Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture.

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by-products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non-toxic, xeno-free, and has good mechanical properties. A recombinant spider silk protein-derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well-suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE-19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

Impact of food processing on rye product properties and their in vitro digestion.

PURPOSE: Rye products have been reported to elicit postprandial insulin and glucose responses which may be beneficial for prevention of type-2 diabetes. However, mechanisms underlying variations in responses related to processing techniques are not fully understood. METHODS: Five differently processed rye products (sourdough-fermented bread, fermented and unfermented crispbread, extrusion-cooked rye, and porridge) and refined wheat bread were characterised. Two in vitro methods, a dynamic method simulating digestion in the stomach and small intestine and a static method, simulating conditions in the stomach were used to determine viscosity development, structural changes and release of glucose during digestion. RESULTS: Structural and compositional differences induced by processing influenced product digestion. Gastric disintegration and digesta particle size were related to characteristics of the starch/protein matrix, while digesta viscosity was reduced due to fibre degradation during fermentation. More cohesive boluses were associated with slower glucose release. Sourdough fermentation increased amylose leakage and appeared to inhibit starch hydrolysis despite low digesta viscosity and rapid disintegration. CONCLUSIONS: The net release of glucose during digestion of foods is determined by several factors which may vary in their importance depending on product specific properties.

Impact of sourdough fermentation on appetite and postprandial metabolic responses - a randomised cross-over trial with whole grain rye crispbread.

Sourdough fermentation is considered to have beneficial effects on postprandial satiety and metabolic responses, but studies demonstrating effects at physiological conditions are lacking. The aim of this acute breakfast intervention study was to determine the effect of consumption of sourdough-fermented and unfermented rye crispbread on self-rated appetite, postprandial glucose and insulin response in healthy subjects. In all, twenty-four Swedish adults were included in a single-blinded, randomised cross-over trial. Three crispbreads (sourdough-fermented and unfermented whole grain rye and yeast-fermented refined wheat as control) were consumed as part of a standardised breakfast. Subjective appetite score, assessed using visual analogue scale, and plasma glucose and insulin concentrations were measured at baseline and postprandially until 360 and 240 min, respectively. Structural changes and viscosity during mastication and gastric digestion were investigated using in vitro methods. Hunger and desire to eat were lower (P<0·05) based on AUC measurements after intake of sourdough-fermented rye crispbread compared with after intake of yeast-fermented refined wheat crispbread. On the basis of AUC (0-230 min), insulin response was lowest after intake of unfermented rye crispbread compared with sourdough-fermented rye and yeast-fermented refined wheat crispbread. Degradation of viscous fibres and faster bolus disintegration for the sourdough-fermented bread may partly explain the less favourable metabolic responses compared with unfermented bread. Our results showed that food processing affects the composition and structural characteristics of rye bread, which has implications for appetite and metabolic responses.

Short communication: Variation in the composition and properties of Swedish raw milk for ultra-high-temperature processing.

The composition and properties of raw milk are of great importance for the quality and shelf life of the final dairy product, especially in products with a long shelf life [e.g., ultra-high-temperature (UHT)-treated milk]. The objective of this study was to investigate the compositional variation in raw milk samples before processing at the dairy plant. Moreover, we wanted to investigate the effect of the UHT process on this variation (i.e., if the same variation could be observed in the corresponding UHT milk). The quality traits analyzed included detailed milk composition, counts of total and psychrotrophic bacteria, proteolytic activity, and color, as well as predictive measures of stability (i.e., ethanol stability and heat coagulating time). Samples of raw milk and the corresponding produced UHT milk were collected and analyzed on a monthly basis during 1 yr. Principal component analysis was used to identify months showing similarities and differences with respect to total variation. In contrast to previous studies, we observed only small variations between months and no clear effect of season for the raw milk. For the UHT milk, July and the winter months (December, January, and February) tended to separate from the other months. Quality traits showing significant variation were only to some extent identical in raw milk and UHT-processed milk. A better understanding of the natural variation in raw milk quality will provide opportunities to improve the shelf life of UHT-treated milk products.

Minimizing anti-nutritional factors in wet protein extraction from Swedish faba beans through the application of response surface methodology.

Faba beans, rich in protein and ideal for Swedish cultivation, are limited in food industry use due to anti-nutritional factors (ANFs) that hinder nutrient absorption. An extraction method was developed in our study to mitigate ANFs in faba beans, using aqueous alkaline methods and isoelectric precipitation with differential salt concentration. This method yielded 15.8 g of protein per 100 g of flour, with a protein concentration exceeding 83% of the total extract. It reduced ANFs like phytic acid (28.0%), lectins (87.5%), vicine (98.5%), and convicine (99.7%). Extraction conditions were optimized using response surface methodology, identifying pH 6, 2 h, and 20 °C as the most effective parameters, achieving an 86% reduction in phytic acid, closely matched the model's predictions (R2 = 0.945). This method effectively reduced ANFs, offering a sustainable approach for producing proteins suitable for diverse food products, including plant-based alternatives.

Feruloylation and Hydrolysis of Arabinoxylan Extracted from Wheat Bran: Effect on Dough Rheology and Microstructure.

Feruloylated arabinoxylan (AX) is a potential health-promoting fiber ingredient that can enhance nutritional properties of bread but is also known to affect dough rheology. To determine the role of feruloylation and hydrolysis of wheat bran AX on dough quality and microstructure, hydrolyzed and unhydrolyzed AX fractions with low and high ferulic acid content were produced, and their chemical composition and properties were evaluated. These fractions were then incorporated into wheat dough, and farinograph measurements, large and small deformation measurements and dough microstructure were assessed. AX was found to greatly affect both fraction properties and dough quality, and this effect was modulated by hydrolysis of AX. These results demonstrated how especially unhydrolyzed fiber fractions produced stiff doughs with poor extensibility due to weak gluten network, while hydrolyzed fractions maintained a dough quality closer to control. This suggests that hydrolysis can further improve the baking properties of feruloylated wheat bran AX. However, no clear effects from AX feruloylation on dough properties or microstructure could be detected. Based on this study, feruloylation does not appear to affect dough rheology or microstructure, and feruloylated wheat bran arabinoxylan can be used as a bakery ingredient to potentially enhance the nutritional quality of bread.

Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH.

BACKGROUND: With the rising demand for osseointegrated titanium implants for replacing missing teeth, often in patients with a history of periodontitis, implant-related infections have become an issue of growing concern. Novel methods for treating and preventing implant-associated infections are urgently needed. The aim of this study was to investigate if different pH, atmosphere and surface properties could restrict bacterial adhesion to titanium surfaces used in dental implants. METHODS: Titanium discs with machined or anodized (TiUnite™) surface were incubated with a co-culture of Streptococcus mitis and Actinomyces oris (early colonizers of oral surfaces) at pH 5.0, 7.0 and 9.0 at aerobic or anaerobic atmosphere. The adhesion was analysed by counting colony forming (CFU) units on agar and by confocal laser scanning microscopy (CLSM). RESULTS: The CFU analysis showed that a pH of 5.0 was found to significantly decrease the adhesion of S. mitis, and an aerobic atmosphere, the adhesion of A. oris. S. mitis was found in significantly less amounts on the anodized surface than the machined surface, while A. oris was found in equal amounts on both surfaces. The CLSM analysis confirmed the results from the CFU count and provided additional information on how the two oral commensal species adhered to the surfaces: mainly in dispersed clusters oriented with the groves of the machined surface and the pores of the anodized surface. CONCLUSIONS: Bacterial adhesion by S. mitis and A. oris can be restricted by acidic pH and aerobic atmosphere. The anodized surface reduced the adhesion of S. mitis compared to the machined surface; while A. oris adhered equally well to the pores of the anodized surface and to the grooves of the machined surface. It is difficult to transfer these results directly into a clinical situation. However, it is worth further investigating these findings from an in vitro perspective, as well as clinically, to gain more knowledge of the effects acid pH and aerobic atmosphere have on initial bacterial adhesion.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.

Nordic Crops as Alternatives to Soy-An Overview of Nutritional, Sensory, and Functional Properties.

Soy (Glycine max) is used in a wide range of products and plays a major role in replacing animal-based products. Since the cultivation of soy is limited by cold climates, this review assessed the nutritional, sensory, and functional properties of three alternative cold-tolerant crops (faba bean (Vicia faba), yellow pea (Pisum sativum), and oat (Avena sativa)). Lower protein quality compared with soy and the presence of anti-nutrients are nutritional problems with all three crops, but different methods to adjust for these problems are available. Off-flavors in all pulses, including soy, and in cereals impair the sensory properties of the resulting food products, and few mitigation methods are successful. The functional properties of faba bean, pea, and oat are comparable to those of soy, which makes them usable for 3D printing, gelation, emulsification, and extrusion. Enzymatic treatment, fermentation, and fibrillation can be applied to improve the nutritional value, sensory attributes, and functional properties of all the three crops assessed, making them suitable for replacing soy in a broad range of products, although more research is needed on all attributes.

Food protein-derived amyloids do not accelerate amyloid ß aggregation.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-ß (Aß), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aß. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aß in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aß pathology and Alzheimer's disease.

Effect of physicochemical properties, pre-processing, and extraction on the functionality of wheat bran arabinoxylans in breadmaking - A review.

Arabinoxylan (AX) is an abundant hemicellulose in wheat bran and an important functional component in bakery products. This review compares preprocessing and extraction methods, and evaluates their effect on AX properties and functionality as a bread ingredient. The extraction process results in AX isolates or concentrates with varying molecular characteristics, indicating that the process can be adjusted to produce AX with targeted functionality. AX functionality in bread seems to depend on AX properties but also on AX addition level and interactions with other components. This review suggests that the use of AX with tailored properties together with properly optimized baking process could help increasing the amount of added fiber in bread while maintaining or even improving bread quality.

The Effects of High Fiber Rye, Compared to Refined Wheat, on Gut Microbiota Composition, Plasma Short Chain Fatty Acids, and Implications for Weight Loss and Metabolic Risk Factors (the RyeWeight Study).

Consumption of whole grain and cereal fiber have been inversely associated with body weight and obesity measures in observational studies but data from large, long-term randomized interventions are scarce. Among the cereals, rye has the highest fiber content and high rye consumption has been linked to increased production of gut fermentation products, as well as reduced risks of obesity and metabolic disease. The effects on body weight and metabolic risk factors may partly be mediated through gut microbiota and/or their fermentation products. We used data from a randomized controlled weight loss trial where participants were randomized to a hypocaloric diet rich in either high fiber rye foods or refined wheat foods for 12 weeks to investigate the effects of the intervention on gut microbiota composition and plasma short chain fatty acids, as well as the potential association with weight loss and metabolic risk markers. Rye, compared to wheat, induced some changes in gut microbiota composition, including increased abundance of the butyrate producing Agathobacter and reduced abundance of [Ruminococcus] torques group, which may be related to reductions in low grade inflammation caused by the intervention. Plasma butyrate increased in the rye group. In conclusion, intervention with high fiber rye foods induced some changes in gut microbiota composition and plasma short chain fatty acid concentration, which were associated with improvements in metabolic risk markers as a result of the intervention.

Robust Assembly of Cross-Linked Protein Nanofibrils into Hierarchically Structured Microfibers.

Natural, high-performance fibers generally have hierarchically organized nanosized building blocks. Inspired by this, whey protein nanofibrils (PNFs) are assembled into microfibers, using flow-focusing. By adding genipin as a nontoxic cross-linker to the PNF suspension before spinning, significantly improved mechanical properties of the final fiber are obtained. For curved PNFs, with a low content of cross-linker (2%) the fiber is almost 3 times stronger and 4 times stiffer than the fiber without a cross-linker. At higher content of genipin (10%), the elongation at break increases by a factor of 2 and the energy at break increases by a factor of 5. The cross-linking also enables the spinning of microfibers from long straight PNFs, which has not been achieved before. These microfibers have higher stiffness and strength but lower ductility and toughness than those made from curved PNFs. The fibers spun from the two classes of nanofibrils show clear morphological differences. The study demonstrates the production of protein-based microfibers with mechanical properties similar to natural protein-based fibers and provides insights about the role of the nanostructure in the assembly process.

Hierarchical propagation of structural features in protein nanomaterials.

Natural high-performance materials have inspired the exploration of novel materials from protein building blocks. The ability of proteins to self-organize into amyloid-like nanofibrils has opened an avenue to new materials by hierarchical assembly processes. As the mechanisms by which proteins form nanofibrils are becoming clear, the challenge now is to understand how the nanofibrils can be designed to form larger structures with defined order. We here report the spontaneous and reproducible formation of ordered microstructure in solution cast films from whey protein nanofibrils. The structural features are directly connected to the nanostructure of the protein fibrils, which is itself determined by the molecular structure of the building blocks. Hence, a hierarchical assembly process ranging over more than six orders of magnitude in size is described. The fibril length distribution is found to be the main determinant of the microstructure and the assembly process originates in restricted capillary flow induced by the solvent evaporation. We demonstrate that the structural features can be switched on and off by controlling the length distribution or the evaporation rate without losing the functional properties of the protein nanofibrils.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to ß-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Effect of mechanical and thermal treatments on the microstructure and rheological properties of carrot, broccoli and tomato dispersions.

BACKGROUND: The food industry has shown an increased interest in the manufacture of healthier and more natural food products. By tailored processing fruit and vegetables can be used as structurants thus reducing artificial gums and stabilisers. The effect of different thermal and mechanical treatments, including high-pressure homogenisation, on the microstructural and rheological properties of carrot, broccoli and tomato dispersions was studied. As part of the rheological characterisation small oscillatory deformation as well as shear flow measurements were performed. RESULTS: Carrot and broccoli showed a different behaviour from tomato under the conditions studied. Changing the order of thermal and mechanical treatment led to microstructures with different flow properties. The resulting microstructures differed in the manner of cell wall separation: either breaking across the cell walls or through the middle lamella. High-pressure homogenisation decreased the viscosity of carrot and broccoli dispersions, while it increased the viscosity of tomato. Cryo-scanning electron microscopy showed that the cell walls of carrot and broccoli remained as compact structures after homogenisation whereas tomato cell walls were considerably swollen. CONCLUSIONS: Based on the type of vegetable, the different processes applied led to microstructures with different rheological properties. This study shows that particle size distribution, morphology and phase volume are important parameters to explain the complex relationship between rheology and microstructure for these types of systems.

A hypocaloric diet rich in high fiber rye foods causes greater reduction in body weight and body fat than a diet rich in refined wheat: A parallel randomized controlled trial in adults with overweight and obesity (the RyeWeight study).

BACKGROUND AND AIM: A high intake of whole grain foods is inversely associated with body mass index (BMI) and body fat in observational studies, but mixed results have been found in interventional studies. Among whole grains, rye is the richest source of dietary fiber and meals containing high-fiber rye foods have shown increased satiety up to 8 h, compared to meals containing refined wheat products. The aim of the study was to determine the effect of consuming high fiber rye products, compared to refined wheat products, on body weight and body fat loss in the context of an energy restricted diet. METHODS: After a 2-week run-in period, 242 males and females with overweight or obesity (BMI 27-35 kg/m2), aged 30-70 years, were randomized (1:1) to consume high fiber rye products or refined wheat products for 12 weeks, while adhering to a hypocaloric diet. At week 0, week 6 and week 12 body weight and body composition (dual energy x-ray absorptiometry) was measured and fasting blood samples were collected. Subjective appetite was evaluated for 14 h at week 0, 6 and 12. RESULTS: After 12 weeks the participants in the rye group had lost 1.08 kg body weight and 0.54% body fat more than the wheat group (95% confidence interval (CI): 0.36; 1.80, p < 0.01 and 0.05; 1.03, p = 0.03, respectively). C-reactive protein was 28% lower in the rye vs wheat group after 12 weeks of intervention (CI: 7; 53, p < 0.01). There were no consistent group differences on subjective appetite or on other cardiometabolic risk markers. CONCLUSION: Consumption of high fiber rye products as part of a hypocaloric diet for 12 weeks caused a greater weight loss and body fat loss, as well as reduction in C-reactive protein, compared to refined wheat. The difference in weight loss could not be linked to differences in appetite response. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov, Identifier: NCT03097237.

Stewardship of Wild and Farmed Edible Insects as Food and Feed in Sub-Saharan Africa: A Perspective.

Edible insects have gained popularity as alternative food resources in the face of climate change and increasing carbon and environmental footprints associated with conventional agricultural production. Among the positive attributes that make edible insects suitable as food and feed substrates include rapid reproduction, high energy conversion efficiency, wide distribution, diversity, reduced greenhouses gases and ammonia emissions, possibility to reduce waste and high nutritional composition. In Sub-Saharan Africa, considerable scientific data exist on use of insects as food and livestock feed. However, coherent policies regarding safety, sustainability, trade and regulation of insects as food and animal feed are lacking. The benefits associated with edible insects are likely to accrue in Sub-Saharan Africa through use of a combination of approaches such as ensured sustainable utilization of edible insects in the wild, preservation of traditional conservation, harvesting and consumption practices, development of captive mass production schemes and strengthening robust value chains to incentivise indigenous participants. Collectively these approaches are referred to as the steward and use of insects as food and animal feed. This paper examines the policy frameworks that exist to support the use of edible insects as food and feed on the African continent. This investigation employed a literature review focussing on national policies in selected African countries to assess the relevance to edible insects. Using a baseline of more than 10 edible insect species consumed, 10 country cases in Sub-Saharan Africa were used to support our in-depth examination of the policy situation that may support good stewardship of edible insects as food and feed. Focus on how policies encompassing biodiversity, natural resources, culture, education, research, technology development, trade, health and nutrition and how that could be improved to support inclusivity of edible insects is discussed. We conclude by proposing a pathway that may accelerate recognition and valorisation of edible insects as important food and feed resources in Sub-Saharan Africa including improving policies to support good stewardship of these resources for sustainability.

Processing of tomato: impact on in vitro bioaccessibility of lycopene and textural properties.

BACKGROUND: Human studies have demonstrated that processing of tomato can greatly increase lycopene bioavailability. However, the difference between processing methods is not widely investigated. In the current study different thermal treatments of tomato were evaluated with regard to their impact on in vitro bioaccessibility and retention of lycopene and beta-carotene as well as textural properties. Thermal treatments used were low (60 degrees C) and high (90 degrees C) temperature blanching followed by boiling. RESULTS: Lycopene was relatively stable during thermal treatment, whereas beta-carotene was significantly (P < 0.05) reduced by all heat treatments except for low temperature blanching. In vitro bioaccessibility of lycopene was significantly increased from 5.1 +/- 0.2 to 9.2 +/- 1.8 and 9.7 +/- 0.6 mg kg(-1) for low and high temperature blanching, respectively. An additional boiling step after blanching did not further improve lycopene bioaccessibility for any treatment, but significantly reduced the consistency of low temperature treated samples. CONCLUSION: Choice and order of processing treatments can have a large impact on both lycopene bioavailability and texture of tomato products. Further investigations are needed, but this study provides one of the first steps towards tomato products tailored to optimise nutritional benefits.

Product Quality during the Storage of Foods with Insects as an Ingredient: Impact of Particle Size, Antioxidant, Oil Content and Salt Content.

To increase the acceptability of insects as food in Western culture, it is essential to develop attractive, high-quality food products. Higher acceptability of insect-based food has been shown if the insects are "invisible". Mincing or chopping the insect material could be a first processing step to reduce the visibility of the insects. In this work, we processed yellow mealworms by using traditional food techniques: chopping, mixing and heat treatment in a retort. The results show that all factors in the experimental design (particle size, oil content, salt content and antioxidant) influenced the products to a larger extent than the storage time. The results, measured by sensory analysis, TBAR values (Thiobarbituric acid reactive substances), colourimetry and viscosity, show clearly that the food products packaged in TRC (Tetra recart cartons) 200 packages and processed in a retort stayed stable during a storage time of 6 months at room temperature.