally detected compounds including 6-n-propylthiouracil (PROP)20, sugar21, and salt22, while others have also noted an association with creamy perception23. PROP supertasters (who presumably have a higher number of fungiform papillae) are able to distinguish high fat from low fat salad dressings24 and are able to discriminate the fat content and creaminess of dairy foods more accurately than non-tasters23,25. At this stage however, the relationship between fungiform papillae density and oral fatty acid &#39;taste&#39; detection is unknown.
At the basis of human oral fatty acid chemoreception research is the application of various sensory techniques. Identifying individual variability in oral fatty acid detection is a major focus and largely depends on the determination of fatty acid detection thresholds, that is, the lowest point at which fatty acid is able to be detected in solution9. While the specific testing method and stimulus vehicle used varies across the literature and between research groups, the typical procedure involves presenting a participant with a set of emulsified fatty acid and control (no fatty acid) solutions and identifying which is the &#39;odd&#39; sample. Here we present an established, reliable and reproducible method for threshold determination10 using emulsified milk solutions and an ascending forced choice triangle procedure.
The extent to which oral fatty acid sensitivity influences diet, namely consumption of fatty foods, and the ability to perceive fat in foods is also of interest and here we also report on two additional established techniques to further extend our understanding of fatty acid chemoreception. Fatty food liking can be identified by providing individuals with samples of commercially available foods, with both a regular and a low-fat option who are asked to indicate liking of each16. In regards to fat perception, a fat ranking task has been developed by our laboratory, designed to evaluate an individual&#39;s ability to detect fat in custard, a typical food matrix16. To evaluate genetic or physiological differences between individuals, a commonly used tongue photography method involves staining, photographing and quantifying fungiform papillae26. While using this technique in fatty acid research is in its infancy, increasing application, especially in both lean and overweight/obese population groups may help to identify inherent causes of excess fat consumption.

<table><tbody><tr><td>Gum Arabic TIC Pretested PRE-HYDRATED FT Powder</td><td>Alchemy Agencies Ltd. NZ</td><td>CFR# 21 CFR 184.1330</td><td>Food grade agrigum</td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Merck</td><td>1.08418 0250</td><td>Disodium salt dehydrate</td></tr><tr><td>L4RT Homogenizer</td><td>Silverson Longmedow, MA</td><td>L4RT</td><td></td></tr><tr><td>Liquid Paraffin</td><td>Fauldings</td><td></td><td>No catalog number as liquid paraffin is a regular consumable product</td></tr><tr><td>Nikon AF-S VR Micro Nikkor 105-mm f/2.8G IF-ED camera lens</td><td>Nikon</td><td>2160</td><td></td></tr><tr><td>SLIK Sprint Pro II tripod</td><td>Slik Corporation</td><td>611-849</td><td></td></tr><tr><td>Nikon D90 Digital Camera with LCD Protector</td><td>Nikon</td><td>BM-10</td><td></td></tr><tr><td>Nitrogen</td><td></td><td></td><td></td></tr><tr><td>Tanita Body Scan Composition Monitor Scales</td><td>Tanita, Cloverdale, WA, Australia</td><td>BC-551</td><td></td></tr><tr><td>Seca Stadiometer</td><td>Medshop Australia, Fairfield, VIC, Australia</td><td>MED435</td><td></td></tr></tbody></table>

measuring oral fatty acid thresholds, fat perception, fatty food liking, and papillae density in humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

The methods detailed above are important as some emerging evidence has indicated that impaired fatty acid chemoreception in the oral cavity and GI tract may be associated with increases in BMI and the development of obesity17. Several studies have used the described protocols to investigate oral fatty acid detection and our recent publication has shown the method is both reliable and reproducible10. Studies using this methodology have been able to reliably determine individual&#39;s oral fatty acid detection thresholds by identifying the point at which participants are able to detect a difference between milk samples9. After three testing sessions utilizing this protocol, Stewart et al.9 found that the mean detection threshold for C18:1 was 2.2 &#177; 0.1, with detection thresholds ranging from 1-6.4 mM (see Figure 5). More recently, we have established a C18:1 detection threshold range from 0.26-12 mM, (mean: 2.64 &#177; 0.7 mM)10. These results support the notion that fatty acids can be detected in the oral cavity, and that marked individual differences in sensitivity to C18:1 exist. Based on these results, we are able to classify individuals as hypersensitive or hyposensitive to C18:1. Hypersensitive individuals are able to correctly identify C18:1 &#60;3.8 mM, while hyposensitive subjects require concentrations &#62;3.8 mM. Research from our laboratory has found oral sensitivity to C18:1 is associated with dietary fat consumption and BMI (Figure 6), where C18:1 hyposensitive individuals consume more saturated and animal fats and have a higher BMI13. Interestingly, in a study conducted by Stewart and Keast16, it was found that consuming a low fat diet resulted in increased sensitivity to C18:1 for both lean and overweight participants (Figure 7). However, this study also found that when participants consumed a high fat diet, lean individuals had reduced sensitivity to C18:1, while overweight individuals had no change in taste sensitivity (Figure 8). This suggests that habitually consuming a high fat diet, which is more likely for overweight individuals, may result in attenuated fatty acid chemoreception28. However, as there were no differences in baseline sensitivity between lean and overweight participants, these results may indicate that lean individuals are simply more susceptible to dietary changes regarding fat intake. This may also suggest that it was the presence of the specific intervention (high- vs low-fat) that may have influenced outcomes, rather than habitual diets, which may or may not have been different to the intervention diet. Despite this, this study indicates that there are some fundamental differences between lean and overweight individuals regarding fatty acid taste sensitivity, which requires further investigation.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig5highres.jpg" src="/files/ftp_upload/51236/51236fig5.jpg" /> 
Figure 5.&#160;C18:1 taste detection thresholds. Marked variability has been shown in sensitivity to C18:1 with participants able to detect C18:1 across a range of concentrations (1 mM-6.4 mM).
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig6highres.jpg" src="/files/ftp_upload/51236/51236fig6.jpg" /> 
Figure 6.&#160;C18:1 taste detection thresholds and association with BMI. An association between the ability to detect C18:1 and body composition has been shown, whereby those with higher detection thresholds (hyposensitive individuals) have significantly higher BMI values (P = 0.002, r2= 0.467).
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig7highres.jpg" src="/files/ftp_upload/51236/51236fig7.jpg" /> 
Figure 7.&#160;C18:1 detection thresholds following a low fat diet. Following 4 week consumption of a low fat diet, C18:1 detection thresholds increased for both lean and obese individuals.
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/51236/51236fig8highres.jpg" src="/files/ftp_upload/51236/51236fig8.jpg" /> 
Figure 8.&#160;C18:1 detection thresholds following a high fat diet. Following 4 week consumption of a high fat diet, lean individuals displayed reduced sensitivity to C18:1 (P = 0.006), while overweight individuals demonstrated no change (P= 0.609).
Similarly to the impact of diet on detection thresholds for fatty acids, there is research to suggest that food liking may be plastic and altered by exposure. For example, it appears that a high fat diet increases preference for a higher fat product, with the opposite occurring following consumption of a low fat diet16. However, these changes have not been consistent in the literature. It appears that changes to preferences are mediated by the length of time the individual has been adhering to a high or low-fat diet. Specifically, Mattes29 found significant changes in participant food preferences after 12 weeks on a reduced fat diet, while Stewart and Keast16 found only sporadic and marginal changes after four weeks on a similar diet. Consuming a high fat diet altered participant preferences for yogurt, with preferences for low-fat yogurt increasing, converse to expected results (Baseline (BL): 19.44 &#177; 5.73, Week 4 (WK4): 21.94 &#177; 5.21, P = 0.046). Further, after four weeks on a low-fat diet, preferences for low-fat butter increased in all participants (BL: 6.23 &#177; 4.26, WK4: 7.32 &#177; 3.04, P = 0.046). Preferences for low-fat yogurt increased for lean participants only (BL: 2.51 &#177; 3.26, WK4: 3.68 &#177; 4.94, P = 0.07) while preferences for low-fat mousse decreased for all participants (P = 0.01).
The ability to detect fat in foods is assessed by asking participants to taste and rank a series of custards with differing fat contents. Fat perception is identified based on how well participants were able to rank the samples. Fat perception has been known to change with diet, for example, following a low fat diet has resulted in improvements to participant&#39;s performance in correctly identifying and ranking the degree of fat in each custard sample11. Furthermore, it appears that there is an association between sensitivity to C18:1 and identification and ranking of fat content4. Indeed, individuals who were hypersensitive to C18:1 performed significantly better on the fat ranking task (4.3 &#177; 0.6) compared to hyposensitive individuals (2.3 &#177; 0.1, P = 0.02) (scores are out of a maximum of five)9. This indicates that individuals who are more sensitive to fatty acids were also better at differentiating between the four varying fat concentrations in custard. While there was a trend for performance to improve after consuming the low-fat diet for four weeks, this was not a significant change (BL: 1.3 &#177; 0.3, WK4: 2 &#177; 0.3, P = 0.077)16.
Papillae density i.e. the number of papillae (and hence taste buds) present on the tongue varies between individuals and is indicative of taste function. Higher fungiform papillae density has been linked to heightened taste sensitivity, for compounds including sucrose21 and the bitter substance PROP20. Tongue papillae density, as determined by tongue photography, varies considerably between subjects. For example, Zhang et al.21 found that there were significant individual differences between participants, ranging from a concentration of 7.07 &#177; 0.35/cm2 to 233.43 &#177; 0.00/cm2 (data was for a single participant), whereas others26 have found a mean fungiform papillae concentration to be 156.00 &#177; 5.86/cm2. Further, it has been found that papillae can appear significantly differently in structure between individuals, with variation in height, width, and shape,21 though limited evidence regarding the possible implications of these differences. Given previous findings linking papillae number with taste sensitivity, it is plausible that a similar relationship may also exist for oral fatty acid sensitivity, whereby those who are more orally sensitive to fatty acids may have a higher density of taste papillae and therefore a higher number of oral fatty acid receptors. While this association is yet to be established, it presents a novel area of research may help implicate the underlying mechanisms guiding the over consumption of fat.

Watch this Scientific Journal Video about Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans at JoVE.com

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans | Neuroscience | Video

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

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SCIENTISTS IN ACTION

20.2K Views.  Deakin University.  Orale chemoreceptie van vetzuren en de associatie met een dieet en vet voedsel voorkeuren kan de identificatie van mechanismen die betrokken zijn bij de ontwikkeling van obesitas en waarom veranderingen in het dieet kan moeilijk zijn voor veel mensen zijn in staat. 

Video: Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 &deg;C and 60 &deg;C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

Behavior

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Medicine

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously published protocols, threshold estimates are determined using a two-interval forced choice (2-IFC) paradigm with a three down/one up approach. Detection threshold estimates determine the mean force in which a participant can identify the presence of pressure. During the detection threshold procedure, the participant is instructed to choose which of two sequentially presented observation intervals contained the tactile test stimulus. If the participant performs three correct detections in a row (i.e., 3 &#39;hits&#39;), the researcher decreases the stimulus to the next lower target force level. With one incorrect detection (a &#39;miss&#39;), the researcher increases the force delivered to the next higher level. This threshold estimation approach is known as a 3-down/1-up adaptive staircase. Reponses are recorded on a paper ballot, and a participant&#39;s estimated threshold is defined as the geometric mean of five reversals. During the discrimination threshold procedure, the participant is asked to make a choice between two serially presented stimuli as to which is the &#34;harder&#34; or &#34;stronger&#34; pressure. The same scoring of &#39;hits&#39;, &#39;misses&#39;, and stopping points are used. Detection and discrimination testing for oral point pressure at tongue midline takes approximately 20 min to complete. Using these commercially-available clinical tools, individual touch sensation profiles for the midline tongue can be achieved in a relatively time and cost effective means.

This work describes a standard method to assess tactile sensation at the midline of the tongue tip. Using Von Frey Hair (VFH) monofilaments, this protocol provides estimates of detection and discrimination threshold estimates for oral point pressure (OPP).

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously ...

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 mm) to assess spatial lingual tactile sensitivity. During the experiment, the subjects are blindfolded and asked to specify the orientation of the grating (either horizontal or vertical) placed on the tongue. R-index is based on Signal Detection Theory (SDT), and it is an estimated probability of correctly identifying a target stimulus (the signal, e.g., the correct orientation) compared to an alternative stimulus (the noise, e.g., the incorrect orientation). Once the R-index values for each subject and each tool dimension are calculated, it is possible to derive the individual threshold by interpolating the two R-indices immediately below and above the established cut-off (typically 75%) based on one-sided R-index critical values. This procedure can be helpful in the medical field to study the association between oral tactile sensitivity, speech clarity, and swallowing disorders, as well as in sensory and consumer studies to explore individual variation in texture perception, food preferences, and eating behavior.

This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test.

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

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