Bovine Serum Albumin Rejection by an Open Ultrafiltration Membrane: Characterization and Modeling.

The classic application of ultrafiltration (UF) is for the complete retention of proteins, and in that situation, the transport behavior is well established. More open membranes with fractional retention are used when separating different proteins. However, protein transport has not been well documented yet in the literature. The bovine serum albumin (∼69 kDa) observed rejection ranges from 0.65 to 1 using a 300 kDa molecular weight cut-off membrane at different pH, ionic strength, and pressure. We demonstrated that, especially with open UF, the transport of proteins through the membrane is dominated by advection, with insignificant diffusion effects (p value > 0.05). We showed that with open UF, retention is not only caused by size exclusion but also to a large extent by electrostatic interactions and oligomerization of the proteins. Mass transfer in the polarization layer was relatively independent of the pH and ionic strength. It was underestimated by common Sherwood relations due to a relatively large contribution of the reduction in the flow turbulence near the membrane by the removal of fluid through the membrane. We propose a model that allows relatively quick characterization of the rejection of proteins without prior knowledge of the pore sizes and charges based on just a limited set of experiments. Therefore, protein rejection with the open UF system can be targeted by tuning the processing conditions, which might be useful for designing protein fractionation processes.

The Effect of Different pH Conditions on Peptides' Separation from the Skipjack Dark Meat Hydrolysate Using Ceramic Ultrafiltration.

The conversion of Skipjack (Katsuwonus pelamis) dark meat into a hydrolysate via enzymatic hydrolysis is a promising approach to increase the value of tuna by-products as a source of bioactive peptides. Skipjack dark meat hydrolysate (SDMH) contains various sizes and sequences of peptides. To obtain and concentrate the targeted small peptides from SDMH, ultrafiltration, a key unit operation process, was employed to fractionate the protein hydrolysate due to its simplicity and productivity. The objective of this study was to investigate the effect of the feed pH on the membrane performance based on the permeate flux and the transmission of peptides. The fractionation of SDMH was performed using a ceramic membrane (molecular weight cut-off of 1 kDa) with three different pH values (5, 7, and 9) at various transmembrane pressures (TMP) (2.85, 3.85, and 4.85 bar). A high permeate flux and transmission were obtained at pH 9 due to the repulsive interactions between peptides and the membrane surface, leading to the reduction in concentration polarization that could promote high transmission. In addition, the combination of low TMP (2.85 bar) and pH 9 helped to even minimize the fouling formation tendency, providing the highest peptide transmission in this study. The fractionation process resulted in the enhancement of small peptides (MW < 0.3 kDa). The amino acid profiles were different at each pH, affirming the charge effect from the pH changes. In conclusion, the performance of the membrane was affected by the pH of the hydrolysate. Additionally, the ultrafiltration method served as an alternate method of peptide separation on a commercial scale.

In silico modelling of protein digestion: A case study on solid/liquid and blended meals.

We present a dynamic, semi-mechanistic, compartmental protein digestion model to study the kinetics of protein digestion. The digestive system is described as a series of eight compartments: one for the stomach, one for the duodenum, two for the jejunum and four for the ileum. The digestive processes are described by a set of zero or first order differential equations. The model considers ingestion of a meal, secretion of gastric and pancreatic juices, protein hydrolysis, grinding, transit and amino acid absorption. The model was used to simulate protein digestion of a meal composed of a solid and a liquid phase or one where both phases are blended into a homogeneous phase. Luminal volumes and pH of gastric and duodenal contents were estimated for both meals. Further, gastric emptying is described as a function of the energy density of the bolus, instead of the more common mass action approach.

In vitro gastro-small intestinal digestion of conventional and mildly processed pea protein ingredients.

We report on the effect of processing, particularly heating, on the digestion dynamics of pea proteins using the standardised semi-dynamic in vitro digestion method. Fractions with native proteins were obtained by mild aqueous fractionation of pea flour. A commercial pea protein isolate was chosen as a benchmark. Heating dispersions of pea flour and mild protein fractions reduced the trypsin inhibitory activity to levels similar to that of the protein isolate. Protein-rich and non-soluble protein fractions were up to 18% better hydrolysed after being thermally denatured, particularly for proteins emptied later in the gastric phase. The degree of hydrolysis throughout the digestion was similar for these heated fractions and the conventional isolate. Further heating of the protein isolate reduced its digestibility as much as 9%. Protein solubility enhances the digestibility of native proteins, while heating aggregates the proteins, which ultimately reduces the achieved extent of hydrolysis from gastro-small intestinal enzymes.

Effect of Fractionation and Processing Conditions on the Digestibility of Plant Proteins as Food Ingredients.

Plant protein concentrates and isolates are used to produce alternatives to meat, dairy and eggs. Fractionation of ingredients and subsequent processing into food products modify the techno-functional and nutritional properties of proteins. The differences in composition and structure of plant proteins, in addition to the wide range of processing steps and conditions, can have ambivalent effects on protein digestibility. The objective of this review is to assess the current knowledge on the effect of processing of plant protein-rich ingredients on their digestibility. We obtained data on various fractionation conditions and processing after fractionation, including enzymatic hydrolysis, alkaline treatment, heating, high pressure, fermentation, complexation, extrusion, gelation, as well as oxidation and interactions with starch or fibre. We provide an overview of the effect of some processing steps for protein-rich ingredients from different crops, such as soybean, yellow pea, and lentil, among others. Some studies explored the effect of processing on the presence of antinutritional factors. A certain degree, and type, of processing can improve protein digestibility, while more extensive processing can be detrimental. We argue that processing, protein bioavailability and the digestibility of plant-based foods must be addressed in combination to truly improve the sustainability of the current food system.

Protein acidification and hydrolysis by pepsin ensure efficient trypsin-catalyzed hydrolysis.

Enzyme-catalysed hydrolysis is important in protein digestion. Protein hydrolysis is initiated by pepsin at low pH in the stomach. However, pepsin action and acidification happen simultaneously to gastric emptying, especially for liquid meals. Therefore, different extents of exposure to the gastric environment change the composition of the chyme that is emptied from the stomach into the small intestine over time. We assessed the susceptibility of a protein to trypsin-catalysed hydrolysis in the small intestine, depending on its pH and hydrolysis history, simulating chyme at different times after the onset of gastric emptying. Isothermal titration calorimetry was used to study the kinetics of pepsin and trypsin-catalysed hydrolysis. Bovine serum albumin (BSA) that was acidified and hydrolysed with pepsin, showed the highest extent and most efficient hydrolysis by trypsin. BSA in the chyme that would be first emptied from the stomach, virtually bypassing gastric acidity and peptic action, reduced trypsin-catalysed hydrolysis by up to 58% compared to the acidified, intact protein, and 77% less than the acidified, pepsin-hydrolysate. The least efficient substrate for trypsin-catalysed hydrolysis was the acidified, intact protein with a specificity constant (kcat/Km) nearly five times lower than that of the acidified, pepsin-hydrolysate. Our results illustrate the synergy between pepsin and trypsin hydrolysis, and indicate that gastric hydrolysis increases the efficiency of the subsequent trypsin-catalysed hydrolysis of a model protein in the small intestine.

Food Matrix and Macronutrient Digestion.

Food digestion may be regarded as a physiological interface between food and health. During digestion, the food matrix is broken down and the component nutrients and bioactive compounds are absorbed through a synergy of mechanical, chemical, and biochemical processes. The food matrix modulates the extent and kinetics to which nutrients and bioactive compounds make themselves available for absorption, hence regulating their concentration profile in the blood and their utilization in peripheral tissues. In this review, we discuss the structural and compositional aspects of food that modulate macronutrient digestibility in each step of digestion. We also discuss in silico modeling approaches to describe the effect of the food matrix on macronutrient digestion. The detailed knowledge of how the food matrix is digested can provide a mechanistic basis to elucidate the complex effect of food on human health and design food with improved functionality.

Separation of Fructose and Glucose via Nanofiltration in Presence of Fructooligosaccharides.

Fructose and glucose are commonly present together in mixtures and may need to be separated. Current separation methods for these isomers are complex and costly. Nanofiltration is a cost-effective method that has been widely used for separating carbohydrates of different sizes; however, it is not commonly used for such similar molecules. Here, we report the separation of fructose and glucose in a nanofiltration system in the presence of fructooligosaccharides (FOS). Experiments were performed using a pilot-scale filtration setup using a spiral wound nanofiltration membrane with molecular weight cutoff of 1 kDa. We observed three important factors that affected the separation: (1) separation of monosaccharides only occurred in the presence of FOS and became more effective when FOS dominated the solution; (2) better separation was achieved when the monosaccharides were mainly fructose; and (3) the presence of salt improved the separation only moderately. The rejection ratio (Rf/Rg) in a fructose/glucose mixture is 0.92. We reported a rejection ratio of 0.69, which was observed in a mixture of 50 g/L FOS with a fructose to glucose ratio of 4.43. The separation is hypothesized to occur due to selective transport in the FOS layer, resulting in a preferential binding towards fructose.

The importance of swelling for in vitro gastric digestion of whey protein gels.

In this paper we report the importance of swelling on gastric digestion of protein gels, which is rarely recognized in literature. Whey protein gels with NaCl concentrations 0-0.1 M were used as model foods. The Young's modulus, swelling ratio, acid uptake and digestion rate of the gels were measured. Pepsin transport was observed by confocal laser scanning microscopy using green fluorescent protein (GFP). With the increase of NaCl in gels, Young's modulus increased, swelling was reduced and digestion was slower, with a reduction of acid transport and less GFP present both at surface and in the gels. This shows that swelling affects digestion rate by enhancing acid diffusion, but also by modulating the partitioning of pepsin at the food-gastric fluid interface and thereby the total amount of pepsin in the food particle. This perspective on swelling will provide new insight for designing food with specific digestion rate for targeted dietary demands.

Protein Oxidation and In Vitro Gastric Digestion of Processed Soy-Based Matrices.

Process conditions that are applied to make structured soy-protein-based food commonly include high temperatures. Those conditions can induce protein oxidation, leading to a decrease in their susceptibility to proteolysis by digestive enzymes. We aimed to investigate the effects of thermomechanical processing on oxidation and in vitro gastric digestion of commercial soy protein ingredients. Samples were sheared at 100 to 140 °C and characterized for acid uptake, carbonyl content, electrophoresis, and surface hydrophobicity. The enzymatic hydrolysis was determined in simulated gastric conditions. Protein ingredients were already oxidized and showed higher surface hydrophobicity and hydrolysis rate compared with those of the processed matrices. However, no clear correlation between the level of carbonyls and the hydrolysis rate was found. Therefore, we conclude that gastric digestion is mostly driven by the matrix structure and composition and the available contact area between the substrate and proteolytic enzymes.

Analysis and modeling of enhanced green fluorescent protein diffusivity in whey protein gels.

During gastric digestion, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food. We hypothesized that the effect of pepsin is limited by its diffusivity, which is co-determined by the food structure and the local pH in the food during digestion. To investigate the principle mechanism of enzyme diffusion in food matrices, we used enhanced green fluorescent protein (EGFP) as probe to study the diffusivity of proteins in whey protein isolate gels, using fluorescence correlation spectroscopy (FCS). Gels made with different ionic strength showed distinctive elastic moduli but did not show differences in diffusivity of EGFP. Some models for diffusion in hydrogels yield good description of the obtained data, and can approximate the enzyme diffusion in diverse food matrices. However, the enzyme pepsin is more complicated than the probe EGFP, to yield more accurate predictions, electrostatic and enzyme-substrate interaction also need to be considered.

Interactions between acid and proteins under in vitro gastric condition - a theoretical and experimental quantification.

The gastric digestion of proteins is influenced by the pH and the gastric pH fluctuates after food consumption. However, the dynamics of gastric pH still need to be quantitatively understood. Proteins in food strongly influences the gastric pH. Therefore, we studied the interaction between acid and proteins, including the buffer reaction and the acid diffusion in protein gels. The buffer capacity of proteins stems from its content of ionizable amino acid side groups. Based on this, we set up a model and method to parameterize the buffer capacity of proteins. Moreover, the liberated carboxyl and amino groups during enzymatic hydrolysis of protein can also contribute to the buffer capacity. While we expected protons to diffuse faster than pepsin, we found that the penetration distance of acid is comparable to that of pepsin. The buffer reaction caused the acid to concentrate tenfold in the gel compared to the bulk acid concentration. Therefore, we postulated that the buffer reaction reduces acid diffusivity in gels.

Revisiting the enzymatic kinetics of pepsin using isothermal titration calorimetry.

Pepsin is the first protease that food proteins encounter in the digestive tract. However, most of the previous studies on the enzymatic kinetics of pepsin were based on the hydrolysis of small synthetic peptides, due to the limitations in methodology and the complexity of protein substrate. To better understand the role of pepsin in protein digestion, we used isothermal titration calorimetry to study the enzymatic kinetics of pepsin with bovine serum albumin as the substrate. We found that pepsin has a higher catalytic rate at lower pH, while its affinity to substrate is lower. At the same pH, pepsin has lower activity and affinity at higher ionic strengths. We found contrasting kinetic parameters for pepsin-catalyzed hydrolysis of bovine serum albumin and of small synthetic peptides. Time-dependent kinetics also showed that pepsin has lower efficiency towards intermediate peptides during hydrolysis.

The Effect of Gel Microstructure on Simulated Gastric Digestion of Protein Gels.

The objective of this study was to analyse the impact of the gel structure obtained by different heat-induced temperatures on the in vitro gastric digestibility at pH 2. To achieve this, gels were prepared from soy protein, pea protein, albumin from chicken egg white and whey protein isolate at varying temperatures (90, 120 and 140 °C) for 30 min. Gels were characterised prior to digestion via microstructure and SDS-PAGE analysis. Subsequently, the gastric digestion process was followed via the protein hydrolysis and HPSEC analysis up to 180 min. Peptides of different sizes (<5 kDa) were gradually formed during the digestion. Our results showed that gels induced at 140 °C were digested faster. The protein source and gelation temperature had great influence on the in vitro gastric protein digestibility.

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH.

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60, 90 and 120 °C for 30 min led to protein denaturation and aggregation, which was enhanced at higher treatment temperatures. The in vitro gastric digestibility measured during 6 h was lower for protein extracts pre-treated at 90 and 120 °C compared to 60 °C. The digestibility decreased with increasing extraction pH, which could be ascribed to protein aggregation. Protein digestibility of the quinoa protein isolates was higher compared to wholemeal quinoa flour. We conclude that an interactive effect of processing temperature and extraction pH on in vitro gastric digestibility of quinoa protein isolates obtained at various extraction pH is observed. This gives a first indication of how the nutritional value of quinoa protein could be influenced by heat processing, protein extraction conditions and other grain components.

Kinetic characterization of galacto-oligosaccharide (GOS) synthesis by three commercially important ß-galactosidases.

Many ß-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various ß-galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the ß-galactosidase of Bacillus circulans than the bgalactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the ß-galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why ß-galactosidase of B. circulans gives higher yields in GOS production than other ß-galactosidases. Many of the reaction rate constants for the ß-galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform ß-gal-A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well.

Effects of carbohydrates on the oNPG converting activity of ß-galactosidases.

The effects of high concentrations of carbohydrates on the o-nitrophenyl ß-d-galactopyranoside (oNPG) converting activity of ß-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70,000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on ß-galactosidases from B. circulans, A. oryzae, and K. lactis are compared.

Comparison of activated chromatography resins for protein immobilization.

The adsorption of bovine serum albumin (BSA) to an immobilized camelid-derived antibody fragment was investigated using six different activated resins, of which two are prototypes. The resins differed in base material, coupling chemistry and particle size. The adsorption, washing and desorption stage of the affinity chromatography process were taken into account. Dynamic binding capacities at 10% breakthrough ranged between 0.76 and 4.8 mg BSA/mL resin. The washing volume ranged between 2.9 and 10 column volumes. One of the resins did not concentrate BSA, while the highest concentration was 13-fold. We present a method to rank and weigh the properties of the resins to find the optimal resin to meet specific requirements. For three of the resins the adsorption flow rate was varied, while the washing and desorption flow rate was kept the same. The dynamic binding capacity decreased with increasing flow rate, as expected. For one resin, the washing volume remained constant, but for the others it decreased with increasing adsorption flow rate. The number of column volumes required to purify a given amount of BSA increases with increasing flow rate, which indicates that higher flow rates do not necessarily speed up the process.

Characterization of ß-galactosidase isoforms from Bacillus circulans and their contribution to GOS production.

A ß-galactosidase preparation from Bacillus circulans consists of four isoforms called ß-gal-A, ß-gal-B, ß-gal-C, and ß-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44 % and 0.68 % (w/w), ß-gal-A showed the least oligosaccharide formation, followed by ß-gal-B and ß-gal-C; most oligosaccharides were formed by ß-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially ß-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44 % and 0.61 % (w/w), respectively, ß-gal-A and ß-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30 % (w/w), except for a slightly higher production of galactose with ß-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that ß-gal-A and ß-gal-B are the best isoforms for GOS production at high lactose concentrations.