Browsing by Subject "protein"

Andean lupin (Lupinus mutabilis Sweet) is a potential oilseed crop, with a very high protein content (40–45%) and 15–20% oil content. It is valued as an alternative protein source for both human and animal consumption, and like several other lupin species, has a potential role in phytoremediation. Previous experience in central and southern Europe has shown the crop to have low and unstable yields with high sensitivity to heat and drought during the grain–filling stage. The species may therefore be more suitable for cool–temperate climates, as it comes from similar altitudes and latitudes as potato. Therefore, an experiment was set out to quantify the responses of Andean lupin to heat stress, using both gradually and suddenly rising temperatures during grain filling. In preliminary tests 60 accessions from 4 germplasm banks were screened for time to flowering and daylength sensitivity. For this experiment, 3 accessions were chosen, 478435, 457972, 457977, with vegetative phase durations of 44, 53, 64 days from sowing to first flower, respectively. Forty two plants of each accession were sown and reduced to 30 on the basis of uniformity. Plants were grown in a glasshouse with 22 °C, 18 h days and 18 °C, 6 h nights until about 25 days after flowering. Ten plants of each accession were subjected to one of the following treatments: control (continuing in the same glasshouse conditions), sudden heat stress (transferred to a growth chamber and subjected to 38 °C from 11:00 to 15:00) or gradual heat stress (transferred to a growth chamber and subjected to temperature increases of 4 °C day temperature and 2 °C night temperature, with the final two days at 38 °C from 11:00 to 15:00). The plants were returned to the glasshouse and when mature, the seeds were harvested and pooled into 3 replicates per accession and treatment for quality analysis. Seed protein, oil, soluble sugar, ash and moisture content were determined. Data were calculated on the basis of percentage of overall seed mass and also on a milligrams per seed basis in order to reflect the seed physiology at grain–filling stage. Sudden heat stress had greater effects on seed composition than gradual heat stress. When compared with control, sudden heat stress resulted in more loss of every component than gradual heat stress, on a per–seed basis, in all 3 accessions and the responses of the accessions to the sudden stress were not statistically different. Under sudden heat stress, mean seed weight declined by 70%, protein content by 70%, oil content by 85%, ash content by 50%, and soluble sugar content by 75%. The accessions responded differently, however, to the gradual heat stress. Accession 478435 experienced significantly greater reduction in seed weight, protein and ash content than accessions 457972 and 457977. Oil content per seed and soluble sugar content per seed were also lower in 478435 than in the other two cultivars, but the difference was not significant. On the flour basis, sudden heat stress increased ash content and decreased oil content and soluble sugar content significantly in all accessions. Accession 478435 had highest value in ash content at significant level. Under gradual stress, protein and ash content were increased while oil mass and soluble sugar mass were decreased. 478435 had significantly higher protein mass and ash mass in flour with respectively 57% and 5.1%, 457977 had significantly higher soluble sugar content with 112 mg/g. The results showed that heat stress can have a significant effect on the quantity and quality of seed yield in Andean lupin. While all tested accessions were severely susceptible to sudden heat stress, gradual stress identified differences between accessions, with one being much more susceptible than the other two. The most susceptible accession was the earliest to flower. Gradual heat stress allows better resolution than sudden heat stress when screening germplasm for heat tolerance.

The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.

Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.

PURPOSE AND GOALS Microalgae are unicellular eukaryotic organisms capable of photosynthesis. They harvest sunlight and efficiently take up carbon dioxide and nutrients such as nitrogen and phosphorus from their environment and use them for their growth. Due to these properties, their rapid growth and ability to survive in a variety of environments, microalgae have potential in biotechnological applications that promote nutrient recovery and recycling, water purification and the carbon neutral production of biochemicals and possibly biofuels. The purpose of this study was to investigate the suitability of a side stream water originating from the production of baker’s yeast (yeastwater) for the cultivation of a species of microalga called Euglena gracilis. The study aimed to determine the capacity of this water to support growth and protein production of E. gracilis as well as the capacity of E. gracilis to remove nutrients from the water. The effect of filtration of the water on these parameters was also studied. Yeastwater contains an organic molecule called betaine in relatively high concentrations. Betaine has previously been shown to boost the production of the important vitamin cobalamin in bacteria. The study aimed to determine the effect of betaine on the growth of E. gracilis and on the production of cobalamin in the algal-bacterial symbiosis. METHODS E. gracilis was cultured in laboratory scale photobioreactors. Its growth, protein production and nutrient uptake capacity was determined. Baker’s yeast production side stream water diluted with MQ-water was used as the growth medium either in filtered or unfiltered form. A control treatment was prepared where no microalgal inoculate was added to the photobioreactor. The same microalga was also grown in a synthetic nutrient medium with and without betaine. The uptake of betaine and biomass concentrations of cobalamin were determined. For the determination of microalgal growth, dry weight determination and flow cytometry analysis were used. Protein production was determined on the basis of total nitrogen concentration in the biomass. Spectrophotometric measuring kits were used for the determination of nutrient concentrations. Liquid chromatography techniques were used for the determination of betaine and cobalamin concentrations. RESULTS Significant microalgal growth was observed in filtered yeastwater, while growth in unfiltered yeastwater was very low. Nitrogen removal was higher in presence of E. gracilis compared to the control treatment. Protein production in yeastwater was comparable to that of microalgae grown in synthetic medium. E. gracilis grew much better in the synthetic media supplemented with betaine than without the addition. Betaine enrichment had no effect on cobalamin production. Cobalamin was produced in unfiltered yeastwater both with and without the presence of E. gracilis. CONCLUSIONS Unfiltered yeastwater does not support growth of E. gracilis possibly due to its high turbidity. Filtered yeastwater, on the other hand can support the production of E. gracilis biomass. E. gracilis can be used to reduce nitrogen concentrations in yeastwater. Yeastwater can support cobalamin production by bacteria, but this phenomenon did not benefit from the presence of the microalga. The effect of betaine on microalgal growth warrants further study to determine whether it is related to the accumulation of intracellular nutrients, storage compounds or to some other phenomenon. Yeastwater is a promising nutrient feedstock for microalgal biomass production. However, the role of filtration and possibility of using other methods for turbidity reduction needs to be further studied.

The aim of the literature review was to examine barley’s (Hordeum vulgare) alcohol-soluble proteins – hordeins and their technological attributes. Additionally, applicability of flow field flow fractionation (FFF) separation method as well as spectrophotometric and light scattering methods for protein characterization was under investigation. The objective of the experimental research was to determine a suitable extraction method for hordeins and subsequent analysis of their molecular weight distribution, size and conformation by the use of AF4 (asymmetric flow field flow fractionation) in combination with MALS-, UV- and RI-detectors. 40 % 1 propanol combined with mild sonication treatment proved to be the most efficient method to extract hordeins from barley flour. In order to prevent deterioration of the FFF channels the solvent had to be diluted to 20 %. Same dilution was used to measure hordeins’ extinction coefficient and to calculate ?n/?c theoretically. Berry plot was found to be the most suitable fit for the data analysis. Extracted hordeins were analysed with SDS PAGE. Extracts contained monomeric C, B and ? hordeins and polymeric B, D and ? hordeins. Also, small amounts of albumins, globulins and hydrolysed proteins were present. Extracts’ fractograms had five distinctive peaks. All of the peaks’ mass fractals and polydispersity indices were above 1, which means analysed aggregates were polydisperse and shaped as complex rods. This can be explained by 1 propanol influenced protein aggregation. Some inference in light scattering was identified in the MALS detector signal. This and the use of measured extinction coefficient and calculated index of refraction caused some errors in the data. The low sample yield (19–26 %) can be explained by the hordeins’ adhering to a syringe filter and adsorbing to the surface of AF4’s ultrafiltration membrane. Also, the use of over simplifying mathematical model to calculate the results and yield could cause some errors in the results. This study showed that it is possible to study Mw, size and conformation of polymeric hordeins with AF4 combined with MALS/UV-detectors and that hordeins form big aggregates in 20 % 1 propanol. For MALS proteins should be extracted in a solvent that will not interfere with subsequent analysis and proteins net charge, which creates a challenge to find proper solvent for hordeins. Nevertheless AF4 proved to be useful and delicate tool for characterizing cereal polymeric proteins.

Finding new plant-based protein sources is important from a sustainable development perspective. Ethanol and starch production from barley results in fiber and protein side-stream fractions that are currently utilized for animal feed. Nonetheless, it would be more profitable if the barley protein side-stream was used as a human food ingredient. The main storage proteins in barley are known as hordeins. They are polymeric proteins and have low solubility, due to their inter-chain and inter-molecular disulfide bridges and hydrophobic side-chains. In food technology applications protein solubility is an important property for emulsifying and foaming functionality. Proteins are sensitive to the surrounding environment, especially to pH, which could be used to alter the solubility. In this thesis the literature review examined barley (Hordeum vulgare) proteins, especially the hordeins, functionality and effect on food materials. The review includes previous studies concerning cereal proteins functionality in general and their effect on food materials and a discussion on protein stabilized emulsions. The aim of the study was to determine the protein composition of two barley protein concentrates, OP1 and OP2, and to evaluate protein solubility and emulsifying properties. Furthermore, the aim of the study was to compare the functionality of the concentrates. According to SDS gel electrophoresis the protein concentrates consisted mostly of hordeins, especially C-hordein and some B-hordein. Minor amounts of other proteins where also identified. The protein solubility was determined by Lowry’s method. The proteins were more soluble in sodium phosphate buffer than in deionized water. In sodium phosphate buffer at pH 3,8 the solubility of OP1 proteins was 100 mg/g ± 11 mg/g and in deionized water at pH 3,9 the solubility was 45 mg/g ± 1,1 mg/g. The solubility of the OP2 proteins in sodium phosphate buffer at pH 3,8 was 47 mg/g ± 1,5 mg/g and in deionized water at pH 3,7 the solubility was 45 mg/g ± 1,1 mg/g. In both samples the proteins solubility increased as the pH increased. Most proteins were solubilized at pH 11: the solubility for OP1 and OP2 proteins were 240 mg/g ± 11 mg/g and 140 mg/g ± 12 mg/g respectively, which is problematic regarding food products, but could be used as a treatment to improve solubility. The smallest oil droplets were formed in emulsions were the pH was adjusted to 7 with sodium phosphate buffer. The stability of emulsions was however poor in all samples because of phase separation, which was already significant after one day of storage. The phase separation was considered to be flocculation which finally resulted in creaming or sedimentation. The flocculation may have occurred due to hydrophobic interactions between the proteins on the oil/water interface. However, shaking of the emulsion causes the flocculated oil droplets to separate, forming an even emulsion. No coalescence was observed in all samples, except for OP1-emulsions with 1 % (w/v) dry matter. These results confirm that these protein concentrates can be used in food products, especially in milk type drinks that need to be shaken before usage.

The literature review related to faba bean proteins, the mechanism of protein oxidation and detection methods for protein oxidation as well as the relationship between lipid and protein oxidation. Native faba bean, faba bean treated by oven and microwave, as well as soy protein isolate were used as materials. The materials differed in their lipoxygenase activity. Oil-in-water emulsions were made of 10% purified (removal of antioxidants) rapeseed oil and 3% water soluble proteins that were extracted from flour samples. The emulsions were prepared by homogenizing using microfludizer. Oxidation took place in the oven at 37 °C in the dark with constant stirring by magnet stirrers. Sampling for measurements was carried out on day 0, 1, 4 and 7 from each replicate. Oxidative stability of was monitored by following lipid oxidation as formation of conjugated dienes and hexanal, and by following protein oxidation by measuring loss of tryptophan, formation of carbonyls and dityrosine through fluorescence detections both in the lipid phase and in the continuous phase. In this study, the degree of protein oxidation was generally consistent with corresponding lipid oxidation. Generally, the degree of protein oxidation in lipid phase of emulsions was higher than that in continuous phase, although the protein content was far less in the lipid phase. Both results implied co-oxidative relationship between lipid and protein oxidation. In addition, the results showed that thermal treatment or microwave irradiation can not only result in retarding or decreasing lipid oxidation by lowing lipoxygenase (LOX) activity, but also affect protein oxidation via lipid oxidation and the conformational changes of proteins.

Variation of the protein and amino acid content of barley, wheat and oats were studied. Diets based on grain samples of different protein content were optimized for pigs and poultry.The study went on to optimize diets for pigs and poultry with grains of different protein contents. The amino acid and raw protein analysis was undertaken on 38 grain samples. Correlations were calculated between different variables in grain samples and linear regression analysis was conducted between the protein and amino acid composition. The best estimate for amino acid concentrations of cereals was the protein content. The relative content of amino acids decreases as protein content increases and especially so in barley and wheat. Most reliable regression equations between amino acid and protein content were made for barley and wheat samples. For oat reliable regression equations could not be made. Oats also differed by other features from barley and wheat as it correlated with different variables compared to barley and wheat. Amount of needed protein concentrate levels decreased when barley and wheat protein and amino acid contents were considered in optimization. Protein concentrate levels did not decrease when used oat sample with highest protein content.

Haemoglobin and globin from blood cells and albumin, fibrinogen and immunoglobulins from blood plasma are the most important proteins in slaughter blood. Different fractions of blood have slightly different technological properties. Adding blood proteins can have an effect on the gelling, foaming and emulsifying properties of foodstuffs. Blood protein hydrolysates have antioxidative and antigenotoxic effects that could be utilised as food preservatives or in functional foods to strengthen health. The nutritional value of food can be improved by adding blood proteins. Blood proteins can be utilized by replacing egg, fat and sodium caseinate in foods, to improve structure and shelf life of food. Blood proteins can also be utilised replacing egg white, milk or soy proteins or replacing fat in light products. Ultrafiltration is a common method in the food industry and it is also suitable for processing blood proteins. The aim of the study was to optimize an ultrafiltration method to concentrate porcine slaughter blood for use in the food industry and to determine the technological properties of plasma concentrate and its suitability for use in the food industry. Response surface methodology was used to create a mathematical model to calculate the optimal ultrafiltration parameters for plasma concentrations. Optimal conditions for the ultrafiltration were an Ultracel PLTK 30 membrane, temperature of 40 ºC and pressure of 2 bar. The technological properties of blood proteins were measured at pH 4.5, 5.5, 6.3 and 7.0. Volume and stability were measured from foams prepared by whipping from plasma concentrate diluted to protein concentration of 5.8 %. Foam volume and stability were greatest at pH 5.5 and weakest at pH 7.0. Emulsifying capacity was measured from plasma concentrate diluted to 0.01 % protein concentration. Emulsifying capacity was weakest at pH 5.5 and increased towards high and low pH. Rheological properties of gels made from 10 % plasma concentrate were measured. The gels were weakest near the isoelectric point of plasma proteins at pH 5.5. Plasma concentrate was used to prepare bologna sausage. The structure and sensory properties of the sausages were evaluated. The sausages containing plasma concentrate were evaluated as equal to control sausages in every category.