<p>  </p> <p>Pacheco, Kathryn Nichole. M.S. Purdue University, December 2022. Snacking Interventions Differentially Influence Saliva, Salivary Alpha Amylase Activity, and Sensation. Major Professor Dr. Cordelia A. Running.</p> <p>Human saliva contains the enzyme alpha amylase, which greatly influences many facets of human health such as digestion, absorption of nutrients, and the sensory perception of certain foods. However, the complex relationships between chewing behavior, food texture preference, and salivary amylase require further investigation. In this study, we aim to observe salivary alpha amylase through a simple assay using pudding, and to examine whether salivary amylase activity relates to diet, the sensory properties of starchy foods, or mouth behavior. We hypothesized that the pudding/salivary amylase activity assay would show more activity (less pudding remaining) 1) at the end of the high dietary starch intervention week, with little or no change from baseline to the end of the low dietary starch intervention week and 2) for people with greater baseline starch consumption compared to less baseline starch consumption. A counter-balanced, crossover design was implemented for the study. 34 participants (11 Men, 23 Women, 0 Other) completed study tasks, consisting of a 3-day dietary recall, 2 separate weeks of dietary intervention consisting of high starch or low starch snacks, and 4 research visits. These research visits included participant taste and smell acuity assessments, sensory ratings of the study foods, a mouth behavior typing test, and our salivary amylase activity assay that determined flow rate of a mixture of participant saliva and starch-containing ready-to-eat pudding. After our higher and lower starch snack interventions, we saw minimal evidence of changes to salivary amylase activity in our assay; the only trend we observed was opposite our expectation (less amylase activity after the low starch intervention). However, we did observe mouth behavior grouping tended to associate with sensory ratings that validate the premise of the mouth behavior typing tool we utilized. Ultimately, more work on the consistency and usefulness of the salivary amylase activity assay will need to be conducted if it is to be utilized for research purposes, but our data do help validate the concept that different people prefer foods due to their preferred methods of orally manipulating foods. r.  </p>

<p></p><p>Phenylketonuria (PKU) is an inherited metabolic disorder affecting 1 in every 10,000 to 15,000 newborns in the United States every year. Caused by a genetic mutation, PKU results in an excessive build up of the amino acid Phenylalanine (Phe) in the body leading to symptoms including but not limited to intellectual disability, hyperactivity, psychiatric disorders and seizures. Most PKU patients must follow a strict diet limited in Phe. The aim of this research study is to formulate, implement and compare techniques for Phe estimation in commercial foods using the information on the food label (Nutritional Fact Label and ordered ingredient list). Ideally, the techniques should be both accurate and amenable to a user friendly implementation as a Phe calculator that would aid PKU patients monitor their dietary Phe intake.</p> <p> The first approach to solve the above problem is a mathematical one that comprises three steps. The three steps were separately proposed as methods by Jieun Kim in her dissertation. It was assumed that the third method, which is more computationally expensive, was the most accurate one. However, by performing the three methods subsequently in three different steps and combining the results, we actually obtained better results than by merely using the third method.</p> <p> The first step makes use of the protein content in the foods and Phe:protein multipliers. The second step enumerates all the ingredients in the food and uses the minimum and maximum Phe:protein multipliers of the ingredients along with the protein content. The third step lists the ingredients in decreasing order of their weights, which gives rise to inequality constraints. These constraints hold assuming that there is no loss in the preparation process. The inequality constraints are optimized numerically in two phases. The first involves nutrient content estimation by approximating the ingredient amounts. The second phase is a refinement of the above estimates using the Simplex algorithm. The final Phe range is obtained by performing an interval intersection of the results of the three steps. We implemented all three steps as web applications. Our proposed three-step method yields a high accuracy of Phe estimation (error <= +/- 13.04mg Phe per serving for 90% of foods).</p> <p> The above mathematical procedure is contrasted against a machine learning approach that uses the data in an existing database as training data to infer the Phe in any given food. Specifically, we use the K-Nearest Neighbors (K-NN) classification method using a feature vector containing the (rounded) nutrient data. In other words, the Phe content of the test food is a weighted average of the Phe values of the neighbors closest to it using the nutrient values as attributes. A four-fold cross validation is carried out to determine the hyper-parameters and the training is performed using the United States Department of Agriculture (USDA) food nutrient database. Our tests indicate that this approach is not very accurate for general foods (error <= +/- 50mg Phe per 100g in about 38% of the foods tested). However, for low-protein foods which are typically consumed by PKU patients, the accuracy increases significantly (error <= +/- 50mg Phe per 100g in over 77% foods).</p> <p> The machine learning approach is more user-friendly than the mathematical approach. It is convenient, fast and easy to use as it takes into account just the nutrient information. In contrast, the mathematical method additionally takes as input a detailed ingredient list, which is cumbersome to be located in a food database and entered as input. However, the Mathematical method has the added advantage of providing error bounds for the Phe estimate. It is also more accurate than the ML method. This may be due to the fact that for the ML method, the nutrition facts alone are not sufficient to estimate Phe and that additional information like the ingredients list is required. </p><br><p></p>

<p>Low calorie sweeteners (LCS) provide sweetness with little to no energy. Each sweetener has a unique chemical structure that possesses unique sensory and functional characteristics. While LCS are generally considered in aggregate, these unique chemical structures have potential implications for sensory, metabolic, and behavioral differences that may impact body weight and glycemia. Therefore, two, twelve-week experiments were conducted to determine the effect of chronic LCS consumption on body weight, glycemia, and ingestive behaviors. </p> <p>The first experiment investigated the differential effects of four LCS (saccharin, aspartame, rebaudioside A, and sucralose) and sucrose consumed for twelve weeks on body weight, glycemia, and ingestive behaviors among healthy adults with overweight or obesity (body mass index (BMI) between 25 and 40 kg/m²). In a parallel-arm design, 154 participants were randomly assigned to consume 1.25 to 1.75L of beverage sweetened with 1 of the 5 sweeteners daily for 12 weeks. Body weight was measured every two weeks; energy intake, energy expenditure, and appetite were assessed every 4 weeks; and glucose tolerance was measured at baseline and week 12. Every four weeks, participants completed 24-hour urine collections to determine study compliance via PABA excretion. Sucrose and saccharin consumption led to increased body weight across the 12-week intervention (Δ weight = +1.85 and +1.18kg, p ≤ 0.02) and did not differ from each other. While there was no significant change in body weight with consumption of the other LCS treatments compared to baseline, changes in weight in comparison to the sucrose treatment (sucrose – LCS) were significantly different for aspartame, rebA, and sucralose after 12 weeks (weight difference = 1.13, 1.25, 2.63kg, respectively; p ≤ 0.03). In addition, change in body weight at week 12 was significantly lower between sucralose and all other LCS (weight difference ≥ - 1.37 kg, p=0.008).</p> <p>The second experiment investigated the effect of daily aspartame ingestion on glycemia, body weight, and appetite. One hundred lean (BMI between 18 and 25 kg/m²) adults were randomly assigned to consume 0, 350, or 1050 mg aspartame/day for twelve weeks in a parallel-arm design. This experiment followed a similar protocol but measured body weight and blood pressure weekly and contained a 240-min glucose-tolerance test (OGTT) with measurements of selected hormones at baseline and week 12. Participants also collected 24-h urine samples every four weeks. There were no group differences for glucose, insulin, resting leptin, glucagon-like peptide 1, or gastric inhibitory peptide at baseline or week 12. There also were no effects of aspartame ingestion on appetite, body weight, or body composition. </p> <p>These trials demonstrate that all LCS contribute negligible energy but should not be aggregated because of their differing effects on body weight. Sucrose and saccharin consumption significantly increased body weight compared to aspartame, rebA, and sucralose. This differential change in body weight among LCS indicates individual LCS likely exert different physiological responses beyond the contribution of sweetness with negligible energy. Saccharin, rebA, sucralose, and aspartame (ingested at three doses) for twelve weeks had no effect on glycemia. These data do not support the view that LCS are problematic for the management of glycemia. If substantiated through additional testing, findings from this trial have implications for consumers, food industry, clinicians, and policy makers. Some LCS may not hold the anticipated beneficial effects on body weight (e.g., saccharin) and positive effects of one LCS (sucralose) may be attenuated if combined with select other LCS. Going forward it will be important to consider each LCS as a distinct entity with respect to its potential health effects.</p>

<p>Obesity and nutrition-related non-communicable diseases continue to be major challenges that are increasing in severity worldwide. Science-centered carbohydrate dietary strategies may be a viable approach to help address such challenges. Recent reports from our laboratory indicate that certain carbohydrates with slow digestion profiles have the ability to trigger the gut-brain axis and reduce food intake and to slow gastric emptying and potentially affect appetite. Slow carbohydrate digestion may have other impacts on energy metabolism that have not been explored. In the current investigations, we sought to better understand the delayed gastric emptying profile of pearl millet-based foods as well as to understand how altering carbohydrate digestion rate impacts substrate utilization for energy.</p> <p>In the first study, the physical breakdown of pearl millet couscous particles in a simulated gastric environment (Human Gastric Simulator) was studied compared to wheat couscous matched in particle size, and select physicochemical properties of each type of couscous were characterized. Because we previously showed that pearl millet couscous had a marked delay in gastric emptying compared to white rice, boiled potatoes, and pasta in a human study in Mali, the objective of the first investigation was to test the hypothesis that pearl millet couscous was more resistant to breakdown in the stomach than wheat couscous and would take longer to empty. Our findings indicated that pearl millet couscous instead broke down into smaller, more numerous particles than wheat couscous. However, pearl millet had a slower starch hydrolysis property compared to wheat couscous per unit surface area. Pearl millet also had a smaller amylose chain length (839-963 DP) compared to wheat (1225-1563 DP), which may enable a denser packing of millet starch molecules that hinders hydrolysis. We also visually observed that the pearl millet particles formed a paste while breaking down that could reasonably generate viscosity in the stomach to potentially delay gastric emptying. </p> <p>Based off the findings from simulated gastric digestion, we next conducted a human study (<i>n</i>=14) in the U.S. to test the hypothesis that pearl millet-based foods (couscous – commercial and self-made, thick porridge) would reduce glycemic response, increase satiety, and delay gastric emptying compared to wheat couscous and white rice. We complemented this human study with additional <i>in vitro </i>work using an advanced gastrointestinal digestion system (TIMagc) to determine if the viscosity of pearl millet couscous particles as they were breaking down in the stomach was contributing to a decrease in gastric emptying. Our findings indicated that all the pearl millet-based foods and wheat couscous had lower overall glycemic response than white rice, but only the self-made millet couscous showed higher satiety through subjective appetitive response ratings. Surprisingly, there were no differences in gastric emptying among the foods. Additionally, the half-emptying times for these foods were all ~3 h, which is similar to the comparably low half-emptying times observed for white rice, boiled potatoes, and pasta in the previous Mali study. We now hypothesize that there may be diet-induced changes in gut-brain axis signaling when slowly digestible carbohydrates are consumed repeatedly over time, perhaps through modulating the number or sensitivity of small intestinal L-cells. We also found that millet couscous did not exhibit high viscosity in the TIMagc, suggesting that viscosity was not impacting its rate of gastric emptying. We conclude that at least some pearl millet-based foods possess a slow digestion property that may act to trigger the gut-brain axis or ileal brake to increase feelings of satiety or slow gastric emptying, but the discrepancy between U.S. and Malian populations requires further study. </p> <p>In the final investigation, we examined how altering carbohydrate digestion affected partitioning of carbohydrate versus fat for oxidation as well as the efficiency of switching oxidation between these two substrates (termed “metabolic flexibility”) in mice. Metabolic flexibility has been associated with good health related to decreased adipose tissue in the body and improved insulin sensitivity and may have implications on weight management. Carbohydrate digestion was adjusted by: (1) testing mice that lacked a complete set of enzymes by knocking out maltase-glucoamylase (Mgam; null) for moderating starch digestion versus testing wild-type mice; (2) using diets in these two groups of mice to moderate starch digestion that had different levels of resistant starch (53%, 35%, and 18%), had only raw corn starch or sucrose, or were high in fat; and (3) providing a supplement of fungal amyloglucosidase (AMG) to the mice treatment groups to increase starch digestion. Respiratory exchange ratio (RER) was measured through indirect calorimetry and mathematical modeling was used to characterize the diurnal shifts in RER (sine equation) as well as carbohydrate versus fat oxidation and metabolic flexibility (percent relative cumulative frequency [PRCF] with Weibull and Mixed Weibull Cumulative Distribution functions). Our results suggest that null mice lacking Mgam had somewhat increased metabolic flexibility than wild-type mice despite exhibiting minimal to no effects on carbohydrate oxidation. Intriguingly, the raw corn starch diet increased fat oxidation and generally promoted metabolic flexibility, although it did not increase carbohydrate oxidation relative to the other carbohydrate-predominant diets. Increasing carbohydrate digestion through AMG supplementation increased carbohydrate oxidation, and generally prompted earlier shifts to carbohydrate oxidation than without AMG supplementation. These findings provide a basis for better understanding the metabolic consequences of altering carbohydrate digestion and establish novel tools that can be utilized in future investigations. Overall, we propose that moderating carbohydrate digestion provides the ideal combination of balancing carbohydrate and fat oxidation while promoting metabolic flexibility. </p> <p>In conclusion, a slow digestion property may enable some types of pearl millet to trigger the ileal brake and gut-brain axis feedback systems to decrease glycemic response and increase satiety. Moreover, consuming carbohydrates with slow digestion may optimize substrate utilization for energy by the body. In addition to triggering the ileal brake and gut-brain axis, modulating carbohydrate digestion to more effectively switch between carbohydrate and fat for oxidation may be beneficial for weight management and metabolic disease prevention.</p>

<p dir="ltr">Newborn animals undergo a lot of early-life stress that heavily impact on their long-term growth, performance, and welfare. Typically, the stress would indirectly interfere with the capacity of these neonates to utilize dietary nutrients and consequently impact tissue growth and development. In piglets, weaning is a stressful situation characterized by disruption of intestinal epithelial cell development which causes poor digestion of solid feed and a negative impact on absorption of nutrients especially in the post-gastric region. In addition, weaning in piglets could cause an increase in cellular assault by reactive oxygen species thereby potentially causing gut leakiness and paracellular loss of nutrients along the intestinal tract. In broiler chickens, access to feed may take up to 72 h following hatching which may affect their gut development as well as their gut microbiota. After the first feed ingestion, there is a sharp increase in the gut microbiota which triggers an increase in the development of the immune system as well as the gut. There is continuous attention on the strategies and nutritional interventions to mitigate or ameliorate the adverse effects of early life stressors in these food animals, especially in broiler chickens and piglets. In the studies described in this dissertation, myo-inositol (purely supplemented or phytase-induced) and luteolin were tested as nutritional strategies to mitigate the effects of early-life stressors on growth and the potential mechanisms by which myo-inositol and luteolin regulate growth were investigated.</p><p dir="ltr">In study I, the effect of myo-inositol on growth in 128 postweaning piglets fed protein-deficient corn-soy diets was tested. There were 4 dietary treatments in a randomized complete block design with body weight as the blocking factor. The treatments consisted of 1 positive control (PC) diet formulated to meet all the nutrient requirements of the piglets with a 20% crude protein (CP); the remaining 3 diets were the negative control (NC) diets with a 3% reduction in CP, a 2 g/kg myo-inositol supplemented negative control diet (NC+INO), and phytase (3,000 FTU/kg) supplemented negative control (NC+PHY) diet. The results showed that phytase enhanced the apparent total tract digestibility (ATTD) of P in the weanling pigs. Myo-inositol supplementation in a protein-deficient diet improved (P < 0.05) porcine plasma myo-inositol concentration while an in vitro myo-inositol incubation with intestinal epithelial cells increased the expression of genes that encode for Claudin-1, Claudin-3, Claudin-4, ZO-1, NaPiIIb, GLUT2, and SLC7A2. The in vitro analysis of tight junction integrity in the IPEC-J2 cells indicated by the transepithelial electrical resistance and FITC-Dextran permeability showed an enhancement in response to myo-inositol treatment. Although the in vivo study found that myo-inositol did not improve growth performance or ATTD, the in vitro myo-inositol enhanced markers of gut health and function.</p><p dir="ltr">In study II, the effect of myo-inositol on the growth of broiler chickens was tested. In this study, there were 6 experimental treatments based on two dietary protein levels (PC and NC) and three supplement types (BASAL, INO, and PHY) resulting in a 2 x 3 factorial arrangement in a completely randomized design. A total of 384 broiler chickens comprising 6 treatments with eight replicates per treatment and 8 birds per replicate were used. The birds were fed a common starter diet for the initial 7 days after they arrived at the poultry unit followed by a 14-day trial. The protein-deficient diet decreased the feed efficiency of the birds. Phytase addition increased (P < 0.05) the apparent ileal digestibility (AID) and ATTD of P and Ca in both PC and NC groups. The jejunal gut morphology was enhanced by supplemental phytase as indicated by an increase in villus height and the ratio of the villus height-to-crypt depth, coupled with an increase in serum myo-inositol concentration caused by both myo-inositol and phytase. In conclusion, myo-inositol showed a differential influence on growth performance, nutrient digestibility, and gut morphology.</p><p dir="ltr">In study III, the effects of luteolin on weanling pigs and IPEC-J2 cells were examined. A total of 48 piglets were randomly allotted to two dietary treatments consisting of a control group and a luteolin (LUT)-supplemented dietary group for a 4-week trial. A weekly assessment of the growth performance and expression of specific proteins in the jejunal mucosa was performed. In each dietary group, 8 piglets were slaughtered at weeks 1, 2, and 4 postweaning to collect blood, jejunal and ileal mucosa, and tissues. Luteolin supplementation numerically improved the ADG and G:F of the pigs. Luteolin feeding altered the jejunal and ileal gut morphology with increased villi height (P < 0.05) and villus height-to-crypt depth ratio (VCR, P < 0.05) in the jejunum and decreased crypt depth in the ileum. The effect of luteolin on IPEC-J2 global proteome and phosphor-proteome showed that luteolin could potentially improve intestinal barrier integrity by enhancing the abundance of proteins important in cell growth and survival. </p><p dir="ltr">In summary, dietary supplementation with myo-inositol and luteolin could regulate growth and nutrient digestibility in broiler chickens and weanling pigs by enhancing the integrity of intestinal cells and facilitating the expression of nutrient transporters that are significant in the uptake of nutrients across the lining of the gastrointestinal tract. Phytase supplementation improves the P release from phytate in the diets thereby alleviating its loss.</p>