Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture.

The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes.

Application of a Parallelizable Perfusion Bioreactor for Physiologic 3D Cell Culture.

It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to ∼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes.

Dietary fiber as a versatile food component: an industrial perspective.

The continued emphasis on the importance of dietary fibers to the Western diet and the need for products with a lower calorific content is pressuring food companies to allocate more resources to the development of fiber-enriched products. The challenge to the industry is to accomplish this goal without sacrificing the organoleptic appeal of some of their core offerings. As future research details specific nutritional benefits of individual components of dietary fiber, food companies will need flexible alternatives in order to validate new 'functional' food claims and to respond rapidly to emerging trends in fiber-enriched products. These objectives will be achieved by understanding the physicochemical basis for the biotechnical functionality of fibers and by developing, and making available fibers which provide a broad spectrum of bioactive and texture modulating properties.

Changes to the galactose/mannose ratio in galactomannans during coffee bean ( Coffea arabica L.) development: implications for in vivo modification of galactomannan synthesis.

Endosperm was isolated from Arabica Caturra coffee beans 11, 15, 21, 26, 31 and 37 weeks after flowering, and the chemical composition and relative solubility of its component polysaccharides determined at each growth stage. Chemical analysis of the total mannan content of the cell wall material was done after solubilisation of galactomannans by alkaline extraction of the cell wall material followed by enzymatic digestion of the alkali-insoluble residue with a mixture of endo-mannanse and endo-glucanase. Eleven weeks after flowering, galactomannans accounted for approximately 10% of the polysaccharides but were highly substituted, with galactose/mannose ratios between 1:2 and 1:7. As the bean matured, galactomannan became the predominant polysaccharide, until 31 weeks after flowering it accounted for approximately 50% of the polysaccharides. However, it was less substituted, possessing galactose/mannose ratios between 1:7 and 1:40. Early in bean growth, up to 50% of the cell wall polysaccharides were extractable but as the galactomannan content of the bean increased there was a reduction in the extractability of all polysaccharides. The decrease in the galactose/mannose ratio of the galactomannans commenced between 21 and 26 weeks after flowering and was in synchrony with a rise in the concentration of free galactose in the beans. The results indicated that the degree of substitution of the galactomannans in coffee beans is developmentally regulated and may result, in part, from the modification of a primary synthetic product by the action of an alpha-galactosidase.

Texture of cooked potatoes (Solanum tuberosum). 1. Relationships between dry matter content, sensory-perceived texture, and near-infrared spectroscopy.

Properties of fresh potatoes, including dry matter (DM) content, starch content, and near-infrared (NIR) spectra, were determined and related to the sensory-perceived texture of the steam-cooked samples. To quantify these relationships, three potato cultivars, respectively representing a firm cooking potato (cv. Nicola), a mealy cooking potato (cv. Irene), and a cultivar (cv. Bintje) with intermediate cooking properties, were classified on the basis of three size categories. For each size category and cultivar a DM distribution was determined on the basis of the underwater weight of the individual potatoes. Each DM distribution was divided into three subcategories based on low, medium, and high DM contents. This categorization was performed for freshly harvested potatoes and for potatoes stored for 3 and 6 months, respectively. In total, this resulted in 27 DM distributions, of which 16 were non-normally distributed, and 81 samples. Linear relationships were established between the DM content, as determined by either underwater weight analysis or oven-drying, and the starch content. On the basis of partial least-squares regression (PLSR), statistical models were developed relating sensory-based texture descriptors with the DM matter content of the samples. It was also shown, by applying PLSR, that the NIR spectra, originating from the potato samples, could be related to the DM content and to the sensory-perceived texture. From the relationships between DM content and sensory-perceived texture, on the one hand, and from the DM content and NIR spectra, on the other, it was concluded that the DM content rather than the cultivar determines the sensory-perceived texture of steam-cooked potatoes. Cultivar-specific elements may also contribute to the perceived texture but are overruled by the DM content. Storage did not affect the mutual relationships between the DM content, the sensory properties, and the NIR spectra.

Texture of cooked potatoes (Solanum tuberosum). 2. Changes in pectin composition during storage of potatoes.

During the storage season changes in the chemical composition of the pectin moiety of the cell walls of raw potatoes were studied. This compositional analysis was performed for the cultivars Nicola and Irene, which represent two extremes with regard to sensory-perceived texture. Both cultivars were divided into three size categories. From each size category a dry matter (DM) distribution was made. From these distributions potatoes at the low and high ends of this distribution were selected for further analysis. In total 12 different samples were analyzed three times during the storage season. The analysis comprised a pectin fractionation study. Pectic fractions were extracted from the cell wall material (CWM) by increasing the harshness of the extraction procedure. This resulted in a calcium-complexed pectic fraction, two pectic fractions weakly bound to the CWM, and a residue fraction, respectively. It was shown that no statistically significant differences (p > or = 0.95), either in yield or in chemical composition, could be observed between the two cultivars studied (Nicola and Irene), between sizes (large, medium, and small), and between potatoes with either high or low DM contents. However, statistically significant effects of storage both on the yield and on the chemical composition of the pectic moiety of the CWM could be observed, irrespective of cultivar, size, and DM content. Despite the substantial changes in the composition of the pectic moiety of the CWM of the raw material, no to minimal changes in the sensory-perceived texture of the cooked potatoes were observed upon storage. This suggests that the observed changes in pectin composition upon storage are overruled by other aspects that contribute more importantly to the sensory-perceived texture of steam-cooked potatoes.

Texture of cooked potatoes (Solanum tuberosum). 3. Preheating and the consequences for the texture and cell wall chemistry.

Two potato cultivars representing extremes with regard to the texture of the cooked product were divided into subcategories based on size and dry matter (DM) content. The effects of the preheating temperature and time on both the instrumentally determined firmness and the sensory-perceived firmness were measured and compared. Increasing the preheating time at 60 degrees C followed by cooking resulted in greater force required to fracture the tissue, an increase in perceived firmness, and a less mashable product. A principal component analysis showed that with higher DM contents of the potato samples, preheating resulted in a larger force required to fracture the tissue and a firmer product. The changes in fracture force were not linearly related with the changes in perceived firmness. The effects of preheating on the pectin methylesterase (PME) activity, the enzyme assumed to be responsible for the firming effect upon preheating, showed that the activity of this enzyme remained rather constant during preheating at 60 degrees C for 1 h. Preheating at 78 degrees C for 10 min abolished virtually all PME activity. To obtain insight into the consequences of preheating and preheating followed by steam cooking on the yield and composition of the cell wall material (CWM) of potatoes, a cell wall isolation followed by a pectin fractionation study was performed. Attention was also paid to the consequences of the processing conditions applied on the chemical composition of the CWM and the sequentially extracted pectic fractions. Preheating resulted in an increase in yield of the CWM of cooked potatoes and, as a consequence, all of the sequentially extracted fractions, including the residue. Preheating did not have a pronounced effect on the composition of the pectin of the sequentially extracted fractions. This altogether strongly indicates that preheating causes a PME-based firming effect, resulting in an decrease in pectin degradation and, as a consequence, a larger yield of CWM. It seems reasonable to assume that this increase in amount of CWM results in a firmer texture. The contribution of starch-based degradation products to the texture after preheating can, however, not be excluded.

Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed.

Effect of roasting on degradation and structural features of polysaccharides in Arabica coffee beans.

The degree and nature of polysaccharide degradation at different roasting levels was determined for three Arabica (Coffea arabica) bean varieties. Between 12 and 40% of the bean polysaccharides were degraded depending on the roasting conditions. The thermal stability of the arabinogalactans, (galacto)mannans and cellulose was markedly different. The arabinogalactans and mannans were degraded up to 60 and 36%, respectively, after a dark roast, while cellulose showed negligible evidence of degradation. Roasting led to increased solubility of both the arabinogalactans and (galacto)mannans from the bean but the structural modifications, which accompanied this change in solubility, were different for each polysaccharide. Despite the moderate degradation of the (galacto)mannans, those remaining in the bean after roasting showed no evidence of change to their molecular weight even after a dark roast. In contrast, arabinogalactans were depolymerised after a light roast both by fission of the galactan backbone and loss of arabinose from the sidechains. The recently discovered covalent link between the coffee bean arabinogalactans and protein survived roasting. The glucuronic acid component of the AG was degraded markedly after a dark roast, but approximately 30% of the original content remained as part of the AG polymer. The results show that polysaccharide degradation during roasting is more marked than previously documented, and points to roasting induced changes to the polysaccharides as major factors in the changing physicochemical profile of the coffee bean during processing.