The Influence of Fat Perception on Satiety From Consumption of Reduced Fat Snacks

University of Sussex, Quadram Institute Bioscience, Unilever R&D, PepsiCo Global R&D, Mondelēz International, Inc., Arla Foods, Mars, Inc.

The present study aims to investigate the effect of fat level and fat type of a snack on self-reported satiety and associated biomarkers. The relevant individual differences will also be investigated.

The aims are: (1) To determine whether reducing fat in a snack leads to rebound hunger and higher food intake at the subsequent meal, (2)To determine whether a low fat snack product matched for expected satiety leads to differences in post-ingestive satiety (i.e. mouth-gut discordance), (2) To determine whether individual differences in sensory perception influence expected or post-ingestive satiety. Stage 1, Characterising Volunteers: Fat is perceived through three sensory modalities; mouthfeel, taste and odour. Humans vary in their perception of fat across all sensory modalities. Volunteers will be characterised on their ability to taste fatty acids and perceive mouthfeel. Stage 2, Establish Sensory Tolerance in Expected Satiety of a fat reduced snack model: Reduced fat products are typically reformulated to match the perceived texture and mouthfeel of the original product. This stage aims to quantify sensory tolerance to fat reduction. Stage 3, Establish Mouth Gut Discordance of a fat reduced snack model: Using a standard preload study design, and the same fat-emulsion snack model from stage 2, the investigators will contrast effects of 3 test samples in a balanced cross-over design.

This is a 3 way crossover design using a pre-load study protocol that is standard for behavioural appetite studies. The study will be conducted single-blind, as it is not feasible to blind the researcher to the pre-load food products. Each participant will attend 3 visits, the pre-load study foods will be presented to them with random blinding codes.

A standard expanded snack will be used in each of the 3 arms, the content and type of fat added to the snack is varied in the two experimental arms.

Cholecystokinin (CCK) is one of the satiety hormones, a peptide hormone of the gastrointestinal system responsible for stimulating the digestion of fat and modulating appetite. Blood sample will be collected in order to analyze the level (pg/ml) of cholecystokinin (CCK).

Peptide YY (PYY) is one of the satiety hormones, acting to reduce appetite. Blood sample will be collected in order to analyze the level (pg/ml) of Peptide YY (PYY).

Glucagon-like peptide-1 (GLP-1) is one of the satiety hormones, modulating appetite. Blood sample will be collected in order to analyze the level (pg/ml) of Glucagon-like peptide-1 (GLP-1).

Ghrelin is termed as the 'hunger hormone' as it stimulates appetite, increases food intake and promotes fat storage. Blood sample will be collected in order to analyze the level (pg/ml) of Ghrelin.

Leptin is a hormone that can regulate energy intake and modulate hunger. Blood sample will be collected in order to analyze the level (pg/ml) of Leptin.

Gastric inhibitory polypeptide (GIP) can modulate appetite. Blood sample will be collected in order to analyze the level (pg/ml) of Gastric inhibitory polypeptide (GIP).

The level (ppm) of metabolomics (e.g. butyrate, propionate, lactate, acetate and 3-hydroxyisovalerate) in un-stimulated saliva will be analyzed using Nuclear Magnetic Resonance (NMR). As all these metabolomics in saliva can be analysed in one run, this is treated as one outcome.

Sample for urinary Nuclear Magnetic Resonance (NMR) metabolic profiles. Spectra will be compared and differences in integrated peak areas compared as AU (arbitrary units)

One sample at start of study day (0 min) and one sample at start of lunch (180 min) on each stage 3 visit day

Satiety Ratings on visual analogue unstructured line scale (from 0 (not at all) to 100 (extremely)). The score obtained from participants only represents the hunger or satiety at that time point, which does not represent "good" or "bad" outcome.

Hunger Ratings on visual analogue unstructured line scale (from 0 (not at all) to 100 (extremely)). The score obtained from participants only represents the hunger or satiety at that time point, which does not represent "good" or "bad" outcome.

Volunteers will have been characterised on their ability to taste the emulsions samples with the added fatty acid at the level of 0.016% and 0.11% (weight by weight) in Stage 1 (in the first visit). A discrimination forced choice test will be used, where the participant is asked to state the odd sample out of a set. If they can detect fatty acid in the samples correctly three times, they will be classified as hyper-sensitivity, otherwise they will be classified as hypo-sensitivity.

A mouthfeel discrimination test will be carried out using savoury biscuits which are constant in overall fat content but vary in mouthfeel characteristics. Four samples will be prepared varying in mouthfeel and participants will be asked to taste the samples and then rate them for the mouthfeel attributes of Crunchiness, Hardness, Greasiness, on visual analogue structured line scale (from 0 (not at all) to 100 (extremely)).

Von Frey filaments will be used to evaluate tactile sensitivity on the tongue, and to relate this to taste sensitivity. Participants will be asked to wear a blindfold, the middle of their tongue is then either stimulated or not stimulated with two Von Frey filaments of 0.008g and 0.02g sizes. The participant responds to say whether they have felt the stimulation and how sure they are (signal sure, signal not sure, no signal sure, no signal not sure). This is repeated 10 times in rapid succession. Responses are analysed using an R-index (%) value which is standard for a signal-noise detection test.

A simple short questionnaire will be used, which has been validated in the USA, to categorise people as "Crunchers", "Chewers", "Suckers" and "Smooshers". Whereas "Chewers" tend to chew foods to a fine particle size before swallowing, "Crunchers" rapidly crunch and swallow. This questionnaire has been validated in the United States.

In order to determine fungiform papillae density on the tongue, a digital camera will be used to record an image of the number of fungiform papillae in two one cm2 areas of the tongue. A small area of the participant's tongue will be temporarily dyed blue using food colour applied by a cotton wool bud. The tongue will then be blotted dry to remove excess moisture prior to recording a digital image. The blue colour will fade after approximately 1 hour and the extent of the colouration is similar to eating certain coloured sweets (e.g. blue Smarties).The number of the fungiform papillae (FP) density will be counted afterwards and quantified as papillae / cm square.

7 snack samples will be presented to subjects in a monadic sequential manner (i.e. one at a time) in a balanced order. After tasting each sample, participants will rate expected satiety ( "If you were to consume a full portion of this product, how full do you think you would feel?"and "How long do you think it would be before you felt hungry again?"). Visual analogue unstructured line scales (from 0 (not at all) to 100 (extremely)) will be used.

Inclusion Criteria: Men and women Aged 18-70 years Body mass index (BMI): 18-32 kg/m2 Fasting glucose < 7 mmol/l Fasting total cholesterol < 7.5 mmol/L Fasting triglycerides < 2.3 mmol/L Weight stable in the last three months Exclusion Criteria: Diagnosed with diabetes or cardiovascular disease (e.g. stroke or heart attack), gastrointestinal (e.g. Irritable bowel syndrome (IBS), inflammatory conditions, gastroenteritis), endocrine or renal diseases Smoker Taking prescribed medications that could influence study outcomes (e.g. lipid lowering medications, anti-depressants, anticoagulants) Food allergies (e.g. gluten, dairy) and intolerances (e.g. lactose) Drug abuse Anaemia (men: haemoglobin<130 g/L and women <115 g/L) Hypertension (systolic blood pressure > 140 mmHg, diastolic blood pressure > 90 mmHg) Planning or currently on a weight reducing programme Pregnancy, planned pregnancy in the next year or lactating Currently taking part or participation in other research studies within the last three months

No personal identification data will be shared. The study is not under an obligation to share data, however it is possible that some of the individual (unlinked / non-identifiable) data will be useful in a meta-analysis and, hence, sharing individual participant data (IPD) will be considered.

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