Browsing by Subject "hydrolysis"
Now showing items 1-9 of 9
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(2010)3-Chloro-1,2-propanediol (3-MCPD) and its esterified forms are food-borne contaminants. The chemical properties of fatty acid esters of 3-MCPD and their formation in foodstuffs as well as the analytical methods used to detect them were reviewed. The aim of the experimental work was to compare the analytical methods used to detect 3- MCPD esters and to obtain a method for detection of 3-MCPD fatty acid esters in Finnish Food Safety Authority (EVIRA). Synthesised 3-MCPD palmitate esters and palm oil samples were used to detect differences between the methods. Decomposition of 3-MCPD during hydrolysis with sodium methoxide was studied as well as the formation of excess 3- MCPD in palm oil samples during acid hydrolysis. 3-MCPD fatty acid esters were determined as free 3-MCPD by GC-MS after hydrolysis and derivatisation with phenylboronic acid. Free 3-MCPD was cleaved from 3-MCPD esters using acidic hydrolysis with sulphuric acid and methanol or transesterification with sodium methoxide. The amount of 3-MCPD obtained after the hydrolysis of synthesised 3-MCPD esters with sodium methoxide was only 45 % of that obtained after acid hydrolysis. There was a statistical difference between the means at a 95 % level of significance. The formation of excess 3-MCPD during acid hydrolysis was not proven from the results obtained from the palm oil samples. Results showed the opposite, as the amount of 3-MCPD obtained was larger after hydrolysis with sodium methoxide. The precision of the results was poor possibly due to a large systematic error and should be renewed in the future. The results showed there is a significant difference between the two methods of hydrolysis and that the choice between the methods can influence the recovery of the 3-MCPD esters.
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Analytical methods for the determination of Novichok nerve agents and their decontamination products (2023)Novichok nerve agents are persistent, highly toxic chemical weapons which were added to the Chemical Weapons Convention in 2020 after their use on civilians in England. The detection and characterization of Novichok nerve agents and the degradation products formed after their exposure to decontamination products can be accomplished through complementary instrumental analyses. Chromatographic methods such as LC-MS/MS can be utilized to qualitatively detect Novichok degradation products such as hydrolysates and LC-HRMS can provide information about their structure via the elemental composition and fragmentation pathways. By contrasting these data to spectroscopic techniques such as 1H and 31P NMR, structural elucidation of decontamination products is possible as well as the determination of the kinetics of the decontamination process itself. The literature review contains a summary of all published instrumental methods with which Novichok nerve agents, degradation products, biomarkers and adducts have been analyzed and the efficacy of those methods. In the experimental research, Novichok nerve agent A-234 was decontaminated via six different decontaminants and analyzed by LC-MS/MS to identify the mass spectra of the degradation products of each, followed by LC-HRMS analysis to determine the elemental composition and fragmentation patterns of the degradation products. The A-234 rate of hydrolysis kinetics were measured by 1H and 31P NMR spectroscopy in three of the decontaminants and when possible, two dimensional analysis was used to correlate the structural data from the chromatographic analysis. Lastly, the A-234 hydrolysate was derivatized via TMSDAM methylation for GC-MS/MS analysis after testing with two silylating and three methylating agents. Decontamination of A-234 was successful within 48 hours with three decontamination agents and complete hydrolysis was observed within 5 hours with an oxidizer-containing quaternary salt based decontamination agent.
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(2014)Oat protein is a valuable cereal protein with high protein content and a good balance of amino acids profile. However, the inflexible molecular structure and poor watersolubility of oat protein limit its application in food industry. To exploit the food use of oat, the functionalities of oat proteins should be improved. α-chymotrypsin, a proteolytic enzyme, has been proven as being able to deamidate some food proteins without severe hydrolysis under alkaline pH conditions, hence to improve the protein functionalities. The aim of this research was to test whether oat protein could be deamidated by α- chymotrypsin under alkaline pH conditions. The extent of α-chymotrypsin-induced proteolysis on oat protein was also studied. Oat protein was treated by α-chymotrypsin under alkaline conditions from pH 8 to 11 at room temperature for 2 hours. The deamidation degrees of the proteins were measured by ammonia quantification. The hydrolysis degrees of proteins were analyzed by trichloroacetic acid precipitation and protein quantification method. The changes of molecular weights were analyzed by SDS-PAGE and SE-HPLC. The action of α-chymotrypsin under alkaline pH conditions induced slight deamidation of oat protein to low deamidation degrees (9-12%). On the other hand, α-chymotrypsin mainly caused hydrolysis of oat protein to hydrolysis degrees between 49% and 62%. SDS-PAGE and SE-HPLC analysis also revealed that significant hydrolysis of oat protein occurred during the reaction. The hydrolysis mainly caused the shift of oat protein fractions from molecular weight above 20 kDa to molecular weight below 15 kDa. Change in pH did not cause significant differences on deamidation degrees and hydrolysis degrees of oat protein In conclusion, oat protein could not be effectively deamidated by α-chymotrypsin under alkaline pH conditions, while the proteolysis was severe.
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(2014)Oat protein is a valuable cereal protein with high protein content and a good balance of amino acids profile. However, the inflexible molecular structure and poor watersolubility of oat protein limit its application in food industry. To exploit the food use of oat, the functionalities of oat proteins should be improved. ?-chymotrypsin, a proteolytic enzyme, has been proven as being able to deamidate some food proteins without severe hydrolysis under alkaline pH conditions, hence to improve the protein functionalities. The aim of this research was to test whether oat protein could be deamidated by ?- chymotrypsin under alkaline pH conditions. The extent of ?-chymotrypsin-induced proteolysis on oat protein was also studied. Oat protein was treated by ?-chymotrypsin under alkaline conditions from pH 8 to 11 at room temperature for 2 hours. The deamidation degrees of the proteins were measured by ammonia quantification. The hydrolysis degrees of proteins were analyzed by trichloroacetic acid precipitation and protein quantification method. The changes of molecular weights were analyzed by SDS-PAGE and SE-HPLC. The action of ?-chymotrypsin under alkaline pH conditions induced slight deamidation of oat protein to low deamidation degrees (9-12%). On the other hand, ?-chymotrypsin mainly caused hydrolysis of oat protein to hydrolysis degrees between 49% and 62%. SDS-PAGE and SE-HPLC analysis also revealed that significant hydrolysis of oat protein occurred during the reaction. The hydrolysis mainly caused the shift of oat protein fractions from molecular weight above 20 kDa to molecular weight below 15 kDa. Change in pH did not cause significant differences on deamidation degrees and hydrolysis degrees of oat protein In conclusion, oat protein could not be effectively deamidated by ?-chymotrypsin under alkaline pH conditions, while the proteolysis was severe.
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(2020)The literature review deals with the status and the causes of bread waste all over the world. More importantly, the current managements of increasing bread waste. Enzymatic hydrolysis by α-amylase and amyloglucosidase is a potential treatment, which transforms bread waste into syrups for further revaluation with functional compounds. The aim of the experimental work was to determine the influence of enzymatic hydrolysis conditions (hydrolysis time, hydrolysis temperature, enzyme dosage of α-amylase and amyloglucosidase) on glucose content and free amino nitrogen (FAN) content of resulting hydrolysate from bread waste. Furthermore, the effect of lactic acid fermentation on glucose content was studied when bread waste was subjected to simultaneous hydrolysis and fermentation with Pediococcusclaussenii (E-032355T). Glucose content varied greatly under different hydrolysis conditions from nearly 17% to only 5%, while FAN content was barely influenced. pH value had slight changes and no Bacillus cereus bacteria was found. A well fitted model for glucose content was obtained with an excellent power of interpretation, prediction and optimization. Enzyme dosage was the principal factor having a significant effect on hydrolysis efficiency, followed by temperature and time. With optimized hydrolysis conditions (50 mg/kg α-amylase and 2500 mg/kg amyloglucosidase, 30℃, 19 hours), the glucose content 16.31% was achieved, and the result was in accordance with the value 16.39% predicted by the model. Moreover, a 2.2% increase of glucose yield was detected when waste bread was subjected to simultaneous hydrolysis and fermentation compared to the control sample (bread waste was treated only with hydrolysis under the same condition). The well growth of used lactic acid bacteria (LAB) strains Pediococcusclaussenii (E-032355T) resulted in lower pH, which further improved enzymes activities and increased glucose content of the hydrolysate.
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(2018)Production of biofuels from non-food-based materials, such as lignocellulose, provides a good alternative for the traditional burning of fossil fuels. Some of the researched and existing biofuel applications are based on the utilization of enzymes. There are multiple cellulolytic enzymes required in the efficient hydrolysis of lignocellulose, and one of the key enzyme group is β-glucosidases. These enzymatic systems are mainly adopted from wood-decaying fungi. The overall enzymatic system consists of different types of cellulases that first degrade the crystalline cellulose to oligosaccharides and cellobiose. In the final step, β-glucosidases hydrolyse the oligosaccharides to glucose (a fermentable sugar). In fact, β-glucosidases are one of the limiting enzyme classes in this process, due to phenomena such as end-product inhibition. β-Glucosidases belong to Glycoside hydrolases (GH), that can be classified into different protein families. In an industrial perspective, the main interest resides in GH1 and GH3 family enzymes. Many industrially relevant extracellular β-glucosidases belong to GH3 family. However, intracellular GH1 β-glucosidases often exhibit higher tolerance to harsh conditions such as high substrate and product concentrations, high temperatures and low pH. The goal of this MSc thesis work was to purify and characterize a novel GH1 β-glucosidase, named NBG. Both GH1 and GH3 family enzymes were used as references for the characterization work. The GH3 reference enzyme was a β-glucosidase from Aspergillus niger (An Cel3A), derived from the commercial enzyme preparation Novozym 188. The used GH1 reference was a β-glucosidase from termite Nasutitermes takasagoensis (Nt GH1). The applicability of NBG β-glucosidase in biomass hydrolysis was also examined, together with possible considerations for applicability by other type of applications. The purification of An Cel3 reference enzyme was performed as described previously in literature. A novel protocol combining thermal treatment and low resolution IEX purification was developed for the NBG enzyme in this study. The enzyme’s activity on various pNP-substrates was determined, followed by pH stability, thermostability and inhibition studies. According to the result, NBG is a potential candidate for industrial use. The enzyme was found to be thermostable and active in a wide pH range when compared to the reference enzymes (stable up to 20 h at +60 ˚C and in pH 3.5 – 6.0). NBG also exhibited wider activity on pNP-substrates than the reference enzymes, highest specific activity being on pNPG, followed by moderate activity on pNPFuc and low activities on pNPGal and pNPXyl. Furthermore, NBG exhibited higher tolerance to inhibitors such as glucose and ethanol. Glucose inhibition was not observed until concentration of 200 mM for NBG, while in the same concentration the reference enzymes were almost completely inhibited. A Clear activation (of +16 %) by 100 mM glucose was observed with NBG. This enzyme also outperformed the An Cel3A-reference in ethanol tolerance, retaining activity better in 15 and 20 % ethanol. Activation by ethanol was also observed for both of the fungal enzymes, the most pronounced effect being observed for NBG in 15 % ethanol (+21 % of initial activity). The hydrolysis of insoluble cellulosic substrate (Avicel) was investigated using a commercial cellulase mixture (Celluclast 1.5L), where a semi-pure β-glucosidase preparation was added: novel β-glucosidase preparation (NBG (2-S2)) or the reference preparation An Cel3A (Nz188). According to the results, the NBG (2S-2) was outperformed by An Cel3A (Nz188) in Avicel 4 – 72 h hydrolysis experiments. The amount of reducing sugars released from Avicel was approximately 18–19 % higher with the commercial Nz188 preparation when compared to the 2S-2 preparation. Further analyses of samples revealed accumulation of cello-oligosacchardes, which may accumulate due to two possible reasons: Either the NBG enzyme does not possess high enough cellobiase activity (needed in biomass hydrolysis to glucose), or accumulation of cellobiose is due to transglycosylation activity of NBG. According to activity (and 3D modelling) data, NBG may not be a true β-glucosidase belonging to the EC 3.2.1.21 (and having cellobiase activity). Further investigation of the possible substrate specificity and transglycosylation activity of the NBG will be needed in assessing its applicability in other types of biotechnical applications.
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(2019)The literature part of the study dealt with frying methods, frying fats and oils, chemical and physical changes of frying fats and oils during the frying and analyse methods of frying fats and oils. The aim of the experimental part was to study changes in rapeseed oil quality during continuous frying process during three different frying periods. Especially the effect of day without production during the frying period was studied. Samples, frying oils, were taken every day during the different frying periods. In period one and two there was one day without production. Oils were stored in storage tanks during that time. In third period, there was production on every day. All samples were inspected for colour, odour, viscosity and clearness by visual methods. Also viscosity by rotation viscometer, smoking point, free fatty acids by titration method, fatty acid content and water content of samples was analysed. Based on sensory evaluation, the quality of oil deteriorated over thedays of use. Colour was darker, viscosity was higher and off-odours were sensed. Instrumental analyses indicate same kind of changes; viscosity and free fatty acid were increased and smoking point and amount of unsaturated fatty acid were decreased while frying time was increasing. In literature, also same changes were mentioned. There was changes in fatty acid content during the frying periods. More fatty acids characteristic to meat was detected in oils at the end of period than in fresh oils. Based on these results, oil quality remained acceptable in everyday use for a typical frying period of one week. Difference between fresh oil and used oils was clear but still acceptable. The amount of free fatty acids, and the level of viscosity and smoking points were under the limits applied in Finland. The day without production deteriorated oils quality substantially Reducing the amount of oil in process and having shorter frying periods or more efficient filtering of oils can help to keep the quality of frying oil better.
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(2014)Divicine and isouramil are the causative agents of favism. The stability of divicine is of vital importance in faba bean detoxification. The literature review of this thesis focused on the hydrolysis of vicine to divicine, the oxidation processes of divicine and its stability studies. The main aims of this thesis were to produce divicine using the hydrolysis of vicine with ?-glucosidase, and study the effects of nitrogen/air atmosphere, reducing agent, pH and temperature on the stability of divicine. The identities of other compounds observed in vicine hydrolysis were also to be investigated. In addition, convicine was also hydrolyzed in the extracts and pure convicine fractions. Vicine and convicine were co-extracted from dehulled faba bean flour and were separated with preparative HPLC-MS. The extracts and the pure vicine and convicine fractions were hydrolyzed with ?-glucosidase to yield divicine and isouramil. The identities of the compounds formed during vicine fraction hydrolysis were studied by MS. In the following stability studies, the pure vicine fractions were hydrolyzed with ?-glucosidase under nitrogen and in the presence of (+)-sodium L-ascorbate. Moreover, the fractions were firstly hydrolyzed under air, next, the formed divicine was incubated at pH 3.0 or 5.0 at 20 or 37 ºC. An analytical HPLC method was used to study the changes during hydrolysis and stability tests. It was found that higher ?-glucosidase concentration and longer incubation period resulted in higher hydrolysis degrees of vicine and convicine. Further, vicine was hydrolyzed more rapidly at pH 3.0 than 5.0. Vicine was hydrolyzed to divicine. Divicine further generated two compounds, named compound 1 and compound 2 in this thesis. Their corresponding retention times and absorption maxima were: 2.15 min, 282 nm; 1.79 min, 262 nm; and 1.94 min, 210 nm. Compound 1 was directly generated from divicine. It was possibly oxidized divicine, but its characterization with MS failed in this study. Compound 1 decomposed to compound 2 at pH 5.0 at 20 ºC, but at pH 3.0 at 20 ºC, divicine might directly decompose to compound 2. Only one compound (named compound 3) was formed during convicine hydrolysis, and its retention time and absorption maximum were 2.50 min and 280 nm. Divicine and compounds 1, 2, and 3 were not stable, they finally decomposed to non-UV absorbing substances. Divicine was more stable under nitrogen than under air, and in the presence of (+)-sodium L-ascorbate than without its presence. Divicine decomposed similarly at pH 3.0 and 5.0 at 37 ºC, but at 20 ºC, divicine was more stable at pH 5.0. At both pH values, the stability of divicine was increased at 20 ºC compared with 37 ºC.
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(2015)The thesis starts with a review of literatures related to the topic. In the beginning, several speculations of water holding capacity (WHC) in meat are reviewed, and the joint effects of salt and phosphate addition on WHC are discussed in detail. In addition, the hydrolysis and diffusion of phosphates in meat are also explained. At the end of the literature review, several methods of phosphate measurement are listed and compared. The aim of this research was to determine an effective pattern for phosphate addition in meat processing by studying the dynamics of the phosphate diffusion in relation to their hydrolysis. To address this aim, the meat stripes were subjected to one-dimensional diffusion in TPP and PP brines (with the same salt and P2O5 content), and the phosphate contents of TPP, PP and MP at three diffusion levels (0.625, 1.875 and 3.125 cm) were analysed after 0.5, 2, 6, 24, or 48 h. Thin-layer chromatography (TLC) was used to determine phosphate contents. In addition, the effects of TPP, PP and MP on WHC of the laboratory scale cooked sausages with 0.5 h standing time were compared as a reference for the impact of different phosphates on improving WHC in meat. It was found that areas on the surface of the meat always had the lowest MP content; in addition, in every diffusion level, the initial MP content always decreased first before an increase took place. These results suggested that MP underwent two-way diffusion under the effect of naturally occurring concentration gradient between the meat and the brine, and the TPP/PP hydrolysis generated MP as products. Moreover, PP displayed higher stability then TPP: PP could be observed in the diffusion level as far as at 3.125 cm, and the PP content at 1.875 cm was stable during 2 days’ PP brine diffusion; on the other hand, no TPP was found at distances of 3.125 cm, and only a small amount of TPP was found in 1.875 cm. In addition, the acidic pH accelerated the phosphate diffusion, while the effect of metal ions on diffusivity was unclear. Within the 1.875 cm diffusion distances, which is the common size of meat cube used in meat industry, TPP did not exert a better effect in enhancing or prolonging the stability of PP, and it is possible that the application of PP can fulfil the task of common production schedules. In the sausage WHC test, MP sausages had the worst appearance, which suggests poor protein extraction and hence poor gelling properties. The appearance of PP and TPP sausages were similarly fine, while PP sausages had the best WHC.
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