The Hypoglycaemic mechanisms of mushrooms and their polysaccharides on gluten-free food models

Gluten-free biscuits are an essential part of the diet for people living with coeliac disease. The high glycaemic index (GI) is one of the main issues in gluten-free products, which poses a higher risk of developing obesity and type 2 Diabetes Mellitus (T2DM). Sorghum is an important gluten-free cereal and its starch is suitable for biscuit-making. Mushrooms contain high-quality fibres and polysaccharides, which can be utilised to achieve anti-hyperglycaemic function. However, to date, most research is focused on them as pharmaceutical ingredients. The utilisation of mushrooms and their polysaccharides in whole food models has many benefits, such as ease of incorporation and commercialisation. Widely cultivated mushroom cultivars such as shiitake (Lentinula edodes), black ear (Auricularia auricula) and silver ear (Tremella fuciformis) have high-quality polysaccharides and can be readily used in gluten-free food formulations (such as sorghum-based biscuits). Most importantly, they can be used to reduce GI. Many studies have emphasised the importance of molecular interactions between mushroom polysaccharides and starch on starch gelatinisation and digestion. However, the mechanisms of their interactions and hypoglycaemic potential have, so far, not well understood. Moreover, the effect of mushroom polysaccharides on starch digestion has not been correlated to the subsequent glucose transport from brush border. Therefore, the main objectives of this study were to (i) investigate the effects of the whole powder of representative mushrooms on starch digestion in gluten-free biscuits, (ii) elucidate the mechanisms of interaction between sorghum starch and mushroom polysaccharides and (iii) determine the effect of mushroom polysaccharides on the transport of glucose from starch digesta. A combined in vitro digestion-Caco-2 cell transwell model was used to quantify this. In the first stage, mushroom powders of three cultivars (shiitake, black ear, and silver ear) were used in gluten-free sorghum biscuits. The biscuits were enriched with mushrooms at 5%, 10%, and 15% substitution levels. Results showed that the incorporation of mushroom powder improved the overall quality of the biscuits, including an increased in vitro bio-accessible phenolic compounds and small peptides and increased antioxidant activity of the digesta. The undigested residue of mushroom enriched biscuits also had a higher percentage of phenolic compounds and β-glucan than the control. The hydration and pasting properties of sorghum flour substituted with 5%, 10%, and 15% mushroom powder had higher water absorption capacity (WAC) and swelling power (SP). The incorporation of shiitake powder decreased the pasting viscosities, while black ear and silver ear increased the viscosity values. The changes in hydration and pasting properties were correlated to the increased dietary fibre content. Mushroom powder enriched biscuits had a lower predicted glycaemic response. They had a denser and stronger structure network and also a slower digestion rate. The hydration of sorghum flour and the in vitro glycaemic response were closely related. Following the above study, the mechanisms of interaction between sorghum starch and mushroom polysaccharides obtained from shiitake (LEP), black ear (AAP), and silver ear (TFP) and the effect of the interaction on starch gelatinisation and digestion were determined and compared. Sorghum starch was gelatinised alone and combined with the LEP, AAP or TFP. The gelatinisation rate, retrogradation energy, and digestibility of sorghum starch were found to decrease in the presence of mushroom polysaccharides in a concentration-dependent manner. The structure of sorghum starch was also altered. The ratio of ordered starch to amorphous starch was increased. TFP (glucuronoxylomannan) had the most substantial inhibitive effect on starch digestion and gelatinisation level due to an appreciable hydrophobic interaction between starch and glucuronoxylomannan. AAP (β-glucan and glucuronoxylomannan) caused a moderate decrease in starch digestion, which was must probably due to weaker hydrogen bonding and hydrophobic interaction. LEP (β-glucan) encapsulated starch granules by electrostatic interaction and decreased their gelatinisation. Moreover, LEP showed a lower efficacy (8.3%) on starch digestion than AAP (19.6%) and TFP (28.7%). However, starch digestion alone cannot determine the final amount of glucose transported across the mucosal brush border. The starch gels containing 0.6% (w/w) acidic polysaccharides were used as the model to determine glucose transport. The effects of AAP and TFP on the maltooligosaccharides profile, glucose dialysis, and glucose transport of digested starch were studied together with the ability to affect the α-glucosidase activity. AAP and TFP were found to decrease the amount of maltose and delay glucose dialysis, and decreased glucose transport by 34.2% and 38.7%, respectively. AAP and TFP inhibited α- glucosidase activity through molecular hydrogen bonding. The former had a greater ability than the latter to inhibit α- glucosidase. Molecular docking results indicated that the β-glucan and O-acetylated glucuronoxylomannan had better binding affinity than glucuronoxylomannan. The presence of AAP and TFP altered the maltooligosaccharides profile of sorghum starch, entrapped glucose, and inhibited α- glucosidase activity, all of which contributed to the inhibition of glucose transport across the brush border. The inhibition of α-glucosidase by LEP (β-glucan) was studied by enzyme kinetic assay, fluorescence quenching and molecular docking. The effect of LEP on the transport of glucose from the digested starch and maltose was investigated via a combined in vitro digestion-Caco- 2 transwell model. LEP was found to inhibit α-glucosidase in a non-competitive manner and exhibited a better inhibition capacity (IC50 = 0.66 mg/mL) than acarbose (IC50 = 1.66 mg/mL). The structure of LEP facilitated binding with α-glucosidase via electrostatic force and hydrogen bonding, where (1→6)-linkages had a higher binding affinity than (1→3)-linkages. LEP was found to be an effective inhibitor of α-glucosidase. It was also able to decrease the transport of glucose and maltose by 33.7%, which augmented the hypoglycaemic effect. The outcomes of this thesis showed that the partial substitution of sorghum flour with mushroom powder lowed the GI of gluten-free sorghum biscuits through molecular level impact on gelatinisation and digestion. The interaction mechanism between mushroom polysaccharides and sorghum starch was found to depend on the nature (structure-function) of polysaccharides which impacted the gelatinisation and digestion and thus gave rise to different hypoglycaemic mechanisms. These findings contribute to the body of knowledge relevant to the structure-function-application of mushrooms and their polysaccharides obtained from different cultivars.