Exploring the nutritional quality and bioactivity of different rice crackers and the influence of oyster/mussel protein fortification on potential health benefits : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

In New Zealand, a quarter of the population faces multiple chronic health conditions (Ministry of Health, 2023). Nutrient-dense foods containing essential constituents such as protein, fibre, vitamins, and minerals are pivotal for enhancing immunity, maintaining health, and preventing chronic diseases. Rice crackers, a popular modern convenience snack, are favoured for their ease of preparation, extended shelf-life, and appealing texture (Patil, Brennan, Mason, & Brennan, 2016). However, these crackers primarily utilise refined white rice flour obtained by removing the bran layer from brown rice, leading to low levels of protein, dietary fibre, and bioactive compounds, alongside high levels of rapidly digestible carbohydrates. This nutritional profile raises concerns about their potential high glycaemic index and potential implications for chronic health problems (Lafiandra, Riccardi, & Shewry, 2014). As a result, ongoing research is directed toward innovative reformulations of rice-based products to address these nutrient deficiencies. To comprehensively evaluate the quality of white rice crackers, this study examined and compared rice crackers produced from different rice varieties: white rice, brown rice, and black rice. Brown rice and black rice, being whole grain foods, retain their bran layer and offer higher protein, fibre content, and bioactive compounds such as polyphenols and anthocyanins, associated with reduced risks of hypercholesterolemia, cardiovascular diseases, and type 2 diabetes (Ali, Wani, Wani, & Masoodi, 2016; D. Zhu et al., 2023). The investigation encompassed assessments of physical characteristics, nutritional composition, in vitro starch digestion, and glycaemic response. Results showed that brown and black rice crackers exhibited significantly higher essential nutrient levels, including protein, ash, fat, dietary fibre, and resistant starch, compared to white rice crackers (p < 0.05). In contrast, white rice crackers exhibited a higher glycaemic response, characterised by a higher reduced sugar release and a greater content of rapidly digestible starch, along with a lower content of resistant starch during the in vitro starch digestion process. Regarding quality evaluation, white rice crackers exhibited the highest values for brightness (L* = 73.00) and yellowness (b* = 30.89), while black rice crackers demonstrated the lowest brightness (L* = 25.12) and the highest redness (a* = 6.31). Furthermore, black rice crackers displayed the greatest hardness (1022.46 g), whereas brown rice crackers showcased the highest fracturability (18.27 mm). To future examine phenolic compounds in rice crackers and establish links between in vitro digestion, antioxidative properties, and enzyme inhibition, this study extended its investigation to evaluate total phenolic content (TPC), total anthocyanin content (TAC), and antioxidant activities of white, brown, and black rice crackers. Additionally, the study evaluated changes in these parameters following simulated in vitro digestion. The identification of specific phenolic compounds within the rice crackers was carried out using high-performance liquid chromatography (HPLC). Moreover, the study explored the inhibitory effects of these phenolic compounds on the enzymatic activities of α-amylase and α-glucosidase. The findings revealed that black and brown rice crackers consistently displayed higher TPC, TAC, DPPH, and FRAP values compared to white rice crackers. In vitro digestion led to substantial TPC release, approximately 3.81-4.95 times higher, significantly increased DPPH values by 14.96-24.14 times, and elevated FRAP values by 4.25-4.90 times (p < 0.05). However, there was a reduction of TAC values following digestion. HPLC analysis indicated the presence of various phenolic compounds, including gallic acid, 2,5-dihydroxybenzoic acid, catechin, vanillic acid, rutin, p-coumaric acid, p-hydroxybenzoic acid, ferulic acid, and quercetin, in brown and black rice crackers, while white rice crackers primarily contained gallic acid and 2,5-dihydroxybenzoic acid. Furthermore, brown and black rice crackers exhibited stronger inhibitory effects on α-amylase and α-glucosidase compared to white rice crackers, with lower IC50 values. These findings highlight substantial variations among the different rice varieties, indicating the potential of brown and black rice for the production of rice crackers with lower glycaemic responses, higher nutritional value and the potential health benefits associated with enhanced phenolic composition and strong inhibitory effect on carbohydrate-digesting enzymes. Protein is essential for tissue growth, repair, and maintenance, as well as the production of enzymes, hormones, and antibodies, contributing to metabolic processes, bodily functions, and immune system function (Wu, Gao, Yu, & Wang, 2022). In response to the prevalent protein insufficiency in white rice crackers, a novel research avenue involves the formulation and advancement of protein-fortified rice crackers. Oysters (Crassostrea gigas) and mussels (Perna canaliculus) are valuable seafood resources consumed widely in New Zealand and contain higher levels of easily digestible protein and iron compared to traditional fish and red meats (Yaghubi et al., 2021). Oyster and mussel proteins are explored for functional foods and supplements, providing essential amino acids and potential health gains. Mussel proteins aid tissue engineering and drug delivery via biocompatibility and adhesion. Oyster proteins offer nutraceutical potential, aiding heart health, immunity, and antioxidants (Harnedy & FitzGerald, 2012; Ulagesan, Krishnan, Nam, & Choi, 2022). Fortifying white rice crackers with oyster and mussel proteins presents an opportunity to boost their nutritional content, lower glycaemic impact, and introduce potential bioactivity, including noteworthy antioxidant capacity and functional effects on inflammatory responses. This study evaluated the overall enhancement in the quality of crackers fortified with oyster and mussel powder at concentrations of 5%, 10%, and 20%, and assessed their impact on glycaemic response and protein bioaccessibility using in vitro starch and protein digestion assays. Additionally, antioxidative capacity was examined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and the Ferric Reducing Antioxidant Power (FRAP) assay. Results indicated that oyster and mussel powders inclusion led to a substantial reduction in starch content, resulting in a decrease of approximately 30% in glucose release and area under the curve (AUC) values during starch digestion compared to the control sample (white rice crackers). Furthermore, the addition of oyster and mussel powders significantly (p < 0.05) increased TPC, with values ranging from 16.74 mg GAE/100g sample to 104.90 mg GAE/100g sample in 20% oyster enriched sample and 77.65 mg GAE/100g sample in 20% mussel enriched sample. Moreover, the release of phenolic compounds during gastric and intestinal digestion was enhanced by nearly 150% and 50%, respectively, compared to the control sample. Antioxidant activity (DPPH and FRAP value) of enriched crackers also exhibited a significant increase corresponding to higher oyster and mussel powder incorporation. However, despite the increase in protein levels from 6.36% to 12.60%, protein digestibility declined from 77.64% to 63.05%. This reduction could be attributed to interactions between phenols and proteins in the samples, potentially affecting overall protein digestion. Furthermore, to evaluate protein availability and bioaccessibility, the research examined alterations in soluble protein content and amino acid composition after in vitro digestion and assessed the protein quality using the Digestible Indispensable Amino Acid Score (DIAAS). Additionally, the study evaluated bioactive peptides by measuring ACE inhibition and anti-inflammatory effects using LPS-stimulated RAW264.7 macrophages. Outcomes revealed heightened protein content and soluble protein content following digestion in oyster and mussel-enriched samples (oyster20, mussel20) compared to the control. Amino acid analysis showcased the increased release of essential and non-essential amino acids during digestion in enriched samples. DIAAS analysis underscored enhanced amino acid bioavailability in enriched rice crackers. Notably, molecular weight distribution exhibited the formation of smaller peptides during digestion. ACE inhibitory activity displayed significantly elevated values in enriched samples, suggesting potential cardiovascular benefits. Anti-inflammatory assessments demonstrated decreased NO production and pro-inflammatory cytokine levels in enriched samples compared to LPS-induced RAW 264.7 macrophages. Overall, this study underscores the significant variations among rice varieties and emphasizes the potential of brown and black rice for producing crackers with enhanced nutritional value, lower glycaemic responses, and health-promoting phenolic compounds. The exploration of marine-based protein fortification further offers a promising avenue for improving rice cracker quality and bioactivity. Through comprehensive analyses, this research contributes to a better understanding of the potential benefits and quality improvements that can be achieved through innovative approaches in rice cracker production.