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

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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.
Listed in 2022 Dean's List of Exceptional Theses
Cysteine proteinases, Gluten, Proteolysis, Kiwifruit, Therapeutic use, Digestive organs, Diseases, Dean's List of Exceptional Theses