DEVELOPMENT AND TESTING OF A SATIETY BEVERAGE FORMULATED WITH FISH AND PALM OILS

DEVELOPMENT AND TESTING OF A SATIETY BEVERAGE FORMULATED WITH FISH AND PALM OILS

OVERVIEW

Obesity has emerged as a serious threat to world health. Though a complex phenomenon, it is basically caused by consuming more calories than are expended.  One area of research being utilized to address obesity involves food substances that can trigger what is called the ileal brake, a feedback mechanism designed reduce appetite by triggering the release of satiety (“feeling of fullness”) hormones in the body.   Our project proposes to develop a food emulsion composed of fish and palm oil, and incorporate it into a beverage to trigger this satiety mechanism.  The emulsions will be tested in mouse cells as well as in human subjects. Emulsions are easy to blend with yogurts and fruits to create smoothie-type beverages, to make them a palatable.

Schematic of the ileal brake (26)
http://www.ifr.ac.uk/info/science/foodstructure/images/digestion.gif

PHASE 1: The first phase of the study consists of determining which specific properties of the oils in the emulsion, such as melting point, affect the satiety hormones in the mouse cells.  Each emulsion sample will be added to cell cultures and the hormone expression levels will be analyzed via Luminex instrumentation.  The expected result is that there will be a correlation between emulsion melting point and hormone expression levels.

PHASE 2: The second phase is the product development phase wherein the sensory tests are conducted to determine if any emulsion flavor identity can be masked vs. a placebo such as milk fat.

PHASE 3: The third phase will consist of a clinical study in which the product developed from the second phase will be fed to approximately 6-10 human subjects (of varying weights, normal/overweight/obese)  over two 4-week periods. Participants will record/report satiety feelings using a standard VAS (visual analog sensory scale).  The expected outcome is that the emulsion will provide a significant feeling of fullness in contrast to a milk fat control.  The overall goal is to aid in the development of commercial food products to address the obesity issue by helping people consume fewer calories.

GOALS/OBJECTIVES/ANTICIPATED

The goal of this study is to develop lipid emulsions composed of various fatty acids and investigate their ability to promote satiety through satiety hormone mechanisms.

The objectives of this study are directly related to the following research questions and hypotheses:

HYPOTHESIS

Emulsions and their specific lipid properties such as melting point and fatty acid composition have a positive correlation on satiety via activation of the ileal brake.

RESEARCH QUESTIONS

1.  Can an emulsion beverage composed of a specific fish:palm oil ratio induce the release of the satiety hormones GLP-1 and Peptide YY in a STC1 cell culture?

2.  Can an emulsion beverage be prepared so that there is no detectable difference in taste or mouthfeel compared to the control?

3.  Do the emulsion beverages induce satiety in humans in clinical trials?

4.  Do the specific properties of emulsions influence the feeling of satiety in human subjects?

The first phase will test the first research question.  This phase will determine if the emulsions will induce the expression of GLP-1 and Peptide YY (satiety hormones).  It will also determine if the specific properties of the emulsions have a correlation with palm:fish oil ratio.  The higher the palm:fish oil ratio, the higher the melting point.  It is hypothesized that a higher melting point will result in a higher level of GLP-1/Peptide YY expression due to the expectation that a higher melting point emulsion will be able to reach the ileum undigested and can therefore trigger the ileal brake.  However, in a cell culture it is not known if this difference will be apparent.  The second phase of the study is to focus on developing an emulsion beverage that is fit for a commercial product and is desirable for consumption using a misture of ingredients including fruits, fat free yogurt and skim milk.  It is somewhat difficult to predict the outcome of this phase, but combinations based upon the research literature and our trials will attempt to make the emulsion beverages indistinguishable from the controls on a visual, taste and texture level. The third and final phase consists of the clinical study in which the emulsion beverages developed in phase II will be consumed by approximately 10 subjects over two separate 4-week periods.  The subjects will be recruited using various methods such as advertised recruitment and class volunteers.  The study will determine which emulsion provides the longest and/or greatest feeling of satiety.

ANTICIPATED RESULTS

1.  It is expected that the cell culture study will show that the emulsions do show a significant level of Peptide YY and GLP-1 expression using a Luminex detection method. It is also expected that there will be a positive correlation between melting point and level of expression.

2.  The product development phase is expected to confirm via triangle test that there will be no difference in taste between the beverage supplemented with emulsions compared to milk fat control.

3.  The clinical study phase is expected to show that there is a significant statistical difference in the feeling of hunger for the subjects that take the emulsion beverages compared to control beverages.  It is also likely that the melting point of the emulsions will influence the level of satiety and that the emulsion with the highest melting point since it is most likely to remain stable once and trigger the ileal brake.

METHODS

FATTY ACID MELTING BEHAVIOR: The fat melt test will be provided by Omega Pure.  When designing a lipid blend of saturated and unsaturated fats and oils for targeted release in the ileum (small intestine), it is necessary to have a melting point slightly higher than body temperature (37oC).  Samples are placed in capillary tubes and then stored in the freezer to solidify.  The tube is removed from the freezer and placed in a beaker (filled with water) in a water bath.  A thermometer is placed in another beaker.  The temperature of the water bath increases slowly.  As soon as the fat melts, the temperature is recorded from the thermometer to determine the melting point.

EMULSION PREPARATION: The emulsions are prepared using the following method provided by Omega Pure Corporation.  Palm-stearin was melted at 40oC and then mixed with already melted fish oil along with a rosemary antioxidant and a mono/diglyceride emulsifier to create the oil phase.  Using a Turrax shearing mixer the oil phase solution was mixed with a gum arabic solution dissolved in water and then passed through a homogenizer at 400 + 50 Bar.  The resulting gum arabic-oil mixture solution was then further mixed using Turrax shearing mixer with sodium caseinate solution to create the final mixture.  The final mixture was once again passed through the homogenizer to minimize the oil droplet size and to generate a homogenized emulsion.  Three emulsions (A, B and C) were prepared using this method.  Emulsion A,B and C had a palm:fish oil ratio of 2:1, 3:2 and 1:1 with melting points of 38.7oC, 37.1oC and 36.2oC.  Each emulsion was stored at 4oC until ready for experiments.

CELL HORMONE EXPRESSION: The cell study will be performed following a protocol established by Geraedts et al using the murine STC-1 cell line obtained from ATCC with permission granted by Dr. Hanahan from the University of California at San Francisco and GLP-1 and PEPTIDE YY expression levels will be tested (19).  The cells will be grown in Dulbecco’s modified eagle medium supplemented with high glucose (1.5g/L), 1mM Sodium Pyruvate, 1X Non-essential amino acids (Sigma), 20% fetal bovine serum with penicillin (100units/mL) and streptomycin (100 g/ml) at 37oC in 5% CO2 passed until there are a sufficient number of cells to seed at least 5 24-well plates at ~105 cells/well.  After 72 hours incubation the cells will be washed 3 times with PBS and then incubated with the following all dissolved in Hanks Balanced Salt System (HBSS): Control (HBSS), 10% Emulsion A,  10% Emulsion B, 10% Emulsion C, with 100mM glucose, and .5M butyric acid serving as positive controls.  Each plate will represent time points of 30, 60, 90 and 120 minutes.  The supernatants will then be collected and frozen until samples are ready to be analyzed.

 

FIGURE 2: Structure of GLP-1 (27)
http://www.ganfyd.org/images/b/b5/GLP-1%287-36%29amideStructure.png

 

FIGURE 3: Structure of Peptide YY (28) http://upload.wikimedia.org/wikipedia/commons/0/09/PBB_Protein_PEPTIDE YY_image.jpg

The samples will be analyzed using PEPTIDE YY and GLP-1 ELISA kits provided by Millipore and followed according to manufacturer’s instructions.  The differences in potency will be analyzed using a one-way analysis of variance test with a p-value of <0.05.

PRODUCT DEVELOPMENT: The product development phase will identify a suitable food vehicle for the emulsion.   The ideal food vehicle will possibly be a fruit smoothie type beverage.  There will be four smoothies total: three supplemented with each emulsion and one supplemented with milk fat to serve as a control.  The emulsions may be mixed bananas, fat free yogurt, skim milk and chocolate syrup.  This mixture must demonstrate uniformity in color, texture, and taste.  A triangle sensory test will need to be conducted to prove that there is no perceptible difference in taste when comparing each emulsion smoothie to the control smoothie.   Roughly 15 subjects will be recruited and will be screened using a basic sensory test in which the screening will require the subjects to identify which one out of three skim milk samples has been supplemented with cod liver oil.  Any subject that is unable to successfully identify the cod liver oil supplemented milk sample will be excluded from the final sensory test.   For the sensory test, each subject will be given three smoothie samples in a randomized order, two control (milk fat) smoothie samples and one smoothie sample with the emulsion.  The subjects will be instructed to drink the smoothie samples from left to right and then pick out the sample that is supplemented with the emulsion.  Using 15 subjects and a significance of (0.05) we will need less than eight correct responses to reject the hypothesis of a detectable difference.   The triangle test will need to be conducted for each emulsion (three times total) to prove that there is no detectable taste difference between treatments and controls.

Triangle Test

CLINICAL SATIETY STUDY: The clinical satiety study will begin after the product development phase is completed and the ideal smoothie and/or yogurt method has been determined.   It is expected to require either 4 or 8 weeks depending on the number of subjects.  Subjects will be recruited from Texas A&M University using advertisements or fliers.  The subjects will need to meet certain criteria before they will be accepted into the study.   These criteria includes anyone of age between 18-60, BMI 18-35, must be in good health, have no food restrictions and have normal physical activity.  The subjects will need to be easy to communicate with via email, text messaging or phone.  Smokers, vegetarians and subjects taking prescribed medication will be excluded from the experiment.  BMI for each subject will be recorded prior to the beginning of the study.  The study will be a randomized placebo-controlled double blind test and will have subjects alternating emulsion products after each week for four consecutive weeks.   The day of the week will remain constant for each patient.  For example if Subject A comes in on a Monday for the first week, that subject will be required to come in on Monday for the three remaining weeks.   Each subject will be instructed to fast and drink only water after 8pm on the night prior to the study day.  On the following morning at 9 am each subject will be given a 200g beverage supplemented with either 6g of emulsion A, B, or C (test) or 6g milk fat (control).  After consumption each subject will be instructed to avoid consuming any food or drinks except water until 12pm.   At 12 pm each subject will be given a very large bowl of spaghetti and meatballs that will far exceed a standard meal size.  Each bowl will be weighed before and after the meal then documented.  Subjects will be given a scorecard and will rate their hunger and desire to eat before and after each meal with the choices being: Very hungry, Hungry, Somewhat hungry, Not Hungry, Full.

Satiety Scorecard

COLLABORATORS

–    Dr. Peter S. Murano, Department of Nutrition and Food Science, Institute for Obesity Research and Program Evaluation, Texas A&M University, College Station, TX (overall project administration/product development/clinical satiety)

–    Dr. Susanne Talcott, Department of Nutrition and Food Science, Institute for Obesity Research and Program Evaluation, Texas A&M University, College Station, TX (cell hormone expression)

–    Dr. Ernesto Hernandez, Scientist, Omega Pure, Houston, TX (satiety ingredients and emulsion preparation)

–    Yuting Wan, Food Research Assistant, Omega Pure, Houston, TX (satiety ingredients, emulsion preparation, fatty acid characteristics)

–    Merrick Gearing, Ph.D. candidate (project researcher, all aspects)

 

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