In the last decades, the consumption of energy-dense diets, primarily consisting of highly digestible starchy foods like bread, along with a global increase in obesity rates and a sedentary lifestyle, has emerged as the main contributors to the development of non-communicable diseases such as cardiovascular diseases (CVD) and diabetes type 2. Therefore, there is a need to reduce the starch digestibility of bakery products, and in turn their glycemic index, with a specific emphasis on wheat bread. Several strategies have been used to decrease the glycemic index and insulin response of bread; however, most of these techniques have a detrimental effect on the texture, volume, taste, and color of bread, limiting the consumer's acceptability. Preservation of the native microstructure (cell wall integrity) and employing processing techniques to create a macrostructure (protein network and food matrix) can be used to influence the product structure and therefore how the product is chewed (oral processing), and how these factors can affect carbohydrate digestion and glycemic response. The aim of this study was to examine the effect of different textural characteristics of bread on oral processing in relation to the glycemic and insulin response of the three breads. In the present study, a total of 16 healthy volunteers will be recruited, and if eligible (they need to meet the inclusion and exclusion criteria), they will attend an oral processing test on three breads, a test to measure the glycemic index (ISO) and insulin response.

The bread sample composition will be as follows:

Bread A is made with 95% durum wheat fine semolina (< 400 micrometer) + 5% gluten+ 1.2% yeast + 1% salt + 59% water Bread B is made with 80% durum wheat fine semolina (< 400 micrometer) + 20% gluten+ 1.2% yeast + 1% salt + 59 % water Bread C is made with 80% durum wheat coarse semolina (> 500 micrometer) + 20% gluten+ 1.2% yeast + 1% salt + 59 % water.

Worldwide, the number of people suffering from type 2 diabetes is around 422 million, and this number is continuously rising. It is globally agreed that, by 2025, serious action against the spread of this disease must be taken, also because diabetes is a major cause of the development of cardiovascular diseases. The spread of diabetes in the last decades is the result of a global rise in obesity, a more sedentary lifestyle, and an energy-dense diet, given the overconsumption of mainly highly digestible starchy food. Among highly digestible starchy foods, bread is a staple food consumed daily in western countries and is characterized by a high glycemic index. Carbohydrate-rich foods can be divided into three categories depending on their glycemic index (GI) (low: GI < 55, medium: 55 < GI < 69, or high: GI > 70). This classification is based on how the consumption of food containing carbohydrates affects the blood glucose level in relation to a reference food (such as glucose solution or white bread), which has the same quantity of available carbohydrates (50 g). Foods with a high GI induce a significant rise in postprandial blood glucose concentration and, consequently, a high insulin response, which could lead to hyperinsulinemia and insulin resistance. For this reason, how to decrease the blood glucose response of starchy foods, such as bread, and consequently its GI, has been extensively studied in the last few decades. In plant food, starch granules are naturally encapsulated in the cell. For cereals, the intact cell could limit the accessibility of starch in flours (wheat, sorghum, and barley) and simple food products such as porridge both in vitro and in vivo. However, when coarse flour containing intact cells is used to produce complex foods, such as bread, this effect of protection is lost. The authors hypothesized that during the long mixing time and fermentation, the cell walls increased their porosity due to the solubilization of the cell wall components, such as beta-glucans and arabinoxylans, increasing the diffusivity of the enzymes inside the cell. Moreover, in bread, the addition of coarse flour could also limit the cohesiveness of the crumb, increasing the disintegration rate and, in turn, the contact surface between the enzyme and its substrate. For these reasons, coarse flour could not efficiently decrease the in vitro starch digestibility of bakery products. Protein, the second macronutrient present in cereals, also has a role in decreasing starch digestibility. Gliadin and glutenin, which are the main proteins of wheat grain, formed a discontinuous network that surrounded the starch granules, named gluten. It has been demonstrated that a dense and compact gluten network could decrease starch accessibility, acting as a barrier between starch and enzyme.More specifically, in pasta, its lower glucose release and, in turn, its lower GI compared to bread must be researched in light of the dense and compact structure given by the strong gluten network. Indeed, the dense structure of pasta limits disintegration during oral processing and gastric digestion. This leads to lower postprandial glycemic and insulinemic responses compared to foods with the same recipe but a more porous and easier-to-disintegrate structure. A strong gluten network in bread could, therefore, change the crumb structure, increasing the crumb cohesiveness and resilience.

There is mounting evidence that demonstrates that food structure plays an important role in the digestion and absorption of nutrients. Bread texture affects bread disintegration during the gastric phase, but it mainly influences oral processing and the mastication rate. Oral processing behavior contributes to individual differences in glycemic response to foods, especially in plant tissue, where chewing behavior can modulate the release of starch from the cellular matrix. The addition of gluten cannot only physically hamper the contact between starch and enzyme and reduce the physical disintegration during gastric digestion, but it was also demonstrated that this protein complex could bind pancreatic alpha-amylase and consequently inhibit starch digestibility. The aim of this study is to examine the effect of different textural characteristics of bread on oral processing in relation to the glycemic and insulinemic responses of the three breads. A total of 16 healthy volunteers will be recruited, and if eligible (they need to meet the inclusion and exclusion criteria), they will attend an oral processing test on three breads, a test to measure the glycemic index (ISO) and insulin response.

The bread sample composition will be as follows:

Bread A is made with 95% durum wheat fine semolina (< 400 micrometer) + 5% gluten+ 1.2% yeast + 1% salt + 59% water Bread B is made with 80% durum wheat fine semolina (< 400 micrometer) + 20% gluten+ 1.2% yeast + 1% salt + 59 % water Bread C is made with 80% durum wheat coarse semolina (> 500 micrometer) + 20% gluten+ 1.2% yeast + 1% salt + 59 % water.

For the determination of glycaemic index and insulin response, one fasting capillary blood sample will be taken within 5 minutes, immediately after the participants arrive in the departments. These blood sample results will be used as the baseline blood glucose concentration, expressed in millimoles per liter (mmol/L), and insulin concentration, expressed in milliliters per liter (mU/L). The bread samples and the glucose solution will contain 50 g of available carbohydrates. The different bread samples and glucose solutions will be served to the volunteers using a randomized schedule, and they will finish the portion within 12 to 15 minutes. Test foods will be served with 250 mL of room-temperature natural water; each subject will be asked to drink the same volume for all the tests. The blood sample will be taken at six points (15, 30, 45, 60, 90, and 120 minutes) after the exact time at which the participant started to consume the sample. During testing, the subjects will rest and stay seated. Capillary blood samples will be taken by finger-prick analysis using a sampling lancet (21G x 1.8 mm, ACCU-CHEK Safe-T-Pro Plus, Roche, Switzerland). The blood will be collected in two tubes. At each time point, 3-4 drops of blood will be collected in one Microvette® CB 300 Fluoride/Heparin (SARSTEDT AG & Co., Nümbrech, Germany) for the analysis of capillary blood glucose, and 6-8 drops will be collected in one Microvette® B 300 K2E (Sarstedt Ltd., Germany) for the analysis of plasma insulin. The tubes collected for blood glucose will be immediately analyzed, while plasma from the second set of tubes will be obtained after centrifugation at 4500 rpm for 10 min at 4 °C (Labnet Hermle tabletop Centrifuge Z 200 M/H, Labnet International, Inc., New York, USA) and stored at -80 °C for insulin determination. Blood glucose analysis will be performed using the YSI 2500 Biochemistry Analyzer (Yellow Springs Instrument Company, USA). Insulin concentrations in plasma samples will be determined using a specific immunoassay test kit (Mercodia Insulin ELISA 10-1113-10, Mercodia AB-Uppsala, Sweden). Hunger, satiety, and gastrointestinal symptoms ratings after the test meal consumption will be evaluated with a self-reported questionnaire administered to the volunteers to check subjective feelings of fullness, hunger, and gastrointestinal symptoms at specific time points (before eating, [T0], and after, 30, 60, and 120 min) using a 10 cm visual analog scale. Oral processing parameters will be evaluated for each bread sample through video recording. During this session, the participants will be seated in a chair, and in front of them, there will be a desk with a camera approximately 50 cm from their faces. Participants will be instructed to place the whole sample in the mouth (e.g., a single bite) and to chew naturally until the bolus is ready for swallowing. The oral processing behavior will be described by the following parameters, which will be extracted from video recordings manually: Number of chews and swallows; total eating duration in seconds; chewing rate (number of chews per minute). The food bolus will be evaluated for each bread sample through image analysis to determine particle size distribution and the number of particles present in the bolus, as well as the moisture content, saliva content (grams of saliva per 100 g of bread), and saliva incorporation rate (grams of saliva per minute). For the determination of reducing sugars, the participants will be asked to chew and spit two bites of bread. In the first bolus, HCl will be added to immediately stop the alpha-amylase activity, and then the reducing sugar content will be quantified. For the second one, the reaction will be stopped after 15 minutes, and then the reducing sugars will be measured. The reducing sugars will be anlyzed by the 3,5-dinitrosalicylic acid (DNS) method.

Inclusion Criteria:

• Age between 18-50 years old;
• Have good general health;
• Have normal smell and taste functions;
• Have a normal body mass index (BMI 18.5-24.9 kg/m2) (based on self-reported weight and height).

Exclusion Criteria:

• Have dental braces or a piercing in or around the mouth (except removable piercings);
• Use medications known to affect glucose tolerance and influence digestion and absorption of nutrients (excluding oral contraceptives) - stable doses of oral contraceptives, acetylsalicylic acid, thyroxin, vitamins, and mineral supplements or drugs to treat hypertension are acceptable.
• Have a known history of diabetes mellitus or the use of antihyperglycemic drugs or insulin to treat diabetes and related conditions;
• Have a major medical or surgical event requiring hospitalization within the preceding 3 months;
• Have any food allergy or intolerance for gluten;
• Being pregnant or lactating (self-reported);
• Use medication that may affect the function of taste, smell, mastication, and salivation;