Glycemic & Insulinemic Impact of FOSSENCE®: A Prebiotic Solution

1. White Paper | May 2021 Glycemic and Insulinemic Response of Prebiotic FOSSENCE®

2. Contents: Introduction 1-2 Understanding Glycemic Index and Insulin Index 3-4 Alternative food-based strategies for Glycemic and Weight Control 5-6 Study details conducted at INQUIS clinical research 7 Study outcomes 8-11 Summary 12 References 13

3. George Bernard Shaw had said, there is no sincere love than the love of food. Food is a major part of one’s life, and the variety of food preparations and products available across the world are testimony to this. cardiovascular diseases, diabetes, osteo- arthritis and some cancers. The global diabetes prevalence estimated to be 9.3% (463 million people) (Saeedi P et al, 2019). in 2019 was It’s no secret that the amount of calories one consumes versus its utilization directly impacts weight and metabolic health. Moreover, the modern culture of foods and its preparations subjects one to calorie dense intakes. When combined with a sedentary lifestyle, it may generally result in weight gain and metabolic disorders. Globally, in 2016, more than 1.9 billion adults were overweight, of which over 650 million were obese (WHO, 2018). Obesity and diabetes are major causes of morbidity and mortality, and impaired quality of life. Therefore, attention has now focused on the management of these conditions, with significant importance being given to food-related strategies. Not only is the calorie count given attention, but also how specific carbohydrates impact the post-meal rise in blood sugar called the glycemic response, on which is based the concept of the ‘Glycemic Index’. Being overweight raises the risk of various non-communicable diseases such as Page 1

4. The glycemic response to a food or meal is the effect that the food or meal has on blood sugar (glucose) levels after its consumption (Sadler M et al, 2011).Normally, blood glucose and insulin levels rise after eating and later revert to fasting levels over a short period. This is especially true after the consumption of meals rich in certain carbohydrates. ‘Glycemic Index’ (GI) is expressed as an increase in blood glucose produced by a specific amount of available carbohydrate in the food (Augustin LS et al, 2015).Foods with a high Glycemic index rapidly raise blood sugar levels and cause substantial fluctuations in blood sugar (Bhupathiraju SN et al, 2014). In simpler terms, the glycemic index (GI) is a rating system for foods containing carbohydrates. It shows how quickly each food affects the blood sugar (glucose) level when it is eaten on its own (Augustin LS et al, 2015). It is defined as the incremental area under the curve (AUC) for the blood response after consumption of a 50 g carbohydrate portion of a test food expressed as a percent of the response to an equivalent carbohydrate amount from a reference food ingested by the same subject, with glucose or white bread as the reference food (Wolever TM et al, 1991). High GI carbs cause blood sugar to spike then crash glucose Blood glucose levels Low GI carbs are digested and released slowly for sustained energy Time 1 hour 2 hours Figure 1: Blood glucose levels after eating high and low GI foods (Rizkalla SW et al, 2002). Page 2

5. Glycemic Index is classified as: Low Medium High 55 or less 70 55-70 and above Foods with a high GI score contain carbohydrate that is rapidly digested and produces a sharp rise and fall in blood glucose levels. In contrast, foods with a low GI score contain slowly digested carbohydrate, which produces a more gradual and relatively low rise in blood glucose and insulin levels. Therefore, when one consumes foods, there is an elevation of the insulin concentration in the blood during the 2-h period after the food is ingested. A direct index of the postprandial insulin response to a test food in comparison with an isoenergetic portion of a reference food (analogous to the glycemic index, either glucose or white bread), is defined as a dietary insulin index (DII) (Mirmiran P et al, 2016). We are aware that the body produces the hormone Insulin post meals to maintain blood sugar within a healthy range. The Insulin Index (II) is a relatively new concept which measures the amount of insulin the body produces in response to a set carbohydrate load in a particular food. This index is not necessarily proportional to the GI, and consumption of large volumes of food with a high insulin index may play a role in the development of insulin resistance, although the link has yet to be conclusively established. Insulin Index is classified as follows: Low Medium High 20 <10 10-<20 and more High glycemic and insulinemic responses can affect appetite and energy metabolism, favouring body weight and body fat gain (Pereira et al, 2015). Page 3

6. Foods that have a high GI, Glycemic Load (GL) and DII are quickly digested and absorbed, resulting in a high glycemic response, which in turn stimulates higher insulin secretion (insulinemic response), leading to obesity and diabetes (Figure 2) (Vidya R et al, 2014). High GI / High GL Meal Glucose load Blood glucose levels Insulin demand from pancreas Oxidative stress Increased inflammation Hunger leads to overeating Hyperinsulinemia Insulin resistance Free fatty acids Overweight/obesity CVD Metabolic syndrome Type 2 diabetes mellitus Figure 2: Physiological effects of High GI / High GL foods and link to diabetes and obesity On the other hand, low GI foods are digested and absorbed at slower rates, leading to lower glycemic and insulinemic responses, which in turn induces satiety, reduces food intake and increases mobilization (which is reflected as reduced waist circumference after the intake of low GI meals; (Figure 3). LGI HGI 4 3 2 1 0 * * * * * adipose tissue -1 -2 -3 -4 Body fat (%) Waist circumference (cm) Figure 3: Changes in Waist circumference (WC) and Body fat (%) Waist circumference and body fat changes (post-intervention) in response to the consumption of two daily LGI or HGI meals for 45 consecutive days. Waist circumference (***p=0.008) and body fat (**p=0.050) reduced after the consumption of the LGI vs. HGI meal (Mann-Whitney Test). Adopted from: Pereira et al, 2015. Hence, the knowledge of the GI index of a food substance may be a guiding factor for those who wish to manage their weight or postprandial rise in blood sugar (Pereira et al, 2015). Page 4

7. Alternative food-based strategies for Glycemic and Weight Control Awareness among people about the role that diet plays in the management of obesity, diabetes and other related health concerns is improving worldwide. Selection of foods based on a glycemic index, glycemic load or insulin index is now gaining recognition in the management of weight and hyperglycaemia. This approach is particularly popular in the “healthy and diabetes-friendly” packaged food category. FOS has beneficial effects on health as it stimulates the growth of some beneficial bacteria such as Bifidobacteria. The use of FOS as a food ingredient has stimulated much research to know its functionality and its effects on human health, especially concerning its bifidogenic character. Its potential preventing and controlling some diseases have been also been studied, especially in conditions where there are conditions of metabolic disorders hyperglycaemia. benefits in In keeping with the diet trend toward high-fibre, low-glycemic carbohydrates and carb-controlled foods, more reduced-sugar products are showing up in groceries. such as Consumer interest in food and beverages with carbohydrates glucose release and lower glycemic index (GI) continues to rise. Slowly digestible carbohydrates (SDC) like FOS offer an ingredient solution to improve carbohydrate quality and meet consumer needs. The newest ingredient on the block is the prebiotic Fructo-oligosaccharide, or FOS, which is now appearing in many food products. Commonly consumed foods such as bananas, onions, garlic, asparagus, wheat, rye, Jerusalem artichoke, contain FOS. Also, it can be produced enzymatically from sugarcane molasses. offering steady Page 5

8. Short-chain FOS being sweet to taste has been frequently used to replace sugars in low-sugar food products to lower the postprandial glycemic response, reduce energy content, or enrich foods with dietary fibres (Lecerf JM et al, 2015; Respondek F et al, 2014). This section shall discuss the studies about acute glycemic and insulinemic responses of FOSSENCE® (which contains short-chain FOS) when administered alone or when added or substituted for a carbohydrate load. Acute Glycemic and Insulinemic responses of FOSSENCE® FOSSENCE® is a short-chain fructo- oligosaccharide (scFOS) that is a sweet-tasting, soluble dietary fibre produced by Tata Chemicals Limited through a patented process. It is a mixture of tri-saccharide (GF2), tetra-saccharide pentasaccharide (GF4) of glucose (G) and fructose (F). (GF3) and Unlike short-chain a naturally occurring oligosaccharide, remains intact through the upper digestive tract but is degraded in the colon by the ‘good’ gut bacteria. most simple sugars, fructo-oligosaccharide, Page 6

9. Study conducted at INQUIS clinical research (Formerly GI Labs, Canada) In a randomized, controlled, cross-over study conducted in 3 phases explored Glycemic Response and Insulin Response to ingestion of FOSSENCE®, when replaced by/added to available-carbohydrates (avCHO) among 25 healthy adults (40±14years). In each phase GR and IR elicited by 3-4 testmeals were measured among the fasted recruited subjects. (Table 1) (Shah P et al, 2020). Table 1: Interventions received by study subjects in three phases of the study Experimental arms Phases 2 3 4 1 10g FOSSENCE® (10FOS**) Water as control Phase 1 10g Dextrose (10Dex*) 50Dex+15FOS 35Dex 35Dex+15FOS 50g Dextrose (50Dex) Phase 2 50WB+15FOS 35WB 35WB+15FOS 50g available carbohydrate (avCHO) from white bread alone 50WB*** Phase 3 *Dex = Dextrose **FOS = FOSSENCE® ***WB = White Bread On each test occasion, samples for fasting blood glucose and insulin were taken. The subjects were then asked to consume a test meal within 10 min. Each test meal was served with a drink of 1 cup of water. Blood samples were collected at fasting and over 2 hours after the start of the test meal and analyzed for glucose and insulin levels. The primary endpoint was differences in incremental glucose area under curve (IAUC). Page 7

10. The Study Outcomes Page 8

11. Phase 1 The results demonstrated that FOSSENCE® does not increase postprandial glucose and insulin levels at a 10g intake compared to 10g dextrose intake and was comparable to the response of water. The absolute and incremental plasma glucose levels of the subjects were significantly lower after the intake of water and water containing 10g FOS compared to water containing 10g of dextrose at 15, 30 and 45 min. 2.5 Plasma Glucose (mmol/L) 2.0 10Dex Control 10FOS 1.5 1.0 0.5 0.0 0 30 60 90 120 -0.5 Time (min) Figure 4: Incremental Plasma Glucose 25.0 FOSSENCE® is resistant to breakdown, not digested by enzymes in the human small- intestine and therefore is an unavailable carbohydrate that does not raise post prandial blood glucose or insulin. 10Dex Control 10FOS 20.0 Serum Insulin µU/ml 15.0 10.0 5.0 0.0 0 30 60 90 120 -5.0 Time (min) Figure 5: Incremental Serum Insulin Page 9

12. Phase 2 Relative glycemic and insulinemic response of a 50g oral glucose challenge with 15g of FOSSENCE® added or when 15g FOSSENCE® (30% carbohydrates) was substituted for 15g of the dextrose was evaluated. Addition of FOSSENCE® to a carbohydrate challenge showed no significant difference in glucose peak levels, incremental glucose levels or incremental insulin levels. 1 Substitution of carbohydrate by FOSSENCE® showed that glucose IAUC and insulin IAUC for was significantly lower than of IAUC by control (p<0.002 and p<0.0003, respectively). 2 3 Incremental insulin levels were significantly lower after substitution of 30% available carbohydrates compared to control (p<0.02). 4 Peak insulin levels were significantly lower after 30% carbohydrates were substituted with FOSSENCE® compared to control (p<0.001). 75 6.0 50Dex 50Dex+FOS 35Dex 35Dex+FOS 50Dex 50Dex+FOS 35Dex 35Dex+FOS Plasma Glucose (mmol/L) a ab Serum Insulin (µU/ml) 5.0 55 4.0 a a 35 3.0 a ab 2.0 a ab 15 a a 1.0 a ab 0.0 -5 0 30 60 90 120 0 30 60 90 120 Time (min) -1.0 Time (min) Figure 6: Incremental Plasma Glucose Figure 7: Incremental Serum Insulin Results demonstrate that adding 15g of FOSSENCE® to glucose load does not significantly change 2h glucose or insulin IAUC, neither does it modulate postprandial glucose or insulin levels but 30% substitution attenuates postprandial glucose and insulin levels. When FOSSENCE® was added to a carbohydrate challenge, peak or absolute glucose levels did not differ significantly after the intake of and 50g Dextrose +15g FOSSENCE® or between the intake of 35g dextrose and 35g Dextrose + 15g FOSSENCE®. When FOSSENCE® replaced 30% of available carbohydrate, IAUC and plasma glucose levels after the intake of 35g Dextrose + 15g FOSSENCE® were substantially lower than that after the intake of 50g dextrose. Page 10

13. Phase 3 IAUC of plasma glucose over 2 hours were compared after the consumption of a white bread portion containing 50g available carbohydrate and the same white bread portion with 15g of FOSSENCE® added or substituted for 30% of the available carbohydrate in the white bread. 50WB 3.5 a a a b Plasma Glucose (mmol/L) 50WB+15FOS a ab bc c 3.0 35WB 2.5 35WB+15FOS 2.0 a a b b 1.5 a a b b 1.0 0.5 0.0 0 30 60 90 120 Time (min) Addition carbohydrate challenge (50WB vs 50WB+15FOS) resulted significant difference in glucose or insulin IAUC, or incremental glucose or insulin levels at any time point. Substitution (30%) carbohydrate by (50WB vs 35WB+15FOS) resulted in significantly IAUC (p<0.0001) as well as insulin IAUC (p<0.0001). FOSSENCE® also reduced incremental plasma glucose at 60, 90 and 120min and serum insulin levels at 30, 45, 60, 90 and 120 min compared to 50WB meal. of FOSSENCE® to Figure 8: Incremental Plasma Glucose in no 60 50WB 50WB+15FOS 35WB 35WB+15FOS 50 a ab Serum Insulin (µU/ml) a ab bc 40 a a of FOSSENCE® available 30 a a b b 20 lower glucose 10 0 0 30 60 90 120 Substitution of Time (min) Figure 9: Incremental Serum Insulin The glucose and insulin IAUCs were significantly lower after the intake of bread portion containing 35g available carbohydrate and 35g available carbohydrate + 15g FOSSENCE® compared to that after the intake of bread portion containing 50g available carbohydrate and 50g available carbohydrate + 15g FOSSENCE®. Page 11

14. Summary To conclude, these studies demonstrate that FOSSENCE®, when consumed alone, does not increase postprandial glucose and insulin levels, suggesting its resistance to breakdown. When added to a carbohydrate load (Dextrose or White Bread), FOSSENCE® does not increase postprandial glucose or insulin levels, of FOSSENCE® with 30% of glycemic carbohydrate reduces glucose and insulin levels, indicating the blood glucose/insulin attenuation after FOSSENCE®-fortified meals. while replacement postprandial FOSSENCE® has a sweet taste with a sweetness of 30-40% to that of sugar. The sweetness profile is without any bitterness or any Hence, FOSSENCE® maybe advised to individuals on restricted sugar intake. after taste. Page 12

15. References Augustin LS, et al. Glycemic index, glycemic load and glycemic response: An International Scientific Consensus Summit from the International Carbohydrate Quality Consortium (ICQC). Nutr Metab Cardiovasc Dis. 2015 Sep;25(9):795-815. doi: 10.1016/j.numecd.2015.05. Bhupathiraju SN, et al. Glycemic index, glycemic load, and risk of type 2 diabetes: results from 3 large US cohorts and an updated meta-analysis. Am J Clin Nutr. 2014 Jul;100(1):218-32. doi: 10.3945/ajcn.113.079533. Epub 2014 Apr 30. PMID: 24787496; PMCID: PMC4144100. Lecerf JM, et al. Postprandial glycaemic and insulinaemic responses in adults after consumption of dairy desserts and pound cakes containing short-chain fructo oligosaccharides used to replace sugars. J Nutr Sci, 2015; 12;4:e34. https:// doi:10.1017/jns.2015.22. Mirmiran P, et al. Dietary insulin load and insulin index are associated with the risk of insulin resistance: a prospective approach in tehran lipid and glucose study. J Diabetes Metab Disord. 2016 Jul 20;15:23. doi: 10.1186/s40200-016-0247-5. PMID: 27446819; PMCID: PMC4955203. Pereira, et al. Effect of glycemic index on obesity control. Archives of endocrinology and metabolism. 2015 Jun;59(3):245-51. Respondek F, et al. Digestive tolerance and postprandial glycaemic and insulinaemic responses after consumption of dairy desserts containing maltitol and fructo-oligosaccharides in adults. Eur J Clin Nutr. 2014;68:575–80. Rizkalla SW, et al. Health benefits of low glycaemic index foods, such as pulses, in diabetic patients and healthy individuals. Br J Nutr. 2002 Dec;88 Suppl 3:S255-62. doi: 10.1079/BJN2002715. PMID: 12498625. Sadler M, et al, Food, Glycaemic Response and Health. ILSI Europe Concise Monograph Series 2011:1-30. Available from ILSI Europe. Saeedi P, et al. IDF Diabetes Atlas Committee. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract. 2019 Nov;157:107843. doi: 10.1016/j.diabres.2019.107843. Epub 2019 Sep 10. PMID: 31518657. Shah P, et al. Comparison of the Acute Glycemic and Insulinemic Response of Fossence™, a Short Chain Fructo-Oligosaccharide, Taken Alone, Added or Substituted into a Carbohydrate Load, Current Developments in Nutrition, 4(S2): 774. Vidya R, et al. Glycemic Index of Indian Cereal Staple Foods and their Relationship to Diabetes and Metabolic Syndrome. InWheat and Rice in Disease Prevention and Health 2014 Jan 1 (pp. 333-346). Academic Press. WHO Factsheet-Obesity and overweight. Updated April 2018. Wolever TM, et al. The glycemic index: methodology and clinical implications. Am J Clin Nutr. 1991 Nov 1;54(5):846-54. Page 13

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