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    Effect of Kiwifruit actinidin on the digestion of gluten proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Jayawardana, Isuri Achintha
    Gluten proteins are resistant to complete proteolysis by the human gastrointestinal tract (GIT) enzymes, due to their high proline- and glutamine-rich peptide sequences. Proline confers resistance to proteolysis by digestive enzymes, producing indigestible proline-rich peptides, some of which can trigger immunogenic reactions that are responsible for gluten-related health disorders such as coeliac disease, wheat allergy and gluten sensitivity. At present, gluten-free diets (GFD) are the only promising therapy for gluten-related health disorders. However, maintaining a lifelong GFD is challenging. As an alternative therapy, gluten-specific enzymes to hydrolyse immunogenic peptides have shown promising results. Most of these are of microbial origin. Identification of natural alternative enzymes is desirable, with fruit-borne enzymes a possible solution. Actinidin, a cysteine protease found in most green kiwifruit (Actinidia deliciosa), is suggested as an effective exogenous enzyme, to be utilized in this category. The objective of this PhD study was to evaluate the effect of actinidin on the digestion of gluten and gluten-derived immunogenic peptides in the GIT. The effectiveness of actinidin was tested using different in vitro GIT models and an animal (pig) preclinical model with purified gluten or whole wheat bread as sources of gluten, and purified actinidin or and fresh green kiwifruit as sources of actinidin. Analytical techniques such as free amino nitrogen determination, enzyme-linked immunosorbent assay and both targeted and untargeted mass spectrometry were used to determine the degree of hydrolysis (DH), R5 gluten epitopes and immunogenic peptides respectively. Actinidin hydrolysed peptide bonds adjacent to proline residues in the 33-mer peptide, one of the most immunogenic gluten peptides. The gastric DH of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05). Actinidin at a concentration of > 2.7 U/mL and pH > 2 during hydrolysis was considered ideal for gluten hydrolysis. Actinidin increased (P < 0.05) the rate of acceleration of DH of gluten and reduced the amount of R5 epitopes present in the small intestine using a semi-dynamic in vitro GIT digestion model. Actinidin also accelerated the gastric hydrolysis of wheat proteins in whole wheat soda bread, which was reflected in a faster reduction of R5 epitopes in the gastric conditions and the rate of reduction (P < 0.05) of most of the immunogenic marker peptides present in the small intestine. In vivo, the presence of dietary actinidin in the form of green kiwifruit significantly (P < 0.01) enhanced the gastric digestion of wheat proteins in whole wheat soda bread fed to pigs as a model of human GIT digestion. The amount of R5 epitopes was lower (P < 0.01) in the stomach, proximal and distal small intestine and terminal ileum of pigs fed diets containing green kiwifruit (P <0 .05). The number of immunogenic peptides in the proximal small intestine was low in the pigs fed green kiwifruit diet compared to that of the pigs fed yellow kiwifruit diet (control). In addition, a diet containing green kiwifruit markedly reduced (P < 0.05) the amount of seven gluten immunogenic marker peptides including the 33-mer peptide in the stomach chyme of pigs. Actinidin was able to survive peptic proteolysis and gastric pH conditions until 300 min postprandial in pigs. Taken together, these results suggest that actinidin enhanced the rate of proteolysis of both purified gluten and gluten in a food matrix and reduced the amount of immunogenic gluten epitopes reaching the small intestine during GIT digestion in vitro and in vivo. Actinidin was able to reduce both the amount of and the time of exposure to immunogenic peptides in the small intestinal lumen, therefore it is a promising candidate to be considered in oral enzyme therapy for gluten-related health disorders.
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    The kinetics of mild acid hydrolysis of gluten and the functional properties of the modified proteins at various levels of hydrolysis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1988) Higgins, John Joseph
    Gluten is the mixture of proteins remaining in wheat flour after starch and water soluble components have been extracted by washing. Its unique dough forming properties are due to the structure of the proteins. A feature of the protein is the high glutamine content, about 30% of the total amino acids. A number of studies have shown that gluten's properties of insolubility and water-binding can be substantially modified by mild acid hydrolysis. The principal effect of the mild acid treatment is to hydrolyse the amide side chain of glutamine such that the amide group is replaced by a carboxyl group. In addition, it is known that hydrolysis of peptide bonds can have a large influence on the functional properties of proteins. The aims of this study were to determine the kinetics of the acid catalysed deamidation and peptide bond hydrolysis reactions, and to comment on the resultant changes in functional properties. A statistically designed experiment was used to determine the effect of temperature, hydrogen ion concentration and gluten concentration. An initial rate analysis of the results showed that reactions could be described by equations of the form: Rate of amide bond hydrolysis = k1.[Amide][H+] and Rate of peptide bond hydrolysis = k2 [Peptide] [H+] where k = koe -E/R.1/T A stoichiometric analysis of the experimental data confirmed that hydrogen ions were consumed in both reactions. A numerical solution was developed to predict the extent of reaction with time. A computer program incorporating the solution was used to simulate the reaction and test the solution. The simulation results appeared to overestimate the progress of the reaction with time. A series of ten gluten powders, hydrolysed to different extents was prepared at small pilot scale. The composition of the samples was determined and compared with the extent of hydrolysis predicted by the reaction simulation. Reasonable agreement was achieved. A selection of the functional properties of the prepared samples was examined. The quantity of alkali required to dissolve each preparation to the extent of its solubility at pH 7.6 increased markedly with the extent of hydrolysis due to the additional carboxyl groups requiring neutralization. The flavour of each preparation was exanined. A cereal flavour was found to decrease with the extent of hydrolysis. A lingering bitter flavour was found to increase with the extent of hydrolysis. The solubility of all preparations at p H 7.6 in 0.1 M phosphate buffer increased with the extent of treatment so that the most hydrolysed samples were almost completely soluble. No (significant) difference was found between freeze dried and spray dried samples. Samples prepared without dialysis showed no solubility difference from those prepared with dialysis at a similar extent of hydrolysis. The hydrophobicity of the preparations was measured using two different fluorescent probes and was found to increase with the extent of hydrolysis. The emulsion-forming properties of the preparations were found to depend on the oil used in the test, as would be expected if hydrophobicity was equivalent to the hydrophile lipophile balance, which is commonly used to classify emulsifying agents. The preparations did not, however, show the additivity properties of emulsifiers. It was also shown that only the soluble portion of the preparations was responsible for emulsion formation. The possibility of achieving deamidation of gluten using the enzymes peptidoglutaminase I and II was examined. No activity against gluten or partially hydrolysed gluten was found.