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Item Understanding the effect of processing and species on milk proteins during digestion : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2025-02-28) Maidment, Catherine AnnMilk is an important source of protein in a balanced human diet. Milk proteins not only have high nutritive value but also have biological properties. Milk composition and structure vary based on factors such as species, processing methods, and lactation stage. These differences are believed to affect digestion by influencing the breakdown of milk proteins, fats, and carbohydrates, as well as the rate and efficiency at which nutrients are absorbed in the gastrointestinal tract. The overall objective of this PhD thesis was to investigate how milk proteins from different species (cow, sheep, goat, and deer) are affected by digestibility under varying processing treatments (heating and homogenisation). Digestibility was assessed by the amount and types of bioaccessible peptides generated during gastrointestinal digestion. A dynamic in vitro digestion model (human gastric simulator (HGS)) was used for this study. Size exclusion chromatography was employed to measure the amount of peptides generated throughout digestion, with significant differences determined by a p-value threshold of 0.05. Mass spectrometry was used to analyse the types of peptides, requiring peptides to be present in at least two-thirds of the samples for inclusion. To assess the validity of the results obtained using the HGS model, comparisons were made with the peptide profiles generated using an in vivo (pig) digestion model. In addition, further work was undertaken looking into the protein composition of deer milk throughout the different lactation stages. This study investigating digestibility found differences in the amount and types of bioaccessible peptides generated throughout gastric digestion in milk from different species. Overall, deer milk produced the most peptides, while goat and sheep milk produced the least. Ruminant species also affected which regions of the parent protein were resistant to digestion as well as their bioactive properties. In contrast, processing treatment did not have as significant an effect on the amount and types of bioaccessible peptides but did affect the digestion kinetics. Differences were only observed during early digestion and appeared to be species dependent. Similarities were found in the peptides released throughout gastric digestion between the HGS model and the in vivo pig model, which suggests that the HGS model is suitable for the study of gastric digestion of protein-rich food. However, the peptide profiles differed during the intestinal stage indicating that the intestinal step attached to the in vitro model needs improving to fully mimic the dynamic nature of in vivo digestion. The study investigating deer milk proteins found that proteins related to transport e.g. apolipoprotein E and vitamin D-binding protein and immunity e.g. osteopontin, immunoglobulin J and lactotransferrin were found to change throughout lactation. This is thought to reflect the changing needs of the newborn as well as the development and protection of the mammary gland over lactation. Proteins were investigated using mass spectrometry, and significant differences throughout lactation were determined using simple linear regression calculations and log fold change calculations, comparing protein levels between week 3 and week 16 of lactation. The results from this thesis will contribute to the knowledge of how milk composition and structure impact protein digestibility throughout gastrointestinal digestion. The information gained from this study may have important consequences for developing dairy products that deliver superior digestive and nutritional outcomes to targeted consumer groups.Item Investigating the health benefits of Monty’s Surprise apple phytochemicals : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand. EMBARGOED until 18 November 2027.(Massey University, 2024) Nezbedová, LindaCancer is a major health concern worldwide, requiring effective prevention strategies to reduce its burden. There is evidence suggesting that diet plays an important role in disease prevention. Apples are an example of a commonly available fruit that can be easily incorporated into a diet to reduce the risk of chronic disease and some cancers. Apples are rich in phytochemicals with chemo-preventative properties. However, assessing their benefits in humans is complex due to various factors affecting phytochemical composition and low bioavailability. Understanding and controlling for these factors is crucial for maximising their potential in preventing cancer and promoting human health. The PhD project presented in this thesis uses a multidisciplinary ‘from orchard to fork to function’ approach to evaluate the health benefits of phytochemicals in a New Zealand heritage apple cultivar known as Monty’s Surprise, with an emphasis on cancer prevention. This thesis particularly focuses on apple’s most abundant phytochemical group known as phenolic compounds. After controlling fruit harvest and maturity, Monty’s Surprise apple was found to contain high concentrations of health beneficial phenolics, especially procyanidins, with apple skin having higher total phenolic content compared to its flesh. Pureeing was identified as a suitable processing technique with minimal impact on apple’s phenolics, allowing long-term preservation and creating uniform material for use in intervention and feeding trials. Moreover, Monty’s Surprise apple extract depleted of sugars inhibited the proliferation of lung, breast, and colorectal cancer cells in a dose-dependent manner in vitro. Lastly, in a human acute feeding trial, Monty’s Surprise apple puree increased plasma antioxidant capacity and decreased postprandial glucose response compared to sugar-matched placebo. The feeding trial also showed that procyanidins were not detected in the participant’s blood, while epicatechin and chlorogenic acid, which are prevalent phenolics in Monty’s Surprise apple puree, were absorbed into the bloodstream. Therefore, these compounds were available to contribute to the improved health parameters measured in the study. This study offers practical recommendations for optimising harvesting, storage, and processing practices to preserve and more clearly elucidate the health-promoting properties of phenolics in apples. Overall, this research demonstrated the potential health benefits of Monty’s Surprise apple and its phenolics, suggesting its value as a functional food for improving human health and well-being.Item Effect of beetroot pomace consumption on acute blood pressure and postprandial blood glucose responses on healthy adults : a thesis presented in the partial fulfilment of the requirements for the degree of Master of Science in Nutrition and Dietetics, Massey University, Albany, New Zealand(Massey University, 2023) Gorbani, ElnazBackground: Nitrate-rich beetroot juice (BRJ) supplementation has been shown to improve blood pressure (BP) levels in younger and older adults via increased nitric oxide (NO) production leading to dilation of blood vessels. Similarly, to some extent a reduction of blood glucose (BG) levels through several different mechanisms. However, limited research exists on the by-product of beetroot juice production, beetroot pomace (BRPOM), and its role in controlling BP and BG levels. Objective: To investigate and compare the changes in BP and BG levels in a 3 h period following the ingestion of BRPOM, BRJ and placebo as part of an oral glucose tolerance test (OGTT). Methods: Following an overnight fast, twelve healthy adults consumed either BRPOM (600 mg nitrate, 10 g fibre, 75 g carbohydrate), BRJ (600 mg nitrate, <1 g fibre, 75 g carbohydrate), or placebo (0 mg nitrate, <1 g fibre, 75 g carbohydrate) 15 minutes prior to the OGTT, separated by 1-week washout periods, in a single-blind, crossover design, pilot study. Blood pressure and BG levels were measured at baseline, then every 15 minutes for BG and every hour for BP during a 3 h OGTT. Results: No significant changes in SBP for BRJ (-2.3 ± 5.56 mmHg, p = 0.174), BRPOM (-3 ± 13.4 mmHg, p = 0.456), or PLA (5.2 ± 11.3 mmHg, p = 0.142) treatment group after 3 h. However, there was a trend towards significance in the differences between the three groups (p = 0.075, η²ₚ = 0.404). Clinically meaningful reductions in SBP were observed for BRJ (-2.3 ± 5.56 mmHg) and BRPOM (-3 ± 13.4 mmHg). There were no differences (p = 0.739, η²ₚ = 0.059) in mean glucose incremental area under the curve (iAUC) between BRJ, BRPOM and PLA (232.8 ± 61.9, 242 ± 54.7 and 252.4 ± 60.6 mmol/L/min, respectively). There were no differences between BRJ, BRPOM and PLA in time to peak (p = 0.269, η²ₚ = 0.313), peak glucose levels (p = 0.241, η²ₚ = 0.247), 2 h glucose levels (p = 0.565, η²ₚ = 0.150), 3 h glucose levels (p = 0.395, η²ₚ = 0.233) and percentage increment of postprandial BG (p = 0.783, η²ₚ = 0.048). Conclusion: This pilot study with a small sample size showed a large effect size and clinically meaningful attenuations for acute SBP changes following the consumption of beetroot pomace. Further investigations would be needed to explore the applicability of these findings to hypertensive individuals. However, its impact on acute BG levels remains non-significant, possibly influenced by the small sample size and participants’ baseline normoglycaemic levels. Further research is needed to explore potential effects in a hyperglycaemic population.Item An audit of ultra-processed vegan food in the major supermarkets of New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutrition and Dietetics at Massey University, Albany, New Zealand(Massey University, 2023) Czifra, AimeeBackground: The rising rates of veganism have led to an increased demand for plant-based meat and dairy analogues (PBMAs and PBDAs). These analogues fall into the category of ultra-processed foods (UPFs). There is an urgent need to obtain an understanding of vegan UPFs available to New Zealand consumers. Aim: To audit ultra-processed vegan-labelled, PBMAs and PBDAs available in New Zealand supermarkets, including comparing their nutrient composition (energy, protein, saturated fat, carbohydrate, fibre, sodium, calcium, iron, zinc and B12) against the products of animal origin that these foods emulate. Methods: The audit was completed during March – June 2022 using a combination of data collected online and directly from supermarkets. Products were chosen that directly emulated foods of animal origin, naturally vegan foods such as Tofu and legumes were excluded. Data were collected from New Zealand’s five major supermarkets, Countdown, Fresh Choice, New World, Pak ‘n Save and Four Square. The data collected included the nutrient information listed on product labels, this information was available on some online shopping sites or taken directly from product labels by visiting the various supermarket stores. The nutrient information of the meat sources was taken from an average derived from no less than eight samples, calculated by the Food Standards Australia and New Zealand (FSANZ) and FoodWorks. The nutrient information for meat burgers was collected from product labels available online or instore. Results: The PBMAs generally had higher energy and lower protein than their meat counterparts., for the vegan burger had a mean of 15.2mg/100g compared to the meat burgers at 18.5mg/100g, the mince showed similar comparisons. The veggie burger had considerably lower protein with a mean of 6.6mg/100g. The vegan chicken breast had higher mean protein (27mg/100g) compared to the meat chicken breast (21.35mg/100g). The PBDA was generally lower in protein. Soy milk was the only plant milk with a similar protein to cow’s milk, plant yoghurt had a range of 1.4 – 2mg/100g compared to dairy yoghurt (5.0-7.92mg/100g). Only one of the plant cheeses had a protein content above 1mg/100g (Vegan Parmesan 17.7mg/100g) when compared to dairy cheese (8.5-35.1 mg/100g). The PBMAs were high in sodium, with a mean across the various products of 351mg/100g to 693 mg/100g, compared to meat which had a mean of 47mg/100g to 796.7mg/100g (excluding bacon). The PBDAs showed a similar trend with sodium across the various analogues with a mean of 22.75mg/100mL to 68mg/100mL. The sodium across the various plant-based cheese analogues (PBCAs) had a mean of 603mg/100g to 4970mg/100g. The calcium of PBDA milk was separated into fortified and unfortified. Fortified PBDAs had a mean of 100.5mg/100mL to 147mL/100mL, 29.4% had no calcium fortification. Of the PBDAs soy milk had the closest protein quality to dairy milk, though the levels of protein were varied. Fortification of calcium was mostly absent from both plant-based cheese and yoghurt analogues (PBYAs). Vitamin B12 had a mean of 0mcg/100g to 0.6mcg/100g across all the PBMAs. Conclusion: The plant-based analogues offered a range of ultra-processed foods, very few of which could be considered in a healthy range of nutrients. The levels of sodium were high across the range of PBMA and PBDAs. The levels of fortification would need to be standardised to offer a product of similar nutritional value to animal-based foods.Item Nitrate-rich beetroot juice and its effects on cardiovascular health and cognition in younger and older adults : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Sport and Exercise Science at Massey University, Auckland, New Zealand(Massey University, 2022) Stanaway, LukeBackground: Evidence suggests supplementation with nitrate-rich beetroot juice (BR) may improve cardiovascular responses and cognition in male and female, younger and older adults. However, there is still limited research in this area, particularly in older adults and with regards to measures of endothelial and cognitive function where equivocal findings have been observed. In addition, it is unclear whether age-related differences impact on the benefits from dietary nitrate supplementation. Older adults tend to have an increase in blood pressure (BP) and decline in cognition with aging, and thus may respond more favourably to BR supplementation compared to younger adults. Additionally, the effects of dose and duration (acute versus chronic) of supplementation with dietary nitrate are still not well known. Overall aims: To 1) investigate the effects of varying doses of nitrate-rich BR on cardiovascular responses and cognition in older adults, and 2) examine the effects of acute and chronic supplementation with BR on cardiovascular responses and cognition in the two age groups. Methods: The “Dose-response study” (Chapter 4) examined the effects of acute supplementation with varying doses of BR (0.15 (placebo; PL), 2.5, 4.9, and 9.8 mmol nitrate) on cardiovascular and cognitive responses in older adults (50-80 years; n=11) in a randomised, double-blind, crossover designed trial. At each visit, resting blood samples for plasma nitrate and nitrite, BP, mean arterial pressure (MAP), systemic vascular resistance (SVR), heart rate (HR), resting metabolic rate, and cognitive function were completed pre- and 2.25 h post-supplementation. The “Acute study” (Chapter 5) was a randomised, double-blind, crossover study. Twenty-four participants, 13 younger (18-30 years) and 11 older (50-70 years), completed resting blood samples, BP, HR, cognitive function, and mood and perceptual measurements pre- and post-supplementation with nitrate-rich BR (10.5 mmol) or placebo (1 mmol). The “Chronic study” (Chapter 6) was a double-blind, randomised control trial investigating acute, acute + chronic, chronic + acute (exercise performance) and chronic supplementation with either nitrate-rich BR (10.5 mmol) or PL (0.15 mmol) over 28 days in 21 younger (18-30 years) and 22 older (50-80 years) adults. On days 0, 14, and 28 resting blood samples, BP, MAP, SVR, HR, and cognitive performance measures were completed pre- and 2.25 h post-supplementation, while oxygen uptake (V̇O₂) and time trial performance were measured 2.5 h post-supplementation. Results: Ingestion of varying doses of dietary nitrate in the Dose-response study increased plasma nitrate and nitrite concentrations in a dose-dependent manner, with a significant difference shown between each treatment for plasma nitrate (p < 0.001). However, plasma nitrite was only significantly increased following consumption of the 9.8 mmol treatment compared to PL (p = 0.004). Systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP were reduced 2.25 h post-supplementation with the 9.8 mmol treatment compared to the PL, 2.5, and 4.9 mmol treatments (p < 0.05). Following supplementation with the 9.8 mmol dose, Corsi span (cognitive function) was improved during the Corsi block tapering test (CBT) compared to PL (p = 0.013) and 2.5 mmol (p = 0.004) treatments. In the Acute study, supplementation with nitrate-rich BR also significantly increased plasma nitrate (p < 0.001) and nitrite (p = 0.003) concentrations in younger and older adults relative to PL. Systolic BP was reduced in both younger and older adults (p < 0.001) 2.25 h post-supplementation with BR compared to PL, while DBP was only reduced in older adults (p = 0.013). Older adults had a greater increase in plasma nitrite (p = 0.038) and reduction in DBP (p = 0.005) compared to younger adults. Cognitive performance measures were also improved in younger and older adults following acute supplementation with nitrate-rich BR, with a reduction in reaction time observed during the Stroop test (p = 0.045). The Chronic study showed a significant reduction in SBP, DBP, and MAP following acute supplementation with nitrate-rich BR on day 0 in older adults (p < 0.001) and following an acute dose in the context of chronic supplementation (acute + chronic) on day 28 in both older (p < 0.01) and younger (p < 0.05) adults. Fourteen days’ chronic supplementation with nitrate-rich BR, in the absence of an acute dose, also reduced SBP (p = 0.019), DBP (p = 0.004), and MAP (p = 0.005) in older but not younger adults. Older compared to younger adults also had a greater reduction in SBP, DBP, and MAP following acute supplementation with nitrate-rich BR on day 0 (p < 0.05) and following chronic supplementation on day 14 (p < 0.05). Acute + chronic supplementation with nitrate-rich BR reduced SVR in older adults on days 14 (p = 0.032) and 28 (p = 0.016), with a greater reduction in older compared to younger adults observed on day 14 (p = 0.015) and a trend for greater reduction in older adults observed on day 28 (p = 0.056). Acute (on day 0) and acute + chronic (on day 28) consumption of nitrate-rich BR improved reaction time during the Stroop test in older adults (p = 0.042 and 0.006, respectively), while older (relative to younger) adults also showed a greater improvement in reaction time during the Stroop test following chronic supplementation on days 14 (p = 0.016) and 28 (p = 0.02) despite no effect of treatment on these days. Older adults also showed a greater reduction in V̇O2 at 20% completion of the cycle time trial following acute supplementation with BR on day 0 (p = 0.044), despite no effect of treatment at this time point. Conclusion: The results from this PhD project showed that acute and daily supplementation with nitrate-rich BR can improve BP and cognitive function in younger and older adults, with greater benefits observed in the older cohort. Furthermore, chronic supplementation with nitrate-rich BR in older adults can improve BP independent of an acute dose. These results suggest that daily supplementation with nitrate-rich BR may have a role in clinical settings for helping maintain healthy cardiovascular and cognitive function. Future research should investigate the use of nitrate-rich BR as a potential preventative therapy for cardiovascular and cognitive diseases such as hypertension and dementia.Item The effect of a 14-day sugar reduction intervention on individuals with a ‘sweet tooth’ on intake, desire, and preference for sweet foods : a thesis presented to Massey University in partial fulfilment of requirements for the degree of Masters of Science (MSc) in Nutrition and Dietetics(Massey University, 2023) Hsiao, Wei-HsiBackground: People who identify as having a sweet tooth, may find it difficult to control their sugar intake. Gymnema sylvestre is a plant that contains a chemical compound called gymnemic acid which can reversibly suppress sweet taste responses. Objectives: The study aimed to investigate whether supplementing Gymnema sylvestre (GS) can reduce sugar cravings, sweet food desire and consumption among adults that identify as high sweet food consumers (sweet tooths). Method: On day zero, 32 healthy participants who self-classified as having a sweet tooth underwent baseline sensory testing for sweet taste perception using the placebo mint (PLAC). Participants then went through the randomisation process into the two groups. On day 15, participants underwent further sensory testing (with GS mint) before embarking on 14 days' supplementation using the GS mints following either a systematic (3 times/day at specified times; SYS) or ad libitum (up to 6 mints/day at times of their choosing; AD-LIB) regimen. On day 30, participants swapped over to the other trial (using the other regimen). On day 45, participants completed final questionnaires and anthropometric measurements. At each visit, participants were required to complete questionnaires (food frequency questionnaire, beverage questionnaire and cravings questionnaire), sensory testing and measurement of anthropometry. At all visits participants completed questionnaires, anthropometric measures, and sensory testing. Sensory testing was not required for day 45. Results: AD-LIB reduced daily sugar-sweetened beverages (SSB) intake by 42% relative to PLAC (p=0.015). AD-LIB reduced overall sugar cravings by 28% relative to PLAC (p=0.045) AD-LIB and SYS reduced pleasantness ratings (p=0.005). Conclusion: Gymnema sylvestre consumption using an ad libitum regimen reduced sugar cravings and changed sweet food desire and consumption.Item Evaluation of the effects of acute caffeine supplementation on selected cognitive domains in older women : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Nutrition and Dietetics at Massey University, Albany, New Zealand(Massey University, 2022) Khindria, NeelamCaffeine is the most frequently consumed psychoactive substance globally and has been shown to enhance various aspects of cognition, particularly in younger adults. Previous research has demonstrated that acute intake of low to moderate doses of caffeine (e.g., 100 mg to 300 mg) significantly improves lower-order cognitive functions such as processing speed and attention in younger adults. The impact of caffeine on higher-order cognitive domains such as memory and executive function remains unclear. Since older women experience age- and hormonal-related changes that may negatively affect cognition, this age group may be more likely to experience greater functional improvements in cognitive performance following caffeine supplementation compared to younger adults. This pilot study aims to evaluate the effect of acute caffeine supplementation on selected cognitive domains including processing speed, sustained attention, memory, and executive function, in post-menopausal women, aged 55 to 79 years. Twelve female participants (mean age ± SD = 63.75 ± 6.81 years) took part in a randomised placebo-controlled, double-blind, crossover study. Participants were asked to abstain from caffeine for 24 hours before each of three repeated cognitive testing sessions (separated by two-week intervals): baseline, 45 minutes post-ingestion of 100 mg caffeine, and 45 minutes post-ingestion placebo. Repeated measures ANOVA (treatment × time) indicated that 100 mg of caffeine supplementation significantly improved movement time (p = 0.04) in a five-choice reaction time (RTI) task, compared to placebo. There was a significant improvement over time for Rapid Visual Information Processing (RVP) sensitivity score (p = 0.04) and correct responses (p = 0.02), and Spatial Working Memory strategy score (p = 0.03). However, post-hoc analysis indicated no significant differences between caffeine and placebo supplementation. The key finding of the current study is 100 mg of caffeine supplementation significantly enhanced processing speed in older post-menopausal women but did not improve other cognitive processes including attention, memory, and executive function. In line with other research, caffeine supplementation may only affect performance on simpler tests requiring lower-order cognitive functions. This pilot study contributes to the growing body of research on caffeine and cognition, through a unique examination of older healthy women across a range of cognitive functions. However, further studies are necessary on a larger population scale and perhaps, utilising different doses of caffeine, to corroborate these findings.Item Effect of New Zealand Greenshell™ mussel on osteoarthritis biomarkers and inflammation in healthy postmenopausal women : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, School of Health Sciences, Massey University, Palmerston North, New Zealand(Massey University, 2023) Abshirini, MaryamNew Zealand GreenshellTM Mussel (GSM) showed chondroprotective effects in a pre-clinical study using a rat model of metabolic osteoarthritis, warranting further assessment in a human study. This PhD project aimed to assess the effect of GSM supplementation on cartilage degradation biomarkers in humans, and to develop novel biomarkers through a metabolomic approach. A double-blind, placebo-controlled, longitudinal clinical trial was carried out in overweight postmenopausal women who were given 3 g/day whole meat GSM powder or placebo (sunflower seed protein) for 12 weeks. Plasma samples from the pre-clinical rat trial were assessed through an untargeted metabolomic approach, followed by metabolomic analysis of plasma samples from the clinical trial. In participants with active knee pain, the cartilage turnover biomarker C-terminal telopeptide of type II collagen was significantly lower in GSM participants compared to placebo at weeks 6 and 12. GSM significantly reduced joint pain and improved knee-related symptoms. GSM but not placebo altered the faecal microbiota population and reduced the rate at which body fat accumulation increased. However, no changes in inflammatory cytokines were found. The metabolomic analysis of rat plasma samples revealed that GSM supplementation regulated the alteration in plasma triglyceride and other lipids caused by a high-fat diet. In the plasma of human participants, GSM supplementation increased long-chain polyunsaturated fatty acids (PUFA), ceramide, and some other lipids. In both rats and humans, GSM suppressed the sphingomyelin synthesis pathway. Polar metabolites including threonine, histidine and pipecolic acid were significantly impacted in both rat and human and are potential metabolic biomarkers for the impact of GSM powder supplementation in metabolic osteoarthritis. In conclusion, consumption of GMS powder may provide cartilage protection and reduce joint pain, particularly in women with symptomatic knees. However, no significant impact was observed on circulating inflammatory cytokines, suggesting that GSM may exert anti-inflammatory effects at the microenvironmental rather than systemic level. The bioactive compounds present in GSM powder such as omega-3 PUFA and chondroprotective glycosaminoglycans may be responsible for the beneficial effect through inhibiting the breakdown of type II collagen in cartilage, regulating gut microbe abundance, improving body composition, and the metabolite profile which needs to be investigated in future research.Item The effects of UV-C on the total polyphenol contents and antioxidant activity of New Zealand grown green asparagus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Human Nutrition at Massey University, Manawatū, New Zealand(Massey University, 2022) Zhu, LiyuBackground: Asparagus is favoured by global consumers for its exceptional nutritional value. New Zealand has a long history of asparagus cultivation and a considerable domestic market. Recently, several technologies such as UV and gamma irradiation have been used to increase the bioactive compounds in plants, resulting in improved plant quality and human health. The main aim of the present study was to study the hoemetic effects of UV-C (0.45- 2.27 kJ/m²) on the total polyphenol contents and antioxidant activity of New Zealand grown asparagus. Methodology: A three-factor-three-level Box Behnken Design (BBD) was used to determine the optimum condition for ultrasonication-assisted extraction (UAE). Three doses of UV-C radiation (0.45, 1.13, and 2.27 kJ/m²) were applied to study the effects of UV-C light on the antioxidant activity of asparagus. The antioxidant assays were evaluated by total polyphenol contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability, and peroxidase (POD) activity. The leading flavonoid of asparagus extracts was analyzed by High-Performance Chromatography (liquid chromatography and mass spectrometry; HPLC-MS). Results: The optimum conditions for extracting asparagus TPC were 60% ethanol (v/v), 20 min extraction time, and a solid to liquid ratio of 1:30 (w/v). Overall, the UV-C irradiated samples showed significantly higher TPC than unirradiated samples (p< 0.05). The highest TPC value (131.37 ± 2.05 mg GAE/g; dry weight) was obtained at 1.13 kJ/m² UV-C exposure, and the TPC started to decrease as UV-C irradiation surpassed 1.13 kJ/m². The DPPH activity showed a similar trend as the TPC assay. The lowest IC50 of DPPH assay was found in the 1.13 kJ/m² group (36.84 µM), followed by the 0.45 kJ/m² group (49.20 µM), 2.27 kJ/m² group (99.21 µM), and unirradiated group (115.01 µM). The effects of UV-C irradiation on POD activity exhibited an opposite trend as that of TPC. The HPLC-MS results showed that rutin was the main flavonoid in asparagus with a content of 14.095 mg/100 g (dry weight). Conclusion: Ultrasonication-assisted extraction (UAE) was more effective than traditional maceration on extracting asparagus TPC. The hormetic effects of UV-C were seen in this study as promoting the plant bioactivity at an optimum UV dose and decreasing such activity when the dose continued to increase. Rutin contains o‐diphenol, benzene ring, and keto functional group (-C=O) and might be the reason for the high antioxidant activity of asparagus.Item 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 AchinthaGluten 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.
