In vitro digestion behavior of complex formulations for clinical nutrition applications based on model systems

[eng] In vitro digestion methods simulating digestion processes are widely used to study the gastro-intestinal behavior of food or pharmaceuticals. In vitro digestion methods typically include the oral, gastric, and small intestinal phases, and occasionally the large intestinal phase. These methods try to mimic physiological conditions in vivo, taking into account the presence of digestive enzymes and their concentrations, pH, digestion time, and temperature, among other factors. In vitro digestion methods have been used to address such diverse scientific questions as the digestibility and bio-accessibility of pharmaceuticals, mycotoxins, and macronutrients such as proteins, carbohydrates, and lipids. Bio-accessibility provides an indication for the maximum of bioavailability via the oral route and is an important parameter. In this master’s thesis two different methods were studied: 1) Static digestion method: Static in vitro digestion models use sequential exposure to simulate digestion in different compartments (mouth, stomach, and intestine). During each step, the substrate is incubated for a specific time with the appropriate simulated digestive fluids. The pH is generally maintained at a fixed value by using a pH-stat or a buffer. 2) Dynamic digestion method: Dynamic in vitro digestion models reproduce the gradual transit of ingested compounds through the gastrointestinal tract more. The system reproduces the temperature, pH changes, gastric emptying, addition of simulated fluids and dialysis of digestion end products. To carry out this thesis three different carbohydrate sources were selected, Maltodextrin DE 11 – 16, Tapioca Dextrin and Modified starch, and all of them are starch derivatives. To carry out different studies like the study of digestibility, bio-accessibility, volume effect, matrix effect, etc. the static and dynamic digestion methods were used. The obtained results show that Maltodextrin DE 11 – 16 liberates more amount of glucose than Tapioca Dextrin and Modified starch and the results also show the dependency between length chain and digestibility. The comparison of the static and dynamic digestion method show that there are no big differences between the recovery obtained from each method. The obtained results of the study of volume effect suggest that is possible use smaller volumes with static digestion method, which is important to save resources. And the results obtained with the study of matrix effect indicate that the matrixes used do not affect the digestibility of Tapioca Dextrin and the pre-treatment of the meal favors the release of glucose. The objectives of this thesis are the following: 1) Study of digestibility and bio-accessibility of three different carbohydrate sources: this is of interest because with this study the amount of released glucose from the different sources (in a time-dependent manner) can be obtained. The released glucose represents available glucose for intestinal absorption. In vivo, this glucose levels would impact on the blood glucose levels and is of special interest for products intended for patients with diabetes.2) Study matrix effect: clinical nutrition products for enteral root are rarely including only polysaccharides, but also contain macro- and micronutrients. In those complex mixtures it is most likely that matrix could affect digestibility of the contained polysaccharides. For this reason, the effect of different matrixes was also studied. 3) Comparison between two different digestion methods: this part of the thesis should reveal advantages and drawbacks of each method, and, those results should serve as the basis of decision for the application of each system in future. 4) Study volume effect: this part of the study is focused on the used static digestion method and intents to determine the impact of the used reaction volume and to explore the potential to save resources

Study of potential alternatives to human digestive enzymes and prediction of digestibility and bioaccessibility of starch containing meals

[eng] This PhD thesis investigates the adaptation of two in vitro digestion methods for glycaemic index prediction and also outlines the effect of the macronutrients on the digestion of two starch sources. Potential alternatives to the main digestive human enzymes (α-amylase, pepsin, trypsin, lipase), which are expensive or not commercially available, have been studied based on their pH and temperature profiles to build a strong in vivo–in vitro correlation. This is important, as commonly used enzyme activity methods are often performed at non-physiological conditions such as 20°C, in the absence of digestive electrolytes and without considering the pH variations that occur during digestion. Therefore, the effect of digestive electrolytes on enzyme activity was also studied. Moreover, the storage stability of enzymatic solutions was studied and the maximum storage time at constant enzyme activity was defined. The alternative sources found in this study mimic the pH profile of the human enzymes at 37°C. The results demonstrated that, for the alternative sources, the effect of the temperature on the activity was similar to that of the human enzyme. In the presence of digestive electrolytes, the results revealed that each enzyme reacts differently. While α-amylase increased its activity in the presence of electrolytes, trypsin activity decreased and for lipase and pepsin no change in activity was found. Furthermore, enzymatic solutions can be used in in vitro digestion methods for a long period when stored at specific conditions (temperature, solvent). The digestion of carbohydrates and the effect of other macronutrients (protein and fat) was studied by determining the amount of glucose released from the different sources. The released glucose represents glucose available for intestinal absorption and in vivo, these glucose levels would impact blood glucose levels, thus being of special interest for products intended for patients with diabetes. The digestion of three foods (rice, potato, cocoa cream) was studied with two in vitro digestion methods (static and dynamic). With the aim of demonstrating that the selected in vitro digestion methods could provide reliable results, the measured glycaemic index (GI) was compared with reported values. The obtained results showed that the in vitro GI measured with both methods was slightly lower than the reported values. However, the reported GI for those meals was reached for most of the meals by applying a correction factor (with the exception of the cacao cream) when the dynamic digestion method was performed. To identify the digestion pattern of different carbohydrate sources, the digestion of two commercial starch sources (starch from rice and starch from potato) was studied with both methods (static and dynamic), the degree of hydrolysis (DH) and GI being measured. Then, the effect of the other macronutrients on the starch digestion was studied by adding protein (egg whites from chicken) or fat (olive oil or butter) to the starch. In general, clinical nutrition products for enteral route include the three macronutrients, thus, the digestion of meals that contains starch, protein and fat was also studied. The main differences obtained in the digestion of the different meals were due to their disintegration and solubilisation profiles, which would affect the time needed for the meal to be reached by the enzymes and subsequent enzymatic starch hydrolysis. Finally, the obtained results (DH and GI) with both in vitro digestion methods were compared, leading to the conclusion that there were no differences between the DH and GI obtained with the adapted static or adapted dynamic digestion methods.[cat] Aquesta tesi investiga l’adaptació de dos mètodes de digestió in vitro per a la predicció de l’índex glicèmic (GI) i descriu l’efecte dels macronutrients a la digestió de dos tipus de midó. S’han estudiat potencial alternatives als principals enzims digestius humans (α-amilasa, pepsina, tripsina, lipasa), que són cars o no estan disponibles comercialment, en funció dels seus perfils de pH i temperatura per a la obtenció d’una bona correlació in vivo-in vitro. Això és rellevant, ja que els mètodes per a mesurar l’activitat enzimàtica es solen dur a terme en condicions que no són fisiològiques, com podria ser a 20ºC, absència d’electròlits digestius o sense considerar les variacions de pH que ocorren durant la digestió. Per aquest motiu, també s’ha estudiat l’efecte dels electròlits digestius a l’activitat enzimàtica. S’ha estudiat l’estabilitat de les dissolucions enzimàtiques durant el seu magatzematge i s’ha definit el temps de magatzematge màxim a activitat enzimàtica constant. Les alternatives trobades en aquest estudi imiten el perfil de pH dels enzims humans a 37ºC. Els resultats han demostrat que, per a les alternatives d’aquests enzims, l’efecte de la temperatura a l’activitat enzimàtica és similar als enzims humans. Els resultats han revelat que en la presència d’electròlits digestius cada enzim reacciona de manera diferent. Mentre l’α-amilasa augmenta la seva activitat amb la presencia d’electròlits, la tripsina disminueix la seva activitat i en el cas de la lipasa i pepsina no es varen observar canvis en la seva activitat. S’ha trobat que les dissolucions enzimàtiques es poden usar en els mètodes de digestió in vitro durant un llarg període de temps quan aquestes es guarden en condicions específiques (temperatura i dissolvent). S’ha estudiat la digestió dels carbohidrats i l’efecte d’altres macronutrients (proteïna i grassa), determinant la quantitat de glucosa alliberada dels carbohidrats. La glucosa alliberada representa la glucosa disponible per a la seva absorció intestinal i in vivo, aquests nivells de glucosa tendrien un impacte en els nivells de glucosa en sang, el que és d’especial interès per a productes per a pacients amb diabetis. S’ha estudiat la digestió de tres menjars (arròs, patata i crema de cacau) amb dos mètodes de digestió in vitro (estàtic i dinàmic). Amb l’objectiu de demostrar que els mètodes de digestió in vitro seleccionats poden proporcionar resultats fiables, els GI obtinguts s’han comparat amb els valors publicats (in vivo). Els resultats obtinguts demostren que els GI in vitro mesurats amb ambdós mètodes són més baixos comparats amb els valors publicats. No obstant, aplicant un factor de correcció (excepte per la crema de cacau en el mètode dinàmic) es varen obtenir els GI publicats per a gairebé tots els menjars. Per a la identificació del patró de digestió d’alguns carbohidrats, s’ha estudiat la digestió de dos tipus de midó (d’arròs i patata) usant ambdós models in vitro i s’ha mesurat el grau d’hidròlisis (DH) i el GI. Es va estudiar l’efecte dels altres macronutrients en la digestió dels midons afegint proteïna (clares d’ou de pollastre) o grassa (oli d’oliva o mantega) al midó. En general, els productes de nutrició clínica enteral inclouen els tres macronutrients, i per aquest motiu també es va estudiar la digestió de menjars que contenien midó, proteïna i grassa. Les diferències principalment obtingudes es deuen als perfils de solubilització dels diferents menjars, el que afectaria el temps necessari per els enzims per arribar al menjar i la posterior hidròlisis enzimàtica del midó. Es varen comparar els resultats obtinguts amb ambdós mètodes de digestió in vitro. Aquesta comparació va conduir a la conclusió que no hi havia diferències entre el DH i GI obtinguts amb els models estàtic o dinàmic[spa] Esta tesis investiga la adaptación de dos métodos de digestión in vitro para la predicción del índice glicémico (GI) i describe el efecto de los macronutrientes en la digestión de dos tipos de almidón. Se han estudiado potencial alternativas a los principales enzimas digestivos humanos (α-amilasa, pepsina, tripsina, lipasa), que son caros o no están disponibles comercialmente, en función de sus perfiles de pH y temperatura para la obtención de una buena correlación in vivo-in vitro. Esto es relevante, ya que los métodos para medir la actividad enzimática se suelen llevar a cabo en condiciones no fisiológicas, como a 20ºC, ausencia de electrolitos digestivos o sin considerar los cambios de pH que ocurren durante la digestión. Por este motivo, se ha estudiado el efecto de los electrolitos digestivos a la actividad enzimática. Se ha estudiado la estabilidad de las disoluciones enzimáticas durante su almacenamiento y se ha definido el tiempo de almacenaje máximo a actividad enzimática constante. Las alternativas encontradas en este estudio imitan el perfil de pH de los enzimas humanos a 37ºC. Los resultados han demostrado que, para las alternativas de estos enzimas, el efecto de la temperatura a la actividad enzimática es similar a los enzimas humanos. Se ha revelado que en la presencia de electrolitos cada enzima reacciona de manera diferente. La α-amilasa aumenta su actividad con la presencia de electrolitos, la tripsina disminuye su actividad y en el caso de la lipasa y pepsina no se observaron cambios en su actividad. Se ha encontrado que las disoluciones enzimáticas se pueden usar en los métodos de digestión in vitro durante un largo periodo de tiempo cuando estas se guardan en condiciones específicas (temperatura y disolvente). Se ha estudiado la digestión de los carbohidratos y el efecto de otros macronutrientes (proteína y grasa), determinando la cantidad de glucosa liberada de los carbohidratos. La glucosa liberada representa la glucosa disponible para su absorción intestinal y in vivo, estos niveles de glucosa tendrían un impacto en los niveles de glucosa en sangre, lo que es de especial interés para productos para pacientes con diabetes. Se ha estudiado la digestión de tres comidas (arroz, patata y crema de cacao) con dos métodos de digestión in vitro (estático y dinámico). Con el objetivo de demostrar que los métodos de digestión in vitro seleccionados pueden proporcionar resultados fiables, los GI obtenidos se han comparado con los valores publicados. Los resultados demostraron que los GI in vitro medidos con ambos métodos son más bajos comparados con los valores publicados. No obstante, aplicando un factor corrector (excepto para la crema de cacao en el método dinámico) se obtuvieron los GI publicados. Para la identificación del patrón de digestión de algunos carbohidratos, se ha estudiado la digestión de dos tipos de almidón (arroz y patata) usando ambos métodos in vitro y se ha medido el grado de hidrólisis (DH) y el GI. Se estudió el efecto de los otros macronutrientes en la digestión de los almidones añadiendo proteína (claras de huevo de pollo) o grasa (aceite de oliva o mantequilla) al almidón. Los productos de nutrición clínica enteral suelen incluir los tres macronutrientes, por lo tanto, se estudió la digestión de comidas que contenían almidón, proteína y grasa. Las diferencias se deben principalmente a los perfiles de solubilización de las comidas, lo que afectaría el tiempo necesario de los enzimas para llegar a la comida y la posterior hidrolisis del almidón. Se compararon los resultados obtenidos con ambos métodos de digestión in vitro y se concluyó que no había diferencias entre el DH y GI medidos con ambos métodos de digestión in vitro

Determination of Dissolution Profile and Bioaccessibility of Ketosteril Using an Advanced Gastrointestinal in Vitro Model

[eng] Ketosteril is an originator drug prescribed for nutrition therapy for patients with chronic kidney disease (CKD). Ketoanalogues (KAs) of amino acids are part of the active pharmaceutical ingredients in the Ketosteril film-coated tablets

INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies

International audienceIn vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 µg) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter-laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol