Toxic Metamorphosis-How Changes from Lysosomal to Cytosolic pH Modify the Alpha-Synuclein Aggregation Pattern.

Alpha-synuclein (aSyn) is a cytosolic, aggregation-prone protein that is associated with neurodegenerative disorders like Parkinson's disease. Interestingly, the protein can appear in different conformations, including monomeric and oligomeric forms as well as amyloid fibrils. Its individual structural constituents seem to be dependent on various factors and the composition of the respective cellular surroundings. Although under physiological conditions, most aSyn is found in the cytosol and synapses of neurons, aSyn can also be found in lysosomal compartments, where it gets degraded. We here compare the assembly speed, morphology, folding state, and spreading of aSyn at cytosolic pH (pH 7.4) and lysosomal pH (pH 5) using Thioflavin T, transmission electron microscopy, circular dichroism, and Fourier transform infrared spectroscopy. Interestingly, we found substantial differences between aSyn aggregation under neutral and acidic pH conditions, like those present in cytosolic and lysosomal cellular compartments. Also, lysosomal aSyn enriched from an aSyn-overexpressing cell line was able to seed aggregation in a concentration-dependent manner. Moreover, we observed that aSyn aggregates formed under in vitro lysosomal pH (pH 5) conditions were not stable at neutral pH and collapsed into partly soluble aggregates with changed structural characteristics. Our findings have meaningful implications in intracellular toxicity events as well as in lysis procedures for molecular and structural characterization of intracellular aSyn conformers.

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation.

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils.In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.

Dairy-Inspired Coatings for Bone Implants from Whey Protein Isolate-Derived Self-Assembled Fibrils.

To improve the integration of a biomaterial with surrounding tissue, its surface properties may be modified by adsorption of biomacromolecules, e.g., fibrils. Whey protein isolate (WPI), a dairy industry by-product, supports osteoblastic cell growth. WPI's main component, ß-lactoglobulin, forms fibrils in acidic solutions. In this study, aiming to develop coatings for biomaterials for bone contact, substrates were coated with WPI fibrils obtained at pH 2 or 3.5. Importantly, WPI fibrils coatings withstood autoclave sterilization and appeared to promote spreading and differentiation of human bone marrow stromal cells (hBMSC). In the future, WPI fibrils coatings could facilitate immobilization of biomolecules with growth stimulating or antimicrobial properties.

Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein ß-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20-48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells.

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component ß-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

Ultrafast dynamics of UV-excited trans- and cis-ferulic acid in aqueous solutions.

The ultrafast UV-induced processes of the neutral, anionic and dianionic forms of trans- and cis-ferulic acid (FA) in aqueous solution were studied by static and femtosecond time-resolved emission and absorption spectroscopy combined with quantum chemical calculations. In all cases, initial excitation populates the first 1ππ* state. For the dianionic cis-isomer cFA2-, electronic deactivation takes place with a time constant of only 1.4 ps, whereas in all other cases, excited-state deactivation happens more than ten times slower, on a time scale of ≈20 ps. The data suggest sequential de-excitation pathways, where initial sub-picosecond solvent rearrangement and structural changes are followed by internal conversion to an intermediate excited electronic state from which deactivation to the ground state proceeds. Considering the time scales, barrierless excited-state pathways are suggested only in the case of cFA2-, where the observed formation of the isomerisation photoproduct tFA2- provides clear evidence for a cis ⇄ trans isomerisation coordinate. In the other cases, pathways with an excited-state energy barrier, presumably along the same coordinate, are likely, given the longer excited-state lifetimes.

Differences in heat stability and ligand binding among ß-lactoglobulin genetic variants A, B and C using (1)H NMR and fluorescence quenching.

The structure of ß-lactoglobulin (ß-LG) is well characterized, but the exact location of binding sites for retinol and (-)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic ß-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and ß-LG A with the most binding sites for EGCG, which include Tyr(20), Phe(151) and His(59). Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of ß-LG as nanotransporter.

Differences in binding behavior of (-)-epigallocatechin gallate to ß-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B).

The lipocalin ß-lactoglobulin (ß-LG) exists in different natural genetic variants--of which ß-LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes--building homodimers of ß-LG A and ß-LG B and heterodimers of ß-LG AB. Although ß-LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero- and homodimers of ß-LG is largely unknown. The present findings revealed significant differences for hetero- and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (-)-epigallocatechin gallate (EGCG)). These findings were confirmed using FT-IR, where the addition of EGCG influenced the ß-sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of ß-LG in the order ß-LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of ß-LG and gives not only important information for ß-LG binding studies, but may also apply for polymorphisms of other self-aggregating lipocalins.

Lipid oxidation induced protein scission in an oleogel as a model food.

Lipid oxidation induced protein scission was investigated in oleogel using beta-lactoglobulin (whey protein isolate) as gelator. Extracted cleaved peptides were measured using high resolution mass spectrometry (FT-ICR-MS), which was provided by an automatically generated annotation list approach to identify relevant masses and sum formula using the isotopic pattern. The identified oxidized peptides were then further evaluated using partial least squares regression to relevant lipid hydroperoxide formation data, which provide the significance and importance of the peptides toward lipid induced scission. Thereby, the most important peptides are located at the surface of the protein in random coil segments and especially at the ends of the protein sequence. The most important amino acids were cysteine and aliphatic amino acids, which undergo scission mostly by the α-amidation pathway. The findings compare well with studies investigating depletion of amino acids initiated by lipid oxidation in systems containing bovine albumin or gamma-globulin.

Engineering artificial casein micelles for future food: Is casein phosphorylation necessary?

Industrial-scale production of recombinant proteins for food products may become economically feasible but correct post-translational modification of proteins by microbial expression systems remains a challenge. For efficient production of hybrid products from bovine casein and recombinant casein, it is therefore of interest to evaluate the necessity of casein post-translational phosphorylation for the preparation of hybrid casein micelles and study their rennet-induced coagulation. Our results show that dephosphorylated casein was hardly incorporated into artificial casein micelles but was capable of stabilising calcium phosphate nanoclusters with an increased size through adsorption on their surface. Thereby, dephosphorylated casein formed larger colloidal particles with a decreased hydration. Furthermore, the presence of increasing amounts of dephosphorylated casein resulted in increasingly poor rennet coagulation behaviour, where dephosphorylated casein disrupted the formation of a coherent gel network by native casein. These results emphasise that post-translational phosphorylation of casein is crucial for their assembly into micelles and thereby for the production of dairy products for which the casein micelle structure is a prerequisite, such as many cheese varieties and yoghurt. Therefore, phosphorylation of future recombinant casein is essential to allow its use in the production of animal-free dairy products.

Trends and prospects in dairy protein replacement in yogurt and cheese.

There is a growing demand for nutritional, functional, and eco-friendly dairy products, which has increased the need for research regarding alternative and sustainable protein sources. Plant-based, single-cell (SCP), and recombinant proteins are being explored as alternatives to dairy proteins. Plant-Based Proteins (PBPs) are commonly used to replace total dairy protein. However, PBPs are generally mixed with dairy proteins to improve their functional properties, which makes them dependent on animal protein sources. In contrast, single-Cell Proteins (SCPs) and recombinant dairy proteins are promising alternatives for dairy protein replacement since they provide nutritional components, essential amino acids, and high protein yield and can use industrial and agricultural waste as carbon sources. Although alternative protein sources offer numerous advantages over conventional dairy proteins, several technical and sensory challenges must be addressed to fully incorporate them into cheese and yogurt products. Future research can focus on improving the functional and sensory properties of alternative protein sources and developing new processing technologies to optimize their use in dairy products. This review highlights the current status of alternative dairy proteins in cheese and yogurt, their functional properties, and the challenges of their use in these products.

Amyloid-like aggregation of recombinant ß-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified ß-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant ß-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.

Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein ß-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native ß-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with ß-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

Functionality of Ingredients and Additives in Plant-Based Meat Analogues.

Meat analogue research and development focuses on the production of sustainable products that recreate conventional meat in its physical sensations (texture, appearance, taste, etc.) and nutritional aspects. Minced products, like burger patties and nuggets, muscle-type products, like chicken or steak-like cuts, and emulsion products, like Frankfurter and Mortadella type sausages, are the major categories of meat analogues. In this review, we discuss key ingredients for the production of these novel products, with special focus on protein sources, and underline the importance of ingredient functionality. Our observation is that structuring processes are optimized based on ingredients that were not originally designed for meat analogues applications. Therefore, mixing and blending different plant materials to obtain superior functionality is for now the common practice. We observed though that an alternative approach towards the use of ingredients such as flours, is gaining more interest. The emphasis, in this case, is on functionality towards use in meat analogues, rather than classical functionality such as purity and solubility. Another trend is the exploration of novel protein sources such as seaweed, algae and proteins produced via fermentation (cellular agriculture).

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.

Alternatives to Meat and Dairy.

The increasing size and affluence of the global population have led to a rising demand for high-protein foods such as dairy and meat. Because it will be impossible to supply sufficient protein to everyone solely with dairy and meat, we need to transition at least part of our diets toward protein foods that are more sustainable to produce. The best way to convince consumers to make this transition is to offer products that easily fit into their current habits and diets by mimicking the original foods. This review focuses on methods of creating an internal microstructure close to that of the animal-based originals. One can directly employ plant products, use intermediates such as cell factories, or grow cultured meat by using nutrients of plant origin. We discuss methods of creating high-quality alternatives to meat and dairy foods, describe their relative merits, and provide an outlook toward the future.

Removal of phenolic compounds from de-oiled sunflower kernels by aqueous ethanol washing.

Selective removal of phenolic compounds (PCs) from de-oiled sunflower kernel is generally considered a key step for food applications, but this often leads to protein loss. PC removal yield and protein loss were assessed during an aqueous or aqueous ethanol washing process with different temperatures, pH-values and ethanol contents. PC yield and protein loss increased when the ethanol content was < 60% or when a higher temperature was applied. Our main finding is that preventing protein loss should be the key objective when selecting process conditions. This can be achieved using solvents with high ethanol content. Simulation of the multi-step exhaustive process showed that process optimization is possible with additional washing steps. PC yield of 95% can be achieved with only 1% protein loss using 9 steps and 80% ethanol content at 25℃. The functional properties of the resulting concentrates were hardly altered with the use of high ethanol solvents.

Characteristics of Soy Protein Prepared Using an Aqueous Ethanol Washing Process.

Currently, the predominant process for soy protein concentrate (SPC) production is aqueous ethanol washing of hexane-extracted soy meal. However, the use of hexane is less desired, which explains the increased interest in cold pressing for oil removal. In this study, cold-pressed soy meal was used as the starting material, and a range of water/ethanol ratios was applied for the washing process to produce SPCs. Washing enriched the protein content for the SPCs, regardless of the solvent used. However, we conclude that washing with water (0% ethanol) or solvents with a high water/ethanol ratio (60% and above) can be more advantageous. Washing with a high water/ethanol ratio resulted in the highest yield, and SPCs with the highest protein solubility and water holding capacity. The water-only washed SPC showed the highest viscosity, and formed gels with the highest gel strength and hardness among all the SPCs at a similar protein concentration. The variations in the functionality among the SPCs were attributed to protein changes, although the effects of non-protein constituents such as sugar and oil might also be important. Overall, the aqueous ethanol washing process combined with cold-pressed soy meal created SPCs comparable to commercial SPC in terms of composition, but with varied functionalities that are relevant for novel soy-food developments.

Changes in Protein Fluorescence in a Lipid-Protein Co-oxidizing Oleogel.

High and low levels of lipid-induced protein oxidation (tuned by the addition of 0%-8.4% water) were investigated in oleogels, using excitation-emission matrix (EEM) fluorescence spectroscopy, coupled with a partial least-squares (PLS) regression and lipid hydroperoxide data. In high-level oxidation models, the intrinsic tryptophan fluorescence decreased and the emission maxima increased from 352.5 to 356.0 nm indicating the presence of protein modifications, which was further supported by size-exclusion chromatography. PLS recognized 3 latent components, with several excitation-emission points of interest. These apparent compounds include a region associated with radical mediated protein modifications (approximately 325 and 410 nm), lipid oxidation product adducts (approximately 350 nm and 420-425 nm), and malondialdehyde adducts (approximately 375 and 425 nm). The separate evaluation of these apparent compounds, at a 420 nm emission, indicated that lipid oxidation promotes protein lipid adduct fluorescence at high water levels, rather than radical mediated protein fluorescence.