Table of contents?Acknowledgement 2Abstract 3Introduction 4Literature review 62.1 How GI concept can help to tackle diabetes 62.2 Glycemic index (GI), Glycemic response and Glycemic load 62.3 Types of sugars 72.3.1 Rapidly digestable starch 72.4 Measurement of GI using In-Vivo methods2.5 Parameters in in-vivo methods2.5.1 Subjects2.5.2 Reference Foods2.5.3 Test Food 72.7 Comparison between In-Vivo vs In-Vitro methods 9Materials and methods 93.1 Preparation of solutions 93.2 Preparation of solid sample 103.3 Enzyme digestion of food sample 10???Introduction Diabetes is a fast-growing global problem with huge social, health and economic consequences (Ahmad, 2013). The prevalence of diabetes has been growing rapidly especially in low-income and middle-income countries. An estimated of 1.6 million deaths worldwide were caused directly by diabetes in 2015 and it is the eighth leading cause of death for both sexes in 2012 (World Health Organization, 2017). In singapore, diabetes is a serious health concern, with over 400,000 Singaporeans being diabetic (Moh.gov.sg, 2017). One in three singaporeans has a lifetime risk of getting diabetes and if measures are not taken, the number of diabetic patients is expected to reach one million by 2050.High glycemic index(GI) food produce a more rapid rise in glycemia and larger overall blood glucose response as compared to low GI food (Atkinson, Ek and Brand-Miller, 2016). Type 2 diabetes is thought to result from with the toxic effects of chronically elevated levels of blood glucose on the pancreatic cells that release insulin(). A high GI diet may result in impaired glucose tolerance(IGT) and in the long term, elevated glucose levels in IGT may Literature review2.1 How GI concept can help to tackle diabetesType 2 diabetes is characterised by high blood glucose and insulin resistance (Nadine R. et al., 2008) and studies have suggested that the cause of it is due long term consumption of high GI foods, leading to increase in insulin resistance and pancreatic exhaustion (Willett, Manson and Liu, 2002). 2.2 Glycemic index (GI), Glycemic response and Glycemic loadGlycemic index (GI) is defined as the incremental area under the blood glucose response curve of a 50g carbohydrate portion of a test food expressed as a percent of the response to the same amount of carbohydrate from a standard food taken by the same subject (BJÖRK et al., 1998). Any food and beverage containing carbohydrates can be classified into low GI, medium GI and high GI categories. Low GI, medium GI and high GI food are are defined as having a GI of less than 55, from 56 to 69 and more than 70 respectively (Atkinson, Ek and Brand-Miller, 2016).Low-GI foods are digested and absorbed slowly and sugars from the food inherently have a smaller impact on glycemia, resulting in a lower glycemic response. In contrast, high-GI foods lead to a rapid rise in glycemia and greater overall blood glucose response (Atkinson, Ek and Brand-Miller, 2016).Glycemic load of a food is defined as the product of the glycemic index of the food and the amount of carbohydrate in a serving (Sheard et al., 2004). The overall glycemic load of a diet can be obtained by summing the glycemic load of each individual food. Glycemic load is a more reliable method than glycemic index in predicting the glycemic response of a given diet as glycemic index does not take into account the effect of a typical amount of carbohydrate in a food portion on glycemia (Salmeron et al., 1997).Glycemic response is defined as the change in blood glucose concentration induced by ingested food (Jones, 2007). Postprandial glycemic response can be used to calculate the glycemic index and glycemic load to assess whether the intake of a carbohydrate product results in a slow or rapid increase in blood glucose concentration (Bellmann et al., 2017).Calculation of GI Using in vivo:Calculation of GI using in vitro: 2.3 Types of sugarsStarch is a polysaccharide that is made up of only glucose molecules and it can often be found in tubers and legumes. It is mainly made up of two polymers, amylose which is a straight chained polymer and amylopectin which is a highly branched polymer.2.3.1 Rapidly digestible starch 2.3.2 Slowly digestible starch2.4 Measurement of GI using In-Vivo methodsIn vivo methods using ISO Method is known as the gold standard that has been used by many research studies. A minimum of 10 healthy volunteers aged 18-65 years of age are recruited for testing. The volunteers are required to overnight fast 10-14 hours prior to the test (Brouns et al. 2005). Two blood samples are taken while volunteers are in fasting state and the average will be taken as the baseline blood glucose of the respective volunteers. The subjects will then consume the reference food or test food at an even pace within 12 to 15 minutes and are served with a drink of one or two cups (volume ranging from 250 ml to 500 ml) of plain water, coffee or tea with 30 ml of milk as well as a non-nutritive sweetener if any is desired (ISO, 2010). The type of drink and volume given to the subjects are kept constant throughout. Blood samples are then taken using capillary (finger prick) or venous whole blood or plasma. Blood samples are taken to be analysed for glucose levels at zero minutes (before consuming any reference or test foods), followed by additional blood samples at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min after eating has commenced (ISO 2010). This is repeated for a total of 3 times as using reference food is repeated twice before the analysis of test food. Subjects are to be rested for a day after testing before they come back for another round of testing. 2.5 Parameters in In-Vivo Method When using in-vivo methods to test the GI of food, there are many requirements that needs to be adhered to such as for subjects, reference food, test food. Many parameters are kept constant so as to prevent any possible outliers in the possible data that are collected.2.5.1 SubjectsAccording to ISO 26642:2010(E), a minimum of 10 healthy individuals must be selected ranging from the ages of 18 to 65 years. These healthy individuals must have no known food allergy or intolerance as well as no medications known that may affect glucose tolerance. Stable doses of medications through oral consumption however, are acceptable such as thyroxine, vitamins, mineral supplements or drugs to treat hypertension or osteoporosis (ISO 2010). Subjects that are excluded in the study are those that have a known history of diabetes mellitus or the use of any anti-diabetic medication or insulin to treat diabetes or any related conditions, a major medical or surgical event that requires hospitalization 3 months before, the presence of any diseases or consumption of drugs that can affect nutrient absorption or the use of steroids or protease inhibitors which have major effects on glucose metabolism as well as the total fat distribution in the body (ISO 2010) 2.5.2 Reference FoodsAs for reference food, it is crucial in repeating the test for reference food in individuals as part of determining the GI value as it acts as a control for determining the glucose tolerance from subject to subject in different days. The reference food by definition has a glycemic index 100 such as glucose or white bread.2.6 Measurement using In-Vitro methodMeasurement of glycemic index (GI) using in-vitro methods has been a vast and growing technology. There has been a wide range of methods for measuring the GI of carbohydrate containing foods since the 1990s (Woolnough et al., 2008).2.7 Comparison between In-Vivo vs In-Vitro methodsIn vivo measurement requires the recruitment of volunteers, availability of medical personnel for blood drawing which can takes a long time. Not only that, in vivo is also complicated and expensive as compared to in vitro which is a simple and inexpensive glycemic index (GI) measurement method (Argyri et al., 2016).Materials and methods3.1 Preparation of solutionsIn this experiment, several solutions are used and these includes 1.0.05N Hydrochloric acid (HCl) solution, Pepsin/Guar Gum Solution,Invertase Stock Solution, 0.5 M Sodium Acetate (NaAc) Solution, 66% Ethyl Alcohol Solution and Enzyme solution.For the HCl solution, 100 ml l 0.5N HCl was added to 1000 ml of deionized water. To prepare the Pepsin/Guar Gum Solution (2195 U/ml pepsin and 0.5% guar gum (W/V) in 0.05 N HCl), 0.5 g of pepsin and 0.5 g guar gum were dissolved in 100 ml of 0.05N HCl.For invertase stock solution (510.7 U/ml in H20) where 100 mg of invertase is dissolved in 42.10 ml of water. Next, 0.5 M sodium acetate solution was prepared, (0.5 M NaAc in water). 4.10 grams of sodium acetate was weighed and mixed in 100 ml of water. Followed by, 66% Ethyl Alcohol Solution (66% EtOH) (V/V). Ethyl alcohol with a volume of 347 ml was mixed with 153 ml of deionized water in a 1 quart mason jar and capped afterward. This is enough solution for one sample. In preparation of enzyme solution (136 mg/ml pancreatin, 13.4 U/ml AMG and 25.43 U/ml invertase), one gram of pancreatin was weighed into a 50 ml centrifuge tube. 6.67 ml of water was then added in and vortex mix for 10 minutes to dissolve thoroughly (a stir bar or glass balls may be needed if preparing solution for use with for multiple samples). The sample was then centrifuged at 2000 rpm for 10 minutes and for each test sample, six ml of supernatant was transferred to another 50 ml centrifuge tube followed by adding 296 l AMG and 330 lof invertase stock solution. 3.2 Preparation of solid sampleThe solid sample was first placed in a Waring Laboratory Micronizer and grounded for 45 seconds until the sample is well mixed. A 3 ounce/100 g of sub-sample from the Micronizer was then cryogenically grounded in the Spex CertiPrep 6850 Freezer/Mill. At the end of the grinding cycle, the sample was emptied into a suitable container and capped.3.3 Enzyme digestion of food sampleFood sample equivalent to 50 mg of available carbohydrate (total carbohydrate – fiber – sugar alcohols other than maltitol) was weighed and added into a 40 ml glass screw cap vial (…). Thereafter, 0.5 ml of water, 1 ml of freshly prepared pepsin/guar gum solution (…) and 5 glass balls (…) were added to the sample. A sample blank consisting of 50 mg of water was ran with each batch of samples. Using a vortex mixer (…), the capped vial was mixed vigorously and then placed horizontally in a 37° C shaking water bath (175 strokes/min). The sample was left to shake in the water bath for 30 minutes for the hydrolysis of proteins by pepsin. After 30 minutes, 0.4 ml of 0.5 M sodium acetate(…) (equilibrated to 37° C) was added into the vial and mixed. Next, 0.5 ml of the enzyme solution was added and inversion mixing was carried out. pH of the digestion mixture was measured to obtain an approximate pH value of 5. After mixing, the sample was immediately placed horizontally back into the 37° C shaking water bath to continue the enzyme reaction for 20 minutes. After 20 minutes, the sample vial was removed from shaker and placed immediately into a ice bath to stop the enzymatic reaction. 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