Source: PURDUE UNIVERSITY submitted to
INTERACTIONS OF DIET, FLAVOR, AND SALIVA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1013624
Grant No.
(N/A)
Project No.
IND030500
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2017
Project End Date
Sep 30, 2022
Grant Year
(N/A)
Project Director
Running, CO, A..
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Nutrition Science
Non Technical Summary
"Good medicine tastes bitter," is a saying that dates perhaps all the way to Confucius. The idea is that things that are good for your health are unpleasant to consume. This phenomenon is actually more than psychology, as many of the chemical compounds in both drugs and foods that are "good for you," also have unpleasant flavors. Indeed, the same properties that make these chemicals unpleasant are also the properties that may drive their contributions to health. In foods, for examples, many of the bitter compounds in vegetables are the exact same chemical compounds that could fight cancer, reduce risk of diabetes, or protect against obesity.This results in a fundamental problem for human nutrition: the foods we should eat are the same foods our mouths tell us are unacceptable. However, there are potential solutions to this dilemma. Research indicates that repeatedly consuming a food, even bitter vegetables, increases its palatability. Some of this is due to simple familiarization with food product; as it becomes more familiar, it becomes more acceptable. However, there may also be actual biological components to this improvement in flavor. Studies in rodents indicate that changing an animal's diet will change its saliva in ways that can make bitter or astringent sensations less aversive3. This seems to be due to the animal being less sensitive to the unpleasant sensations, making the food more acceptable.While research is progressing in rodent studies demonstrating these saliva, diet, and palatability interactions, data are not available in humans. Thus, we propose to study the characteristics of human saliva that may be influencing flavor perception, and how dietary changes could alter the experience of those flavors. We will test fatty, spicy, and bitter compounds in food that could be bound by specific proteins in saliva that are likely to alter their potential as flavor compounds. The compounds responsible for these flavors (unsaturated fatty acids, capsaicin, and polyphenols) all have potential health benefits, such as reducing inflammation, protecting against diabetes and obesity, or reducing cancer risk. Additionally, we will develop a database to collect salivary protein profiles of individuals participating in dietary intervention studies. The database will provide knowledge of new potential target proteins in saliva that may change in response to specific dietary behaviors.The outcomes of this work will be foundational data on how diet influences human saliva, and how that saliva influences flavor perception. If specific proteins in saliva are identified that make unpalatable healthy foods more appealing, then we may be able to intentionally change saliva or foods to mimic that binding of unpleasant flavor compounds. These data may allow us to design functional foods that would bind unpleasant flavors in healthy foods, or to make oral rinses that could be used to enhance a person's salivary profile to block unpleasant flavors from healthy food.Additionally, the patterns of salivary changes in response to diet will allow new opportunities for monitoring human food intake. This could be a valuable tool for verifying dietary compliance. Personal reports of dietary intake are notorious for their variability, as well as for their inaccuracy. We hope that the database we are establishing will one day lead to clear patterns in salivary profiles that link to dietary intake of foods, which could be used to verify dietary self-reports or to estimate of dietary intake of particular food items across a population.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7015010309050%
7026010309025%
7046010309025%
Goals / Objectives
What people eat and what they should eat are often very different. A principle for this is that many individuals prefer the flavor of less healthy foods. Thus, our overarching goal is to find ways to improve healthy food flavor. One unexplored avenue of this may be through modification of saliva. Preliminary data already indicate that saliva influences flavor perception for specific, potentially healthy food compounds (unsaturated fat, spicy compounds, polyphenols), and may be useful for making unpleasant sensations less intense. Thus, our objectives are to:Determine salivary proteomic changes before and after dietary addition of target bioactive flavor compounds (unsaturated fat, spicy compounds, polyphenols). Determine whether flavor perception is altered by changes in salivary proteome.Secondary objective: Some food compounds are structurally similar to salivary proteins that influence flavor. We will also determine whether such food constituents interrupt changes in salivary proteome and flavor perception.Determine changes in the salivary proteome during broader dietary interventions. Such interventions (e.g., low sodium diets, non-nutritive sweetener diets, etc.) are ongoing for multiple other projects in the Purdue Nutrition Science Department, and we will collaborate with the principle investigators for those projects on adding collection of saliva both before and after the dietary intervention. These data will be used to start a database on human salivary proteome and dietary behaviors, which will provide the basis for future work.
Project Methods
Objective 1: Determine how dietary exposure to influences salivary composition and flavorThis objective will be addressed through 3 experiments, one for each bioactive flavor type (unsaturated fat, spicy compounds, bitter polyphenols). Each experiment will be a counter-balanced crossover design with three interventions: 1) beverage containing bioactive flavor, 2) beverage containing bioactive flavor and food-based analog, and 3) control beverage (no bioactive flavor, no salivary analog). Each experiment will begin with preliminary sensory tests and salivary collection, then move into a dietary intervention stage.Preliminary sensory tests: Participants will attend a single visit where they will taste the three types of intervention beverages ("Flavor": bioactive flavor, "Analog": bioactive flavor with food-based analog, "Control": no bioactive flavor nor analog). Participants will first provide baseline saliva: unstimulated (spit into collection tube), and stimulated (chew on 1g piece of wax and spit into collection tube) for 60 seconds each. Then, they will taste the Flavor, Analog, and Control beverages in duplicate, presented in counterbalanced order. For each beverage, participants will first taste and expectorate one sip of the beverages. We will keep this expectorate sample for salivary analysis. Then, they will taste and swallow the rest of the sample. Samples will be rated for the flavors of interest as well as overall liking. We will test 3 different bioactive flavors: Polyunsaturated fatty acids (stimulate oleogustus/fatty acid taste and may interact with salivary lipocalin-1 [LCN1]or β-lactoglobulin [β-Lg]); capsaicin (stimulates spiciness and may interact with LCN1 or β-Lg); and polyphenols (stimulates bitterness and astringency and may interact with salivary proline rich proteins or milk casein).Using data collected during the preliminary sensory tests, we will identify groups of participants with: 1) high and low sensitivity to the flavor of interest and 2) high and low expression of the salivary protein of interest. We hypothesize that these groups will overlap, with those who have high expression of the salivary protein giving lower ratings for the flavor intensity. We also hypothesize that the presence of the analog will reduce the variability in the flavor ratings, resulting in lower correlation of the flavor intensity with the salivary protein of interest.Dietary intervention: For this portion of the experiments, participants from the groups identified in the sensory tests (high/low flavor sensitivity, high/low expression of salivary proteins of interest) will be invited back for a 6-week study. The first week will be a washout week, where participants avoid foods containing the bioactive ingredient. The next week, the participant will drink three 12 oz. beverages per day. This will be repeated for each beverages type (Flavor, Analog, Control), in counterbalanced order. At the end of each week (including both washout and beverages weeks), saliva and sensory ratings will be collected.We hypothesize that intensity of the flavors will decrease and salivary proteins of interest will increase during the Flavor beverage week, but that during the Analog beverage week these changes will not occur. We expect no changes in either flavor or salivary proteins during the control week. We also expect that changes will be larger for participants who initially rate the intensity of the flavors as high, or who initially had low expression of the salivary proteins of interest. The control beverage week will help us control for psychological confounders and novelty of the beverages. By comparing data on the control beverages at the beginning and end of the control week to data from the test beverage weeks, we can establish how much of the changes are due to the psychological exposure and how much do to the actual flavor.Salivary analysis: Saliva will be frozen at -80°C until subjected to proteomic analysis. We will collaborate with the Purdue Proteomics Facility to perform global analysis of saliva proteome by quantitative label-free protein abundance profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Reproducibility of LC/MS platform will be evaluated by determining the coefficient of variation (CV) of peptide intensities and retention time (RT) using 3 technical replicates. From this information we will be able to precisely identify all the proteins expressed in the saliva, and their relative abundances. Further, we will compare the outcomes of proteomics to outcomes from western blots to verify our targeted proteins. However, using the proteomic approach will allow us to explore other potential protein targets, yet unidentified in current literature.Data analysis: We will use mixed models to analyze data from sensory ratings and compare them to salivary profiles. This approach allows for repeated measures analysis of variance with additional consideration for repeated samples or treatments.Participants and sample size: Participants will be healthy adults, age 18-45, with no food allergies. We will aim for 96 participants in the preliminary sensory tests, and 32 participants in the intervention tests (equal proportions of males/females). This will allow for 80% power to find: a 5pt difference in sensory ratings (SD=10), or a 41mg/mL or ng/mL difference for sPRP or LCN1 respectively (SD=50mg/mL or ng/mL).Objective 2: Develop a database of proteomic data on saliva and dietary behaviors.This aim will be addressed by a prospective, hypothesis generating study. The aim is to collect saliva and dietary data during screening visits for studies in the PI's lab and to facilitate saliva collection and analysis in conjunction with dietary intervention studies ongoing at Purdue University. As this database expands, it may allow us to uncover more relationships between dietary behaviors and salivary profile. In addition to our primary goals of identifying new flavor/diet/saliva interactions, analyzing patterns of salivary profiles and diet records may identify new relationships between diet and saliva. If these patters between saliva and diet can be defined, then salivary profiles may be useful in verifying diet records or dietary compliance in future studies in the field of nutrition. Knowing current projects at Purdue system, we expect we will be able to collect data on changes in saliva: with low-calorie sweetener consumption, during low-salt diets, and during high protein diets. These are only some examples based on current work, and we will consider other dietary patterns as the studies are established.Participants: Anyone may participate in this study, pending IRB and (for studies under the purview of other investigators) principle investigator approval. Data will be collected on each individual's health status and dietary habits. The goal is to include as diverse a population of race, ethnicity, health status, age, and sex as possible.Saliva collection: Saliva will be collected after rinsing with water. Both unstimulated (passive drool) and stimulated (chewing wax) saliva will be collected, each for 60 seconds. Tongue scrapings will also be collected. This will allow for analysis of both "free" saliva as well as adsorbed proteins on the surface of the tongue.Analysis: Saliva will be stored and analyzed as described in Objective 1.Expected outcomes: This is an exploratory objective based on preliminary data in animals that indicate salivary expression changes with dietary interventions (personal communications Dr. Ann-Marie Torregrossa, SUNY Buffalo). We expect salivary proteins are modifiable by diet, but this has yet to be established in humans. By starting a database on dietary behavior and salivary proteomes, we can uncover correlations in these factors for future studies on saliva as a tool to predict flavor sensations or to monitor dietary intake of certain foods.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Scientists, dietitians, nutritionists, food industry representatives ? Changes/Problems:As noted previously, the pandemic has completely disrupted this project. Even though in-person human studies have resumed at Purdue, we expect ongoing disruptions to this over the next year. Moreover, as we are collecting saliva, that puts our personnel at risk unless we have a validated way to deactivate the virus in all samples. That certainly could be achieved, but would require external oversight in order to make us comfortable with the approach. Thus, we have shifted to virtual visits. We have developed "assays" that can assess the properties of an emulsion (fat project) that are disrupted/augmented by human saliva, and are currently assessing how the outcome of that saliva-emulsion visual "assay" relates to sensory and dietary data. We have also developed an "assay" to assess the inlfuence of salivary amylase on starch, which can also be done remotely through images/video. Using these tools we will continue the project, shifting away from capsaicin (original proposal) to a starch focus. Thus, we can still assess properties of human saliva and how they relate to sensory properties of foods as well as dietary intake of these food constituents, but we can conduct the project remotely over web conferencing platforms. What opportunities for training and professional development has the project provided?One graduate student has completed part of her thesis on this project. One other graduate student is currently collecting and analyzing data, and a third is preparing the final arm of the study. Two undergradaute students have worked on the project, as well as one postdoc. All have been trained in research methodology, salivary biochemistry, responsible conduct in research, and biohazard handling. How have the results been disseminated to communities of interest?One manuscript has been published, and shared on our social media and website. We have also presented the data to academic and industry representatives through conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to collect and analyze data through our new methodology. We are focusing on: 1-dietary fat intake, pairing that with how fatty acid and triglyceride texture is sensed as well how saliva influences in emulsions; and 2) dietary starch inteake, pairing those data with how starch/oligo-/mono-saccharides of glucose are sensed as well as how saliva influences breakdown of starch. Note: the starch project is a shift due to the pandemic, as we can assess that virtually where we could not assess binding of capsaicin by saliva virtually (at least not through non-sensory methods); the starch approach is also arguably more relevant to human health. We anticipate disseminating results on the fat project in mid 2021,while data collecton for the starch project will begin in mid 2021.

Impacts
What was accomplished under these goals? Current preliminary findings indicate that exposure to polyphenols does suppress later reports on bitterness from the same polyphenols. Analysis of salivary proteins indicates some changes in some salivary cystatins, and very small potential changes in a limited number of salivary proline rich proteins. However, the effect sizes are small than we have observed in the past. We are concerned this may be due to either the liquid nature of the stimulus (minimal time in the mouth) or due to the non-caloric nature of the stimulus (which could have implications for any results of this project). We plan to test a minimally caloric, solid chew containing polyphenols to give an indication of whether the solid/liquid nature of the stimulus influences the salivary response. We will also consider future proposals to test whether co-ingestion of energy influences salivary responses. Because of the pandemic, we also chose to investigate some preliminary data on overall dietary correlations with salivary protein expression (this was an alternate approach/secondary aim of our proposal, and we were unable to continue collecting saliva for the primary aims). Initial results indicate potential relationships between dietary fat and lipocalin-1, which we had hypothesized. We have also successfully revamped our strategy for collecting/assessing saliva, making it possible to continue during the global pandemic. We have deveoped two visual/textural"assays" based on properties of saliva that we are currently assessing via remote visits with participants. We expect these approaches to be very valuable for future efforts, even after the pandemic, as they are relatively simple in execution and yet still display a variety of individual characteristics of saliva.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Crawford C.R. & Running C.A. (2020) Addition of chocolate milk to diet corresponds to protein concentration changes in human saliva. Phys Beh, 225: 113080.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Davis L. & Running C.A. 2020. Exposure to polyphenols alters sensation and salivary protein profile. International Symposium on Olfaction and Taste. Portland, OR. Virtual oral presentation.
  • Type: Other Status: Published Year Published: 2020 Citation: Running C.A. 2020. Sensation, Diet, and SalivaHow oral biochemistry and eating habits may influence individual flavor experiences. Department of Food Science and Nutrition Seminar, University of Minnesota.
  • Type: Other Status: Published Year Published: 2020 Citation: Running C.A. 2020. Making bitter better. Indiana HORT Conference and Expo, Wine and Grapes. Indianapolis, IN, USA.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists, dietitians, nutritionists, food industry representatives Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student, one postdoctoral fellow, and one undergraduate student arecurrently working on the project, being trained in sensory evaluation, saliva processing, data analysis, statistics, and computer coding for data management. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will complete the second study focusing on bitterness, and begin the studies on fattiness and spiciness. We will observe whether changes in saliva are specifc to the bitter compounds, or if other sensations from foods can change spit as well. We also intend to compare larger datasets we have accumulated on dietary behaviors (general dietary patterns, like amount of vegetables, starchy foods, etc.) and salivary protein concentrations.

Impacts
What was accomplished under these goals? We have sucessfully completed one study demonstrating that exposure to polyphenols (the bitter stuff in cocoa products) in a chocolate milk mixture caused people's saliva to change. Many of those changes could potenially reduce the intensity of the bitterness of the chocolate. We are currently working on a second study, which is looking at bitter compounds from green tea and whether those also alter saliva and change the intensity of bitterness that people experience. Knowing that diet changes saliva, in ways that influence the flavor of that diet, is critical. Not just because we as scientists will better understand why some people find certain foods very bitter and palatable and others done, but because of the message that can send folks when they try to improve their diets. Changing your diet to a healtheirdiet is hard, especially if you don't like the flavor of the healthy diet. We hope our work will help send a message to folks trying to swtich to a healthier diet, which typically includes of a lot foods with bitter flavors. That message is simple: stick to it, and you body will help you adapt to like that diet better. Our work, at least initially, confirms this, as we have shown thatsaliva adapts to diet to reduce the bitterness of certain foods. Another accomplishment is in the deveolopment of standardized coding for processing the results we get when we analyze all the proteins in saliva. With our pending publication on the first study mentioned above (about chocolate milk and bitterness), we will publish Python code that allows for easier and faster processing of these large datasets (we have have gigabytes of text filesfrom this work; most standard office computers simply crash when we try to open these files using more typical data analysis approaches). This code may be useful for other scientists in the processing of their own data, and the postdoctoral scholar who is working on this project is adapting that code to be used for teaching purposes as well. Finally, we have collected a few hundred saliva samples and diet records from participants in various studies. These data will be important to address our second goal, understanding broader relationships among diet and saliva. The saliva samples have all been analyzed, and work to compare those to diet records is underway.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Running, CA. (2019) Diet and dietary context influence expression of salivary proteins that may modulate oral sensation. Association for Chemoreception Sciences Annual Meeting. Bonita Springs, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Running, CA. (2018) Diet influences saliva. Sensorium 2018. West Lafayette, IN.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists, dietitians, nutritionists, food industry representatives Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?2 undergraduates, 1 graduate student, and 1 postdocwere trained in biochemical techniques related to analyzing saliva as well as sensory evaluation tools. The graduate student and postdoc also were trained in ethics for human subjects clinical trials, as well as statistical techniques related to analyzing the data. The graduate student and postdoc also benefited from attending national/international conferences to present the work, gaining networking and presentation skills. How have the results been disseminated to communities of interest?Results have been published in a thesis, available from Proquest soon, as well as presented to audiences of scientists, nutritionists, dietitians, and food industry representatives. What do you plan to do during the next reporting period to accomplish the goals?Repeat the trial with polyphenols in a more controlled setting to truly evaluation whether changes in saliva cause changes in flavor. We may also begin a trial with spiciness.

Impacts
What was accomplished under these goals? We showed that consumption of chocolate milks (both almond and cow's milk) alters the proteins found in saliva,in ways that should influence flavor. This indicates that polyphenols (the healthy but bitter compounds in chocolate) may indeed influence saliva. We showed that the milks themsevles influence the saliva differently, indicating that the compounds in the food do indeed influence how saliva responds to the diet. We also showed that high vs. low fruit and vegetable consumers exhibit different types of saliva, particularly the proteins in their saliva.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Crawford, Ciera. 2018. Chocolate almond milk consumption changes salivary proline-rich protein expression, which alter astringency and bitterness. Master's thesis. Purdue University, West Lafayette IN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Running, CA. 2018. Diet changes saliva. Oral and poster presentation. 256th American Chemical Society National Meeting & Expo, Fall 2018. Boston, MA.