Application of Filtration to Recover Solubilized Proteins During pH-Shift Processing of Blue Whiting (Micromesistius poutassou); Effects on Protein Yield and Qualities of Protein Isolates

Previous studies of the pH-shift protein isolation process have shown that substantial amounts of solubilized proteins can be trapped in the sediments formed in the first centrifugation step of this process. As a strategy to improve the protein yield during pH-shift processing, the aim of this study was to evaluate how filtration as an alternative to centrifugation in the first separation step of pH-shift processing of blue whiting affected proteins yield and protein isolate characteristics (basic composition, polypeptide profiles, surimi gel quality and color attributes). The study comprised both the acid and alkaline versions of the method, and also fresh as well as frozen fish raw material. Results showed that the replacement of centrifugation with filtration substantially improved the protein yield by from about 38% to 62%, but also reduced the removal of lipid. There were no significant effects on gel quality. Protein isolates from fresh raw material were about 5 % whiter and frozen raw materials about 3% whiter with centrifugation as compared to filtration in the pHshift process. For surimi from fresh raw material centrifugation gave about 2 % whiter gels, while the gels from frozen raw material were about 3% whiter for filtered compared to centrifuged material. The whitest isolates and gels were obtained with acid processing of fresh blue whiting. Slight proteolytic breakdown resulting in fragments of 83 and 152 kDa was however noted with the acid process, especially when centrifugation was used

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa)

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats

MRI diffusion-based filtering: a note on performance characterisation

Frequently MRI data is characterised by a relatively low signal to noise ratio (SNR) or contrast to noise ratio (CNR). When developing automated Computer Assisted Diagnostic (CAD) techniques the errors introduced by the image noise are not acceptable. Thus, to limit these errors, a solution is to filter the data in order to increase the SNR. More importantly, the image filtering technique should be able to reduce the level of noise, but not at the expense of feature preservation. In this paper we detail the implementation of a number of 3D diffusion-based filtering techniques and we analyse their performance when they are applied to a large collection of MR datasets of varying type and quality

Protein isolation from herring (Clupea harengus) using the pH-shift process - Protein yield, protein isolate quality and removal of food contaminants

Herring (Clupea harengus) contain valuable proteins but is difficult to process into high-quality foods due to its small size and high content of bones, heme-proteins and lipids. Herring is among the most abundant fish species in the world, but is currently utilized largely for fish meal and oil production.The work presented in this thesis has aimed at evaluating pH-shift processing as a method to isolate proteins from herring and thereby increase its potential as a food raw material. The pH-shift process solubilizes muscle proteins at low or high pH (pH ≤3 or ≥10.8) whereafter impurities can be removed and the solubilized purified proteins are precipitated near the isoelectric point (~pH 5.5). The focus has been the yield and quality of the proteins. Specific aims have been to investigate: i) possible differences between the acid and alkaline version of the pH-shift process, ii) the possibility to remove dioxins and PCBs, and iii) the effect of alkaline pH-shift processing on the microstructure, salt solubility and in vitro digestibility of the proteins.The acid and alkaline versions of the pH-shift process performed similarly when applied to gutted herring, with protein yields of 57-59%. The protein isolates had significantly higher protein concentration and less ash and lipids than the gutted herring, and also a significantly improved color and a well-balanced amino acid profile. The two process versions isolated proteins with similar ability to form a gel, but the acid process version induced proteolysis. Furthermore, the pH-shift process was highly efficient at removing dioxins and PCBs from contaminated Baltic herring, which was correlated to the removal of lipids. The microstructure analyses of the alkali-processed herring proteins revealed a loose protein network, with no remaining myofibrillar structure. The salt solubility of the proteins was significantly decreased after processing, and this was mainly due to exposure to low pH (5.5) during precipitation of the proteins. Precipitation at pH 6.5 was therefore evaluated and resulted in higher protein salt solubility, less lipid oxidation and higher gelation ability of the proteins compared to precipitation at pH 5.5. Despite the changes in protein salt solubility and microstructure, the in vitro digestibility of the alkali-processed proteins precipitated at pH 5.5 remained the same as that of the herring raw material.To conclude, pH-shift processing is a promising tool to isolate proteins from herring and other small pelagic fish species, resulting in high protein yield and an isolate with good gelation capacity, nutritional characteristics, and low content of lipophilic contaminants. Protein isolation using the pH-shift process therefore has the potential to enhance the value of small pelagic fish species and increase their use for human consumption

Protein isolation from herring (Clupea harengus) using the pH-shift process - Protein yield, protein isolate quality and removal of food contaminants

Herring (Clupea harengus) contain valuable proteins but is difficult to process into high-quality foods due to its small size and high content of bones, heme-proteins and lipids. Herring is among the most abundant fish species in the world, but is currently utilized largely for fish meal and oil production.The work presented in this thesis has aimed at evaluating pH-shift processing as a method to isolate proteins from herring and thereby increase its potential as a food raw material. The pH-shift process solubilizes muscle proteins at low or high pH (pH ≤3 or ≥10.8) whereafter impurities can be removed and the solubilized purified proteins are precipitated near the isoelectric point (~pH 5.5). The focus has been the yield and quality of the proteins. Specific aims have been to investigate: i) possible differences between the acid and alkaline version of the pH-shift process, ii) the possibility to remove dioxins and PCBs, and iii) the effect of alkaline pH-shift processing on the microstructure, salt solubility and in vitro digestibility of the proteins.The acid and alkaline versions of the pH-shift process performed similarly when applied to gutted herring, with protein yields of 57-59%. The protein isolates had significantly higher protein concentration and less ash and lipids than the gutted herring, and also a significantly improved color and a well-balanced amino acid profile. The two process versions isolated proteins with similar ability to form a gel, but the acid process version induced proteolysis. Furthermore, the pH-shift process was highly efficient at removing dioxins and PCBs from contaminated Baltic herring, which was correlated to the removal of lipids. The microstructure analyses of the alkali-processed herring proteins revealed a loose protein network, with no remaining myofibrillar structure. The salt solubility of the proteins was significantly decreased after processing, and this was mainly due to exposure to low pH (5.5) during precipitation of the proteins. Precipitation at pH 6.5 was therefore evaluated and resulted in higher protein salt solubility, less lipid oxidation and higher gelation ability of the proteins compared to precipitation at pH 5.5. Despite the changes in protein salt solubility and microstructure, the in vitro digestibility of the alkali-processed proteins precipitated at pH 5.5 remained the same as that of the herring raw material.To conclude, pH-shift processing is a promising tool to isolate proteins from herring and other small pelagic fish species, resulting in high protein yield and an isolate with good gelation capacity, nutritional characteristics, and low content of lipophilic contaminants. Protein isolation using the pH-shift process therefore has the potential to enhance the value of small pelagic fish species and increase their use for human consumption

Protein Isolation from Gutted Herring (Clupea harengus) Using pH-Shift Processes

Herring (Clupea harengus) and other pelagic fish species are mainly used for fish meal and oil production and not for human consumption. In this study, acid pH-shift processing and alkaline pH-shift processing were used to isolate proteins from whole gutted herring with the aim to investigate the potential use of herring proteins as a food ingredient. The acid and alkaline processes gave rise to similar protein yields, 59.3 and 57.3%. The protein isolates from both processes had a significantly (p < 0.05) whiter color and higher protein and lower lipid contents than the starting material. The removal of ash was >80% for both processes, with a trend (p = 0.07) toward higher removal during the alkaline process. Also, Ca and Mg removal was significantly (p < 0.05) higher during the alkaline process. The isolated proteins from the acid process contained myosin degradation products and had a lower salt solubility than proteins from the alkaline process. Both protein isolates had an amino acid profile meeting the recommendations for adults according to FAO/WHO/UNU and could produce a surimi gel of medium strength. The results show that pH-shift processing could be a valuable method for the production of functional food proteins from gutted herring

Effect of alkaline pH-shift processing on in vitro gastrointestinal digestion of herring (Clupea harengus) fillets

The effect of alkaline pH-shift processing on herring (Clupea harengus) protein oxidation, salt solubility and digestibility, has been evaluated. For the latter, herring mince and pH-shift produced herring protein isolate, both raw and heat-treated, were digested using a static gastrointestinal in vitro model. The pH-shift process resulted in drastically lowered protein salt solubility and increased lipid oxidation while protein carbonyl formation was unaffected. Yet, no significant differences in the degree of hydrolysis (DH) were observed between mince and isolates after completed gastrointestinal digestion, something which was confirmed by a similar release of proteinaceous materia

Removal of lipids, dioxins and polychlorinated biphenyls during production of protein isolates from Baltic herring (Clupea harengus) using pH-shift processes

Dioxins and PCBs are toxic, lipophilic, and persistent substances that impose a serious health threat. A major risk of exposure to these toxic substances is consumption of fish from polluted waters, such as the Baltic Sea. The aim of this study was to investigate if pH-shift processing of Baltic herring with elevated toxicity levels could be used to produce a protein isolate with low fat content and, thereby, reduced dioxin and PCB levels. Both acid (pH 2.7) and alkaline (pH 11.2) pH-shift processing were investigated and resulted in efficient reduction of fat, dioxin, and PCB levels. A reduction of 70-80% per amount of protein was determined for all of these parameters. The amounts, and thus the removal, of lipids and dioxins (R(2) = 0.952) as well as lipids and PCBs (R(2) = 0.996) were highly correlated (

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

Camelina sativais an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuringin vitrosubstrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically,C. sativaacyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase inC. sativaseeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressingC. sativaseeds. Thus, thein vitrodata correlate well with the changes in the overall fatty acid profile and TAG species inC. sativaseeds with altered DGAT1 and PDAT activity. Additionally,in vitrostudies ofC. sativaphosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previousin vivostudies regarding this enzyme