Extraction of β-glucan from Saccharomyces Cerevisiae and its Assessment in the Management of Type-II Diabetes and Oxidative Stress Through Albino Rats Model Studies

Abstract

The aim of the thesis was to extract β-glucan from yeast, its characterization as bioactive component for potential health benefits against diabetes & oxidative stress and to use it in baked products. The noteworthy outcomes of the current investigations are summarized hereafter. The first experiment involved extraction of β-glucan from yeast, its chemical and biological characterization. Yield of β-glucan was determined at different concentration of sodium hydroxide, at different pH, and at different temperature. The effect of 1.0 M NaOH, 90 0C temperature and a pH 7.0 were the best conditions at which the extractability of β-glucan was maximum (P≤0.0001), approximate yield was 66%. Regarding β-glucan chemical characterization i.e FTIR analysis confirms that β-glucan is similar as commercial β-glucan. The SEM analysis show spongy, porous, ordered and uniform structure of extracted β-glucan, and suggesting better and protective extraction process. The extracted β-glucan exhibited melting peak around 125 ºC versus 116 ºC in yeast cells and 122 ºC in case of commercial β-glucan, indicating better thermal stability of extracted β-glucan. In general, thermal analysis clearly indicated strong thermal behavior of the β-glucan. In comparasion with commercial β-glucan, the extracted β-glucan has significantly lower loose and packed bulk density; however, water absorption capacity, swelling behavior and oil absorption capacity of extracted β-glucan were comparable with commercial β-glucan. Extracted β-glucan formed complete gel at 4%, which was better than commercial β-glucan (8%), showing better functional properties of extracted β-glucan and its potential use where more absorption, swelling behavior and oil absorption is required. Total antioxidant capacity (TAC) of commercial (0.229 µg/mg) and extracted β-glucan (0.240 µg/mg) was similar, but remained superior (p≤0.05) when compared with yeast cell wall (0.113 µg/mg). During in vivo experimentation that involved inflammatory response showing that rats given 4% β-glucan added diet has significantly lower (4.465±1.450) myeloperoxidase activity (µmol/min/mg DNA), nitric oxide level (10.5±0.834 µmol/g) and MDA contents (5.84±0.349 mmol/L), indicating that the effect of 4% β-glucan addition is more protective against inflammation and free radical production. Further, total cholesterol (mg/dL) remained unchanged in response to 4% β-glucan added diet (701±19.849) or 2% β-glucan added diet (698±35.440) versus control positive rats (769±23.537), showing cholesterol lowering effect of β-glucan is not concentration dependant. Both 4% and 2% β-glucan diets have similar effect on white blood cells count and platelet. However, 4% β-glucan added diet has significantly reduced neutrophil (18.204±1.376%) versus rats given 2% β-glucan added diet (20.292±1.524) and control positive rats (21.694±2.416). In general, the efect of 4% β-glucan added diet was more effective and plausible. The effect of β-glucan on glucose control and oxidative stress management (measured in enzyme of oxidative stress, and MDA contents) was carried out in streptozotocin induced diabetic Wister rats. Catalase activity across rats groups was similar (P≥0.05). However, 4% and 2% β glucan diets significantly increased superoxide dismutase (SOD) activity, which is a strong antioxidant enzyme and indicating beneficial effect of β-glucan against oxidative stress. The MDA (malondialdehyde, lipid peroxidation product) was significantly low (65.963±51.210) in case of 4% β-glucan added diet comapared with 2% β-glucan added diet (73.018±51.318). furthermore, protein contents in liver homogenate was significantly reduced (5.753±0.377) in 4% β-glucan diet versus 2% β-glucan added diet (7.401±0.303), suggesting weight loss management at 4% β-glucan addition in diet. In conclusion, 4% β-glucan reduced oxidative stress reduced MDA contents and induced weight loss. Regarding glucose control in diabetic rats the effect of 4% β-glucan was more significant (P≤0.05) on glucose reduction (45427.129) versus 2% β-glucan diet (54003.181). Also, 4% β glucan diet reduced significantly lipid synthesis (fad pad), 1.255±0.512g fat pad versus other groups. Its effect on energy metabolism was measured; diabetic rats has significantly increased (AUC, 59512.5 mg*min/dL) glucose synthesis, reflecting high gluconeogenesis due to insulin deficiency in this group. However, this effect is significantly reversed in 4% β-glucan diet (38111.25 mg*min/dL), demonstrating positive effect of β-glucan on glucose metabolism. In conclusion, the effect of 4% β-glucan was more potent on glucose control, lipid synthesis, and energy metabolism. Wheat flour added with β-glucan (0, 1%, 2% and 4%) was characterized for their properties. Protein, ether extract and ash cotent was higher in 4% β-glucan added flour. Similarly, water holding percentage range was 139±3.68 for control flour to 174± 3.265 for 4% β-glucan added folur. The lowest swelling percentage can be seen in control flour (3.033± 0.416) versus 4% β-glucan added flour (4.166± 0.262). Both 2 and 4% β-glucan fortified flour contained more crude fiber (3.966± 0.418, 4.766± 0.205 repectively,) versus 1% β-glucan fortified flour (3.033± 0.169) and control flour (2.6± 0.489). Wheat flour (100%) released significantly higher (p≤0.05) sugar versus β-glucan added flour specially 4% (in vitro glucose release study). These results are highly encouraging as β-glucan added flour mix has retarded the digestion of sugar and therefore, their utilization in foods could help in glucose control or diabetes management. Bakery products such as cookies, cake and chapati was prepared from β-glucan fortified (0, 1%, 2% and 4%) wheat flour. Sensory scores for overall quality, taste, flvour and color depicted significant variations in cookies, while crispiness explicated non-significant differences with treatments. A cookie prepared with 2% β-glucan addition has maximum scores for overall quality. The TPC ranged from 4.29±0.767 (wheat flour) to 6.25 ±0.261mg GAE/g (4% β-glucan added cookies) wherease, the range of iron binding activity was 0.816±0.062 (wheat flour) to 1.6±0.081 mg EDTA/g, suggesting that β-glucan addition in cookies improve antioxidants level and that 2% β-glucan made cookies has low peroxide values and has low absorbance spectra with β-glucan addition, suggesting cookies added with 2 or 4% β-glucan reduces freshness losses versus others. In case of cake, 4% β-glucan fortifications reduced color score (8.313±2.224) while at 2% the color was not affected (10.313±2.210). Similarly, flovour and taste preferences were also high in 2% β-glucan added cake. Texture score was affected (10.053±1.686) beyond 1% β-glucan addition. Overall quality of 2% β-glucan added cake (10.806±1.863) was not different than 1% β glucan added cake or control cake. In general sensory quality of 2% β-glucan added cake was better. Regarding the antioxidant capability, 2% β-glucan added cake showed more TPC while ferric reducing antioxidant power and iron binding capacity was high in 4% β-glucan added cake. Regarding the peroxide value in cake, overall the effect of β-glucan to reduced peroxide values was not better at 30 days of storage. Regarding the sensory analyses of chapati it was found that color, flavor, taste, chewing aility score and overall quality was better in 2% β-glucan added chapati compared with 4% β glucan made chapati. Antioxidant (TPC, FRAP and iron inding activity) and lipid peroxidation inhibition (PV reduction) was also better in both 2% and 4% β-glucan prepared chapati versus other treatments. Last experiment was on chemical modification of β-glucan that involved the addition of phosphate. The chemical modification is confirmed through XRD, DSC, FTIR, SEM and brightfield microscopy. Phosphorylated β-glucan has more viscosity and has successfully formed gel, indicating cross-linked network formation relatively to control β-glucan which failed to make hydrogel. Phosphorylated β-glucan has improved antioxidants activity (p≤0.05) versus the un modified β-glucan.