Food Science Dossier Technical and Applications Data

Pharmachem Laboratories, Inc. 265 Harrison Avenue, Kearny NJ 07032 USA 201-246-1000 • 800-526-0609 www.phase2info.com [email protected]

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Table of Contents

• Safety and Efficacy White Paper “A Proprietary Alpha-Amylase Inhibitor from White Bean (Phaseolus Vulgaris): A Review of Clinical Studies on Weight Loss and Glycemic Control.” Jay Udani, MD, Nutrition Journal.

• Evaluation of Phase 2 Carb ControllerTM vs. Hi-Maize Resistant Starch – A Market Opportunity “Review and Analysis of Hi-Maize Resistant Starch Product from National Starch Company and Evaluation of Potential Benefits for Phase 2 Products Applications.” Kanak Udani, PhD, Food Scientist.

• Studies and Applications Data for Phase 2 Carb Controller/StarchLite Technical Over view Stability and Activity in Fresh Pasta (France) Stability and Activity in White Bread (France) Stability in Instant Mashed Potatoes (United States) Inhibitory Action in Chewing Gum (United States) Stability in Orange Drink (United States) Inhibitory Action in Pasteurized Milk (United States) pH and Thermal Stability (France) Stability in Microwave Cooking (Japan) Amino Acid Sequence of Inhibitor 1 Amino Acid Sequence of Inhibitor 2 Disorder Probability AI 1 Disorder Probability AI 2 Homology of Inhibitor 1 and 2

• Functional Seasoning Application Product Overview Nutritional Fact Sheets Product Specifications Allergen Data

• Sensory Evaluation and Product Recipes “Product Development of Baked Goods with a Proprietary Fractionated White Bean Extract” (includes consumer sensory studies on a variety of baked goods with Phase 2) Wheat Bread Formulation Blueberry Muffin Formulation Cheese Pizza Formulation Coffee Cake Formulation Confidential; for authorized distribution, only. • Product Documents and Technical Specifications Product Data Sheet Certificate of Analysis Material Safety Data Sheet Process Flowchart GMO Statement Irradiated/ETO Statement Kosher Statement Ifanca Halal Product Certificate GRAS Statement Nutrition Journal

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A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): A review of clinical studies on weight loss and glycemic control

Nutrition Journal 2011, 10:24 doi:10.1186/1475-2891-10-24

Marilyn L Barrett ([email protected]) Jay K Udani ([email protected])

ISSN 1475-2891

Article type Review

Submission date 17 September 2010

Acceptance date 17 March 2011

Publication date 17 March 2011

Article URL http://www.nutritionj.com/content/10/1/24

This peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below).

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© 2011 Barrett and Udani ; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A proprietary alpha-amylase inhibitor from white bean

(Phaseolus vulgaris): A review of clinical studies on weight loss and glycemic control

Marilyn L Barrett1, Jay K Udani 2,3§

1Pharmacognosy Consulting, Mill Valley, CA 94941, USA

2Medicus Research LLC, Northridge, CA 91325, USA

3UCLA School of Medicine, Department of Medicine, Los Angeles, CA 90024, USA

§Corresponding author

Jay K. Udani, MD

Medicus Research, LLC.

18250 Roscoe Blvd., Suite 240

Northridge, CA. 91325

Phone: (818) 882-9442

Fax: (818) 479-9373

Email: [email protected]

Abstract Obesity, and resultant health hazards which include diabetes, cardiovascular disease and

metabolic syndrome, are worldwide medical problems. Control of diet and exercise are

cornerstones of the management of excess weight. Foods with a low glycemic index may reduce

the risk of diabetes and heart disease as well as their complications. As an alternative to a low

glycemic index diet, there is a growing body of research into products that slow the absorption of

carbohydrates through the inhibition of enzymes responsible for their digestion. These products

include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller

(Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels.

Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of

Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity.

2

Review

Obesity is a major health hazard, with increased risk for cardiovascular disease (mainly heart disease and stroke), type 2 diabetes, musculoskeletal disorders (especially osteoarthritis) and certain types of cancer (endometrial, breast, and colon) [1]. The World Health Organization

(WHO) estimated that in 2005, approximately 1.6 billion adults worldwide were overweight and at least 400 million were obese. Further, the WHO estimated that at least 20 million children under the age of 5 years were overweight. The projected numbers for 2015 are larger, with 2.3 billion adults expected to be overweight and 700 million expected to be obese [1].

The cause of excess body weight is an imbalance between energy intake and expenditure. The

WHO has identified a global shift in diet towards increased intake of energy-dense foods that are high in fat and sugars but low in vitamins, minerals and other micronutrients. At the same time there is a trend towards decreased physical activity due to the increasingly sedentary nature of many forms of work, changing modes of transportation, and increasing urbanization [1].

Control of diet and exercise are cornerstones of the management of excess weight. A number of nutritional approaches and diets with difference proportions of lipids, proteins and carbohydrates have been prescribed for weight loss. Initial guidance on weight loss was a restriction in saturated fats. However diets low in saturated fats did not necessarily result in weight loss as expected. More recently there has been a shift towards a reduction in carbohydrates, particularly refined carbohydrates, as an approach to reduce weight and the incidence or related disease risk

[2].

In most diets, carbohydrates are the greatest source of calories. Carbohydrates are polyhydroxy aldehydes, ketones, alcohols and acids that range in size from single monomeric units

(monosaccharides) to polymers (polysaccharides). Before being absorbed by the body, carbohydrates must be broken down into monosaccharides. This breakdown occurs due to two major enzymes: amylase and glucosidase [3].

Digestion of carbohydrates begins in the mouth, with amylase secreted by salivary glands. This action accounts for only about 5% of the breakdown of carbohydrates. The process is halted in the stomach due to the high acid environment destroying the amylase activity. When the food enters the intestine, the acidic pH is neutralized by the release of bicarbonate by the pancreas and by the mucous that lines the walls of the intestine. Amylase is secreted into the small intestines by the pancreas. Alpha-glucosidase enzymes are located in the brush border of the small intestines. Amylase breaks down the carbohydrates into oligosaccharides. The glucosidase enzymes (including lactase, maltase and sucrose) complete the breakdown to monosaccharide units. It is only the monosaccharide units that are absorbed into the body. Glucose and other monosaccharides are transported via the hepatic portal vein to the liver. Monosaccharides not immediately utilized for energy are stored as glycogen in the liver or as fat (triglycerides) in adipose tissue, liver and plasma. Carbohydrates that are resistant to digestion in the intestine enter the colon, where they are fermented by colonic bacteria to produce short-chain fatty acids, carbon dioxide and methane.

4 Dietary carbohydrates that are composed mostly of monosaccharide units are absorbed quickly and are said to have a “high glycemic index”. Carbohydrates in polymeric form are absorbed more slowly and said to have a “low glycemic index”. The glycemic index (GI) is defined as the incremental area under the blood glucose curve following ingestion of a test food, expressed as a percentage of the corresponding area following an equivalent load of a reference carbohydrate, either glucose or white (wheat) bread [4]. Factors that influence the GI besides the composition of the carbohydrate are the fat and protein content of the food, the acidity of the food and the presence of fiber [5]. Low GI foods (< 55) include vegetables, unsweetened yogurt and protein- enriched spaghetti. High GI foods (> 70) include white bread, baked potato and dates.

After consumption of high GI foods, there is a large, rapid increase in blood sugar levels and in response a rapid increase in insulin levels. Insulin promotes the uptake of glucose from the blood into cells in the liver and skeletal muscle tissue, storing it as glycogen. Insulin also increases fatty acid synthesis and can result in the accumulation of lipids. Accumulation of lipids in skeletal muscle and the liver is associated with a decrease in insulin sensitivity. Insulin resistance increases the chance of developing type-2 diabetes and heart disease. Post-prandial hyperglycemia and insulin resistance are thought to play a central role in the development and progression of cardiovascular disease in subjects with impaired glucose tolerance. Post-prandial hyperglycemia is associated with endothelial dysfunction and an increase in intima-media thickness as well as a higher prevalence of atherosclerotic plaques. High glucose levels have been shown to stimulate expression of adhesion molecules (intercellular adhesion molecule-1, vascular adhesion molecule-1, E-selectin) and cytokines in in-vitro models. Hyperglycemia causes an increase in oxidative stress with associated oxidation of low-density lipoprotein,

platelet activation and thrombin generation [5,6]. A body of evidence, including prospective cohort studies, randomized controlled trials and mechanistic experiments, support a role for low

GI diets in the prevention of obesity, diabetes and cardiovascular disease [7-9]. Three large-scale epidemiological studies on women reported a correlation between a high glycemic index diet and the incidence of type 2 diabetes [10-12]. The populations studied were 59,000 US black women,

65,000 Chinese women and 91,249 US nurses, who were each followed for periods of time of 5 to 8 years. Another prospective cohort study in Europe, which included 25,000 men and women, concluded that high cereal fiber was inversely associated with the risk of developing diabetes

[13].

As previously indicated, the choice of the type of carbohydrate foods in the diet, with their varying glycemic properties, with determine the rate of absorption of sugars into the body. One means of reducing the GI of a meal is the inclusion of resistant starches. Resistant starches are those that resist digestion in the small intestine, thereby passing into the large intestine, where they act like dietary fiber [14]. These starches are naturally found in seeds, legumes and unprocessed whole grains. The amount of resistant starch in food is influenced by processing, which can either increase or decrease the amounts found in the raw substance. Resistant starch can be added to foods such as bread, biscuits, sweet goods, pasta, nutritional bars and cereal, in order to lower their GI index, without affecting taste or texture [15,16].

An alternative to a low GI diet are products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. Acarbose (Prandase®, Precose®) is a prescription drug, which inhibits

6 alpha-glucosidase enzymes in the brush border of the small intestines and pancreatic alpha- amylase. Other drugs that belong to this class are miglitol and voglibose. Acarbose reduces post- prandial hyperglcemia and is used to treat diabetes type-2. Clinical studies with subjects with impaired glucose tolerance have demonstrated not only an improvement in post-prandial hyperglycemia but also cardiovascular benefits. Acarbose has been shown to slow the progression of thickening of the intima-media in the carotid arteries, reduce the incidence of cardiovascular disease and reverse newly diagnosed hypertension. Recently acarbose has been reported to improve insulin resistance in subjects with impaired glucose tolerance or diabetes type-2. Due to these findings, acarbose has been suggested as treatment to reduce cardiovascular risk in subjects with metabolic syndrome (a cluster of risk factors including high triglycerides, low high-density lipoprotein cholesterol and hypertension) [6].

Alpha-amylase inhibitors with activity against mammalian forms of the enzyme are present in plants and it is suggested that they were developed by plants in order to strengthen their defense against predators. Plant constituents with enzymatic inhibitory activity include polyphenolic compounds and glycoproteins [17]. For example, anthocyanins and ellagitannins present in raspberries and strawberries have been reported to inhibit alpha-glucosidase and alpha-amylase activity, respectively [18]. In addition, theaflavins and catechins present in green and black teas have been reported to inhibit alpha-amylase and alpha-glucosidase activity as well as retard starch digestion in an in-vitro model [19]. Alpha-amylase inhibitors are also present in grains, including wheat and rice [17]. However, the greatest body of research has gone into glycoproteins extracted from kidney beans (Phaseolus vulgaris) and more specifically on the proprietary Phase 2 product.

Properties of alpha-amylase inhibitors from beans

Common beans have 3 isoforms of alpha amylase inhibitor (alpha-A1, alpha-A12, alpha-AIL).

The alpha-AI isoform has anti-amylase activity in humans. This enzyme is found in the embryonic axes and cotyledons in the seed and not in other organs of the plant. It is not active against plant alpha-amylases and is therefore classified as an anti-feedant or seed defense protein

[20].

The alpha amylase inhibitor prevents starch digestion by completely blocking access to the active site of the alpha-amylase enzyme. Factors that affect the activity of the alpha-AI isoform inhibitor are pH, temperature, incubation time and the presence of particular ions. The optimum pH for the inhibitor is 4.5 to 5.5 and the optimal temperature is 22 to 37oC. There is no activity at

0oC and the inhibitor is completely inactivated by boiling for 10 minutes. The ideal incubation period has been recorded as 10 minutes, 40 minutes and 120 minutes by three different researchers [3]. The different incubation times are thought to be due to the use of different test conditions; namely a pH of 6.9 for the longer incubation periods and a pH of 4.5 for the shortest

[3].

Background Experiments in Humans

In the early 1980’s several products containing crude preparations of bean amylase inhibitors were marketed in the United States. However early clinical studies were disappointing and it was discovered that the preparations had insufficient enzyme inhibiting activity, as well as issues

8 with potency and stability. Subsequently, a research group at the Mayo Clinic developed a partially purified white bean product and published a series of studies exploring the activity of inhibitor in human clinical studies. The test product was described as a concentrate: 6 to 8-fold by total protein content and 30 to 40 fold by dry weight [21]. The product was found to inactivate salivary, intraduodenal and intraileal amylase activity in vitro. Its activity was not affected by exposure to gastric juice and only minimally by duodenal juice (by 15%). In vitro studies demonstrated that the inhibitor decreased digestion of dietary starch in a dose-dependent manner [21]. Perfusion of the white bean product into the duodenum of human subjects completely inhibited the activity of intraluminal amylase activity (5.0 mg/ml at 5 ml/min) [21].

Subsequent experiments were conducted with volunteers intubated with an oroileal tube in order to obtain duodenal, jejuna and terminal ileal samples [22]. After intubation the subjects ingested

50 g rice starch and on the subsequent day they ingested starch with the amylase inhibitor (5g or

10g white bean extract). The white bean extract significantly reduced duodenal, jejunal, and ileal intraluminal amylase activity by more than 95%; it acted as quickly as 15 minutes, and for as long as 2 hours. It increased the post prandial delivery of carbohydrates to the distal small bowel by 22 to 24% (as measured by oroileal tube aspiration) and increased hydrogen concentrations in the breath from 30 to 90 minutes after the meal. Hydrogen breath testing is an accepted method of determining carbohydrate malabsorption as colonic bacteria ferment carbohydrates into organic acids, carbon dioxide and hydrogen. A percentage of these gases are absorbed into the portal blood stream and subsequently expired through the lungs [23-25]. The white bean extract also reduced the postprandial plasma glucose rose by 85% and eliminated the subsequent fall of glucose level to below fasting levels. The extract significantly lowered the postprandial plasma levels of insulin, C-peptide and gastric inhibitory polypeptide [22].

A follow-up study using subjects with diabetes mellitus demonstrated a decrease in the postprandial increases in plasma glucose and insulin levels [26]. Further studies revealed that a dose of 3.8 g white bean inhibitor could cause more than twice the amount of hydrogen in the breath following a standard spaghetti meal. The percentage of malabsorbed carbohydrate increased from 4.7% to 7.0% (p<0.05). Also, the form of the inhibitor (powder, tablet) had no effect on the activity when taken with the spaghetti [27]. Follow-up studies found that a dose of

2.9 g was sufficient to significantly inhibit the postprandial increases in blood glucose, C-peptide and gastric inhibitory polypeptide following 650-calorie meal containing carbohydrate, fat and protein [28]. A longer-term study was conducted over 3 weeks with 6 non-insulin dependent diabetics. The subjects were given sufficient white bean inhibitor to reduce the increase in postprandial plasma glucose by more than 30%: a dose of 4 to 6 g with each meal. As a result there were significant decreases in postprandial glucose, C-peptide, insulin and gastric inhibitory polypeptide along with a significant increase in hydrogen excretion in the breath. Diarrhea and gastrointestinal symptoms occurred the first day of administration of the inhibitor and resolved over the next couple of days [29]. A further experiment with 18 healthy subjects reported that carbohydrates perfused into the ileum delayed emptying of a meal infused into the stomach. In one half of the subjects, the amylase inhibitor was added to the ileum perfusate. The inhibitor significantly reduced the absorption of carbohydrate from the ileum and enhanced the delay in gastric emptying. Plasma concentrations of C-peptide, glucagon, motilin, gastrin and human pancreatic polypeptide were not influenced by changes in gastric emptying or by the ileal perfusates. However the delay in gastric emptying was significantly associated with a decrease in plasma concentrations of gastric inhibitory polypeptide and neurotensin along with an increase in

10 concentrations of peptide YY. This effect was caused by the delay in gastric emptying, rather than the other way around. Human polypeptide levels were not changed and the authors concluded that the hormonal changes were not mediated via the vagus nerve [30].

Phase 2 specifications

The Phase 2® product is a water extract of the white kidney bean (Phaseolus vulgaris) standardized to alpha-amylase (8;12;15;39) inhibiting units (Pharmachem Laboratories, Kearny,

NJ). Phase 2 is produced from non-GMO whole white kidney beans, which are ground and then extracted for 4 hours. The liquid is filtered and concentrated under vacuum. The extract is filtered again, and then pasteurized before being spray dried. Phase 2 is odorless and tasteless.

Each lot of Phase 2 has at least 3000 alpha amylase inhibiting units (AAIU) per g when tested at a pH 6.8 using potato starch as the substrate and pancreatin as the enzyme source. The Phase 2 extraction process was designed to make it more potent and stable than the white bean product tested by the Mayo clinic.

Phase 2 is used as a dietary supplement in various forms, including powders, tablets, capsules and chewables. There are approximately 200 brands of nutritional supplement / weight loss products in the worldwide market that contain Phase 2. A typical dose is 1 to 2 capsules, each containing 500 mg, taken before each of 3 daily meals, for a total of 1500 to 3000 mg per day. A private safety panel approved a maximum daily intake of 10,000 mg (10 g) [31].

Experiments conducted incorporating Phase 2 into food products have found that it can be incorporated into chewing gum, mashed potatoes, yeast-raised dough (bread, pizza, etc) without losing activity or altering the appearance, texture or taste of the food [32-34].

Clinical studies conducted with Phase 2

Ten clinical studies have demonstrated weight loss over time following administration of Phase

2. Three studies demonstrated significant loss of body weight with Phase 2 compared to a placebo control in people who are overweight or obese. The doses ranged from 445 mg for 4 weeks to 3000 mg for 8 to 12 weeks .[35-37]. A placebo controlled study by showed a comparative loss in body weight only when subjects were stratified by dietary carbohydrate intake. Those who consumed the greatest amount of carbohydrate, lost significant body weight in comparison to the placebo group [38]. Six additional studies reported a loss of weight over time

[39-44]. Three clinical studies reported a reduction in serum triglycerides over time [40,41,44]

(Table 1).

Weight loss – compared to placebo

A 12 week randomized double-blind placebo controlled trial included 60 overweight individuals

(BMI between 24 and 32 kg/m2). The subjects consumed 2 soft chews before each meal containing either Phase 2 (500 each mg) or placebo for 12 weeks. The Phase 2 group consumed a total of 3000 mg Phase 2 per day. A total of 88 men and women enrolled in the study, while 60 completed the study and were included in the analyses. There was a statistically significant weight reduction in the active group compared with the placebo group at weeks 6, 8 and 12. The amount of weight lost by the active group at 12 weeks was 6.9 ±7.9 lbs (average of 0.575 lbs per

12 week) while the placebo group gained 0.8 lbs ±6.1 (p=0.029 between groups). There were no significant differences between groups in body fat, lean body mass or body measurements

(waist/hip circumferences). No adverse events were reported [36].

A randomized, double-blind, placebo-controlled study was conducted with 60 slightly overweight subjects (5 to 15 kg overweight). The subjects were required to have a stable weight for the past 6 months and underwent a 2-week single-blinded, run-in period prior to randomization. The subjects took 1 tablet (active or placebo) per day for 30 consecutive days before a meal rich in carbohydrates (2000 to 2200 calorie diet). The active tablet contained 445 mg Phase 2 and 0.5 mg chromium picolinate (≈55 mcg elemental chromium). After 30 days, the active group had a significant reduction in body weight, BMI, fat mass, adipose tissue thickness and waist/hip/thigh circumferences while maintaining lean body mass. The active group lost an average of 2.93 kg (6.45 lbs) in 30 days compared with an average of 0.35 kg (0.77 lbs) in the placebo group (p<0.001). BMI in the test group was reduced from an initial 25.9 ± 2.0 (SEM) to

24.9 ± 1.9 (p<0.01). The placebo showed no significant change from the initial 26.0 ± 2.3

(SEM). Body composition was measured with bioelectrical impedance. The active group demonstrated a 10.45% reduction in body fat compared with a 0.16% reduction in the placebo group (p<0.001). Waist and hip circumferences measured in a standard way, showed the same pattern as well. The active group demonstrated 2.93 cm and 1.48 cm reductions respectively, compared with 0.46 cm and 0.11 cm reductions in the placebo group (p<0.001). No adverse events were reported [35].

A randomized, double-blind, placebo-controlled study was conducted in China with 101 volunteers who had a BMI between 25 and 40. The subjects were given a single capsule containing 1000 mg Phase 2 or placebo three times per day, just before meals, for 60 days. The active group ingested a total of 3,000 mg Phase 2 per day. As a result, there was significant weight loss in the active groups compared to the placebo group after 30 and 60 days. After 60 days, the average weight loss in the active group was 1.9 ± 0.15 kg compared to 0.4 ± 0.13 kg in the placebo group (p<0.001). There was also a significant reduction in waist measurement in the active group compared to the placebo group (1.9 ± 0.32 cm compared to 0.4 ± 0.26 (p<0.001).

There was no effect on hip measurements. Blood chemistries did not change significantly over the 2 month study and no adverse side effects were reported [37].

A 4-week, randomized, double-blind, placebo-controlled study conducted with 25 healthy overweight (BMI 25-30) subjects [38]. The subjects took 1000 mg of Phase 2® or an identical placebo twice a day (before breakfast and lunch) as part of a weight loss program which included diet, exercise and behavioral intervention. The subjects were given nutritional guidelines to standardize their caloric intake at 1800 Kcal/day. Breakfast and lunch were provided to increase compliance. In addition, subjects met with a personal trainer to establish an exercise program and had a counseling session with a behavioral psychologist to identify psychological barriers to weight loss. As a result of this intervention, both groups reduced weight and waist size significantly compared to baseline, but there were no significant differences between groups.

After 4 weeks, the active group lost 6.0 lbs and the placebo group lost 4.7 lbs compared to baseline (p=0.0002 active and p=0.0016 placebo). The active group lost a mean of 2.2 in inches from their waists and the control group lost 2.1 inches from their waists compared to baseline

14 (p=0.050 and 0.0001, respectively). For exploratory analysis, subjects were stratified by dietary carbohydrate intake. In this analysis, the tertile that took in the most carbohydrates demonstrated significantly greater loss of body weight compared with the placebo group (8.7 pounds vs. 1.7 pounds, p=0.04). This group also had a significantly greater loss in inches around the waist (3.3 vs 1.3 inches; p=0.01). There were no significant changes from baseline in hip circumference, triglycerides, fasting glucose, total cholesterol, appetite control, hunger, energy level, and percent body fat, neither were there any significantly differences between groups. No side effects or adverse events were reported [38].

Weight loss – over time

In a randomized double blind placebo controlled trial, forty healthy overweight (BMI 27.5 to

39.0) were randomized and instructed to take 2 tablets of the test product immediately after all 3 meals (breakfast, lunch and dinner) for 12 weeks [39]. Subjects were also instructed to follow a

1200 kcal/day low-fat diet. The tablets, 650 mg each, contained a proprietary blend (Suco-

Bloc®) including 200 mg of Phase 2 (Phaseolamin®, Leuven Bioproducts, Belgium), 200 mg of inulin (from chicory root), and 50 mg of Garcinia cambogia extract. The remaining 200 mg in the tablets were not described. All subjects were included in an intent-to-treat analysis, including

7 subjects who dropped out of the study (6 in the placebo arm, 1 in the active arm). After 12 weeks, the active group had a significant reduction in weight, BMI and percent body fat compared to baseline, whereas there was no significant change in the placebo group. The active group lost an average of 3.5 kg (7.7 lb; p=0.001) and the placebo group lost 1.3 kg (2.9 lb). BMI decreased by 1.3 kg/m2 (p=0.01) in the active group and by 0.5 kg/m2 in the placebo group.

Percent body fat (measured by bioelectrical impedance) decreased by 2.3% (p=0.01) in the active

group and by 0.7% in the placebo. Body mass analyses showed that the weight loss in the active group consisted mainly of fat loss as >85% of the weight loss was accounted for by fat. Between group analyses was not provided for any of the variables. No adverse events were reported in either group [39].

A 12 week double-blind, placebo-controlled study was conducted and this period was followed by an additional 12 weeks were in all the participants received the active treatment [40]. In the first part of the study, the subjects took 2 capsules twice a day of placebo or Thera-Slim™.

Thera-Slim™ capsules contained 500 mg Phase 2 plus 250 mg fennel seed powder. The placebo contained cellulose and fennel seed powder. The active group received a total of 2000 mg Phase

2 per day. The subjects were asked to eat a diet in which lunch and dinner contained 100 to 200 g of carbohydrates. Sixty overweight and obese adult subjects (BMI 24-36) were randomized and

54 completed the study. After the first 12 weeks, the active group lost a average of 1.4 lbs and the placebo gained an average of 0.6 lbs. Serum triglyceride levels dropped by almost 3.3 times in the active group compared to the placebo group (-38.1 vs -11.9). The levels of total cholesterol and HDL were similar in both groups. No between group analyses were included in the report.

There were no adverse events reported after 24 weeks of usage.

A randomized double blind placebo controlled study was conducted with 39 obese subjects (BMI

30-43) who were randomly allocated to receive either 1500 mg of Phase 2 or an identical placebo twice daily with lunch and dinner for 8 weeks [44]. The active group received a total of 3000 mg

Phase 2 per day. Subjects were instructed to consume a controlled high fiber/low fat diet that provided 100 to 200 g of complex carbohydrate intake per day. Subjects were also instructed to

16 eat the majority of their carbohydrates during lunch and dinner since those were the meals at which the Phase 2 or placebo were taken. The amount of carbohydrate intake was determined for the subjects on the basis of their estimated daily maintenance carbohydrate requirement. Twenty seven subjects completed the study (14 active and 13 placebo). After 8 weeks the active group lost an average of 3.79 lbs. (an average of 0.47 lbs. per week) compared with the placebo group which lost an average of 1.65 lbs. (an average of 0.21 lbs. per week). The difference was not statistically significant with a two tailed p-value of 0.35. Triglyceride levels in the Phase 2 group were reduced by an average of 26.3 mg/dl. This reduction was more than three times the average reduction of 8.2 mg/dl seen in the placebo group (p=0.07).

Several secondary outcomes were measured during the study including body fat percentage, waist and hip circumferences, energy level, hunger, appetite, HbA1c, and total cholesterol. For each of these secondary measures, no clinically or statistically significant differences were identified between the active and the placebo group. No adverse events occurred that were felt to be due to the active product. One placebo subject experienced abdominal pain, bloating and gas while one active group subject complained of an increased incidence of tension headaches. There were no clinically significant changes in biochemical indicators of safety, including serum electrolytes, and markers of kidney and liver function [44].

Weight loss – open studies

An open study was conducted with 10 healthy subjects (5 men and 5 women) with a BMI between 23 and 30 and a body fat ratio of over 25% for men and over 30% for women [41]. The subjects took 3 capsules of Phaseolamin™ 1600 diet twice a day, 30 min before lunch and

dinner, for 8 weeks. The six capsules (1.5 g) contained 750 mg Phase 2, 200 mg clove, 20 mg lysine, 20 mg arginine, 20 mg alanine.

Over the course of 8 weeks, caloric intake decreased from 1742 ± 254 kcal/day to 1525 ± 249 kcal/day (p=0.01) and the subjects lost a significant amount of weight (2.4%; 74.5 ± 7.3 to 72.7 ±

7.8; p=0.002). There were also a significant reduction in body fat (p<0.001) and BMI (p=0.002).

There were reductions in waist and of hip circumferences, without a significant change in the ratio of waist to hip circumference. Over the 8 weeks there were significant reductions in systolic and diastolic blood pressure (p=0.01 and p<0.001). There were also significant reductions in triglycerides (p=0.019) and HDL cholesterol (p=0.001), but not in total cholesterol or LDL cholesterol. There was no change in blood glucose levels and no adverse events were reported

[41].

An open study was conducted with 50 healthy adult subjects who were overweight or obese.

They were given Precarb (Natrol’s Carb Intercept 500 mg capsules) containing

Phaseolamin/Phase2 [42]. The subject took 1 g Precarb (2 capsules, 3 times daily with high carbohydrate meals) for 30 days. A per-protocol analysis was conducted on the 37 to 39 subjects who completed the study. There was a significant reduction in mean body weight of 2.34 ± 2.21 kg (n=37; p<0.001) and a significant reduction in mean waist-to-hip ratio 2.77 ± 2.55 (n=39; p<0.001).

In an open label study 23 adult men and women (BMI 22) took “Super Bows Diet Type B”, a granular food available in Japan that contains 500 mg Phase 2, Coleus forskohlii extract and

18 mushroom chitosan (Plus fort Barrious®) for a period of 8 weeks [43]. Bows Diet Type B was taken as 1 packet of powder in a glass of water 20 minutes before lunch and dinner. After 8 weeks, the product caused a significant decrease in body weight (0.78 ± 0.20 kg, p<0.01) and percent body fat (1.19 ± 0.37%, p<0.01). There was no change on calorie intake during this period. In 10 subjects who had a BMI over 24 and a total cholesterol over 220 mg/dl, there was a significant decrease in cholesterol after 4 and 8 weeks (25.3 ± 7.1 mg/dl and 11.3 ± 4.0 mg/dl, respectively; both p<0.05). There were temporary gastrointestinal symptoms such as bloating and constipation but these symptoms disappeared following a few days of continuous intake of the product.

Glycemic Index (GI)

Four cross-over clinical studies addressed the potential effect of Phase 2 on post-prandial increases in blood sugar. All four studies indicated that Phase 2 could reduce post-prandial spikes in blood sugar with a suggestion that the effect is dose-related.

In the first study, a placebo-controlled, cross-over study, eleven fasting subjects (men and women aged 21 to 57) were given 4 slices of white bread and 42 g (3 Tbs) of margarine with or without 1500 mg of Phase 2 (the Phase 2 was added to the margarine) [45]. The food contained a total of 610 calories, 60.5 of which came from carbohydrate. The tests were administered a week apart. Absorption and metabolism of carbohydrate was measured as levels of plasma glucose over time. In comparison to control, the glucose levels following consumption of Phase 2 returned to baseline 20 minutes earlier. The area under the plasma glucose vs. time curve was

66% lower with Phase 2 compared to the control (p<0.05). The authors concluded that this

indicated that 1/3rd of the carbohydrate in the bread was absorbed. However, actual absorption and subsequent excretion was not measured.

The second study, published in the same paper, was also a placebo-controlled, cross-over study.

Seven subjects (men and women 23 to 43 years old) were given a frozen dinner containing country fried steak, mash potatoes, green beans and cherry-apple pie (630 calories with 64 g carbohydrate) with and without 750 mg Phase 2. In this study, the Phase 2 was mixed with the gravy. The effect of Phase 2 was to reduce the average plasma glucose vs. time curve by 28% and the authors concluded that 2/3rds of the carbohydrate in the meal was absorbed. The authors noted that there appeared to be a dose-related effect with the 1500 mg dose of Phase 2 being twice as effective as the 750 mg dose [45].

A 6-arm crossover study was conducted with 13 randomized subjects (BMI 18-25) to determine whether the addition of Phase 2 would lower the GI of a commercially available high glycemic food (white bread) [46]. Standardized GI testing was performed using capillary blood glucose measurements following ingestions of white bread with butter, with and without the addition of

Phase 2 in capsule or powder form. In both formulations, Phase 2 was given in dosages of 1500 mg, 2000 mg, and 3000 mg. The powdered form was mixed with the butter. Statistical analysis was performed by one-way ANOVA of all seven treatment groups using unadjusted multiple comparisons (t tests) to the white bread control. For the capsule formulation, the 1500 mg dose had no effect on the GI and the 2000 mg and 3000 mg capsule doses caused insignificant reductions in GI. For the powder, the 1500 mg and 2000 mg doses caused insignificant

20 reductions in the GI, while the 3000 mg dose caused a significant reduction in post-prandial glucose levels (a reduction of 34.11%, p=0.023).

A single dose double-blind cross-over test was conducted on the effects of Super Bows Diet

Type B on blood sugar levels [43]. As previously stated, this product is a granular food available in Japan that contains 500 mg Phase 2, Coleus forskohlii extract and mushroom chitosan. The experiment included 13 men and women with a fasting blood glucose level above 126 mg/dl. In two test periods 1 week apart, the subjects took a packet of product or placebo along with a glass of water 5 minutes before eating 300 g polished rice. Blood samples were taken before the intake of the rice and 30, 60, 90 and 120 minutes afterward. Blood sugar levels 30 minutes after eating the rice were significantly lower with the test product (p<0.01). Plasma insulin levels were significantly lower compared to the control at 30 and 60 minutes after consuming the rice

(p<0.01).

Safety

In the human clinical studies reviewed above there were no reports of serious side effects resulting from ingestion of white bean extracts. Clinical efficacy studies using doses of Phase 2 up to 3000 mg per day in divided doses for periods of 30 days to 24 weeks also reported no significant adverse events. An acute animal toxicity study was conducted in rats with Phase 2 at doses of 500 to 5000 mg/kg body weight along with a subchronic study of 90 days with doses of

200 to 1000 mg/kg. In response, there were no adverse reactions and signs of toxicity in biochemical and histopathological analysis [47]. A 28-day toxicity study conducted with male and female rats reported a no-adverse-effect level (NOAEL) of 2500 mg/kg/day [48]. Cantox

Health Sciences International conducted a safety review of published and unpublished data on

Phase 2 and the panel of experts concluded that it could be safety consumed at doses up to 10 g per day [31].

Since alpha-amylase inhibitors prevent the degradation of complex carbohydrates into oligosaccharies, those carbohydrates will pass through the intestine into the colon. In the colon, bacteria will digest the complex carbohydrates, and this may initially cause gastrointestinal side- effects such as flatulence and diarrhea. In the study conducted with Super Bows Diet Type B which contained Phase 2 along with other ingredients, there were temporary gastrointestinal symptoms including bloating and constipation but these symptoms resolved with continued intake of the product [43].

Raw beans contain phytohaemagglutinin (PHA) at high levels which have been associated with toxic effects in animals and severe gastrointestinal disturbances in humans [3]. However, PHA levels in beans are drastically reduced by cooking. In addition, white beans have negligible amounts of PHA compared to colored beans. Phase 2 is a standardized white bean extract prepared using a specialized process which substantially inactivates haemagglutinating activity

(HA) and trypsin inhibiting activity (TIA). The finished product contains less than 700 HA units per g and less than 20 TIA units per mg dry weight [31].

Product equivalency

This review is focused on the development and clinical research of a proprietary product, Phase 2

Carb Controller (Pharmachem Laboratories, Kearny, NJ). We felt it was important to focus on

22 this product as there is no evidence that carbohydrate blockers are equivalent. Early studies on the alpha-amylase inhibitor from white bean indicated that enzyme stabilization through specific manufacturing processes was key to an active product. The challenge of establishing biological equivalency for protein biopharmaceuticals has been highlighted in recent publications. The complexity of these molecules, and their production in living cells, makes the final product sensitive to changes in manufacturing conditions. Because of this, the European Medicine

Agency has introduced a new regulatory pathway for biosimilars (also known as follow-on biologics) which mandates clinical trials in order to show therapeutic equivalence [49]. In the

US, companies are expected to use the approval process for new branded drugs [50]. While the

Phase 2 product is a dietary supplement, and not a pharmaceutical, we believe similar principals apply.

Conclusions

Experiments conducted with the Phase 2 alpha-amylase inhibitor indicate that it reduces the rate of absorption of carbohydrates, thereby reducing the GI of foods. The evidence also indicates that Phase 2 promotes weight loss when taken concurrently with meals containing carbohydrates.

The importance of reducing the GI of foods in weight management and type 2 diabetic control is indicated by an emerging body of evidence. Reducing the post-prandial spikes of glucose and insulin following a high GI meal may also reduce the risks of developing insulin resistance, which can lead to cardiovascular disease.

Competing Interests

Medicus Research has received research support grants from Pharmachem Laboratories. JKU has provided consulting services to Pharmachem Laboratories. MLB has provided consulting services to Medicus Research. The authors and Medicus Research do not endorse any brand or product.

Authors’ Contributions

MLB and JKU were both involved in writing this review. They both read and approved the final manuscript.

Acknowledgements

The authors would like to acknowledge Pharmachem Laboratories for sponsoring this review.

24

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32 Table 1: Phase 2 Clinical Data

Reference Study Subjects Purpose Preparation/ Dose Main Results

(Author, Design /

date) Duration

Thom, 2000 RPCT, 12 n=40 (BMI weight 400 mg Phaseolamin® body weight, BMI &

[39] weeks 28- 39) loss 3X after meals, total %body fat in active group

1200 mg/day (other (p<0.05), no effect in

ingred. inulin & placebo group; no between

Garcinia extract) group analysis

Erner, 2003 RPCT, 12 n=54 (BMI weight Thera-Slim: 1000 mg trend toward body

[40] weeks, then 24-36) loss Phase2 before 2 meals, weight, 3X decrease in

open 12 total 2000 mg/day triglycerides; no between

weeks group analysis

Rothacker, RPCT, 12 n=88 (BMI weight StarchAway chews: 1000 body weight comparison

2003 weeks 24-32) loss mg Phase2 before 3 to placebo (p<0.05)

[36] meals, total 3000 mg/day

Udani, 2004 RPCT, 8 n=39 (BMI weight Phase 2 1500 mg 2X, body weight comparison

[44] weeks 30-43) loss 3000 mg/day to placebo (ns)

trigylcerides (ns)

Koike, 2005 Open, 8 n=10 (BMI weight 3 capsules Phaseolamin body weight (p=0.002),

[41] weeks 23-30) loss 1600 diet 2X daily; 750 calorie intake, BMI,

mg Phase 2 daily triglycerides & HDL (all

p<0.05)

Osorio, Open, 30 n=39 Weight PreCarb capsules: 1000 body weight &

2005 days (overweight loss mg Phase3 with meals, waist to hip ratio over

[42] & obese) total 3000 mg/day time (both p<0.001)

Celleno, RPCT, 30 n=60 (BMI Weight Phase 2 + chromium; body weight (p<0.001),

2007 days avg 26) loss 445 mg extract daily BMI, body fat (both

[35] p<0.01)

Vinson, PCT, X- Part 1: n=11, Plasma Phase 2 mixed with AUC post prandial blood

2009 over, single Part 2: n=7 glucose margarine or gravy. 750 glucose; higher dose

[45] dose or 1500 mg. (p<0.05).

Udani, 2009 RPC, X- n=13 (BMI Plasma Phase 2 capsules or AUC post prandial blood

[46] over, single 18-25) glucose mixed w/butter. 1500, glucose; 3000 mg w/butter

dose 2000, 3000 mg (p<0.05).

Wu, 2010 RPCT, 60 n=101 (BMI Weight Phase 2; 1,000 mg 3X body weight, waist

[37] days 25-40) loss daily circumference (both

p<0.01)

DB = double-blind, PCT = placebo-controlled trial, RPCT = randomized placebo-controlled trial, X-over = crossover

34

Hi-maize Resistant starch report by Kanak Udani, Ph.D. June 15, 2011

Review and analysis of Hi-maize resistant starch product from National Starch Company and evaluation of potential benefits for Phase 2 products applications

Introduction:

National Starch company (Now part of Corn Products) has been promoting their resistant corn starch product Hi-maize resistant starch as a dietary fiber ingredient for incorporating in baked goods, pasta products and for incorporation in wheat flour for general uses. In March 2010, the European Food Safety Authority (EFSA) issued an opinion on dietary fiber. In this opinion, resistant starch is included in their definition of what constitutes a dietary fiber defined as non- digestible carbohydrates. In April 2011, EFSA issued an opinion on claims related to resistant starch and reduction of post-prandial glycemic responses and concluded that resistant starches reduces post-prandial responses when it replaces digestible starches in baked goods. To bear this claim, high carbohydrate baked goods should contain at least 14% of total starch in replacement to digestible starch. The claimed effect is “digestive health benefits” and “favors a normal colon metabolism”.

The purpose of this report is to review Hi-maize resistant starch product, its properties, uses, benefits and limitations. The report also compares them with Phase 2 and identify opportunities for Phase 2.

General background:

Starch is a carbohydrate produced by green plants as a energy source and it consists of two molecules: the linear molecule Amylose (20-25%) and the branched molecule Amylopectin (75- 80%). Amylose is more resistant to digestion than amylopectin and is therefore an important form of resistant starch.

Resistant starch is defined as the amount of starch and the products of starch degradation that resists digestion in the small intestine of healthy people. Resistant starch is considered a functional fiber.

Resistant starches are classified as type 1 – 4 according to their physical and chemical characteristics. Only type 1, 2 and 3 resistant starches are naturally present in foods. Hi-maize resistant starch is made from fractionation of high amylose corn and it is classified as type RS2 – natural granular starch (no chemical modification).

Foods containing resistant starches have been consumed for thousands of years and this resistant starch has recently been rediscovered as a beneficial ingredient. As the foods have become more processed and packaged for convenience, the amount of resistant starch in our diets has decreased. Individuals in developed countries such as U.S. Consume about 5 grams of resistant starch per day. In less developed countries, individuals consume 15-20 grams of resistant starch in their diet – the amount recommended by health experts for full physiological benefits.

Resistant starch is naturally found in common foods such as legumes beans (including white bean - source of Phase 2), peas, whole grains, potatoes and bananas. For years, health professionals have recommended increased use of complex carbohydrates (whole grains, legumes, fruits etc.) in the diet. Resistant starch is part of complex carbohydrates.

Since Phase 2 delays the digestion and absorption of carbohydrates, reduces caloric impact of starchy foods and lowers glycemic index, a comparison with Hi-maize resistant starch would be useful in identifying new potential opportunities to Pharmachem in expanding their Phase 2 business.

Hi-maize 260 resistant starch product data:

High-maize 260 resistant starch is made from high amylase corn Starch, a natural food source that resists digestion in the Small intestine.

Label: Resistant Corn starch (dietary fiber)

FDA status: GRAS

GMO-free

Reduces calories when it is used to substitute digestible Carbohydrates.

Reduces glycemic response when substituted for digestible Carbohydrates.

Increases insulin sensitivity

Acts as fiber. Total fiber 60% min (DSB)

Recommended for use in baked goods, past and snacks

Color: white/off-white

Taste: Bland

Particle size: 10 – 15 microns

Moisture: 10 – 14%

Phase 2 product data:

Phase 2 is a white bean extract (Phaseolus Vulgaris) that delays Digestion and absorption of carbohydrates

Label: white bean extract

FDA status: GRAS

GMO – free

Assists in weight control with sensible diet

Reduces glycemic index when taken with carbohydrates

Recommended for baked goods, pasta, pizza and snack products

Color : white to beige

Taste: Bland

Mesh: 100% through US 60 mesh

Moisture: <10%

Hi-maize in food applications:

The main nutritional benefits claimed by use of Hi-maize resistant starch when used to replace digestible carbohydrates include: Reduced calories Reduced glycemic response Increased insulin sensitivity Increased satiety – post meal and 24 hours Prebiotic fiber Contributing to regularity

The Hi-maize resistant starch is being recommended for use in baked goods, pasta and snacks to replace flour in the formulations primarily to reduce the digestible starch portion in the flour. Recommended use levels are from 5 to 20% of dry mix.

Phase 2 and Hi-maize – Prs and Cons.:

Product formulations and processing considerations:

Incorporating Hi-maize in in existing baked goods would be challenging. Replacing up to 20% flour in breads with Hi-maize could significantly affect organoleptic attributes. Especially, a high level of resistant starch could produce a dry sensation in mouth. Additional water may be required in the formulation to counter the dryness. In contrast, incorporation of 1 – 2 % Phase 2 in baked goods requires minimum adjustment in the formulations. In terms of preparation procedures, use of both Hi-maize and Phase 2 would require adjustments.

Phase 2 – Opportunities:

Hi-maize is being positioned as an alternate to other fiber products such as wheat fiber, oat fiber and inulin to reduce the digestible starches in baked goods. These other fiber products do not have benefits of weight, glycemic and energy managements and as such it is not competing against Phase 2. In many aspects, Hi-maize and Phase 2 provide similar benefits:

Both reduce absorption of digestible starches present in foods consumed. Both lower glycemic index. Both help weight control – with sensible diet and exercise. Both have prebiotic benefits as undigested starches ferment in large intestine. Thus indirectly, Phase 2 functions as Hi-maize which is classified as a fiber. Both are well tolerated.

The greatest advantage Phase 2 may have over Hi-maize would be the ease of incorporating Phase 2 in baked foods, pastas, pizzas and snack products. Phase 2 incorporation would not require major reformulations of the products and would not affect sensory attributes such as dryness in mouthfeel.

While Hi-maize replaces part of digestible starch from the food product, a large portion of digestible starch still remains in the product. Inclusion of Phase 2 in these products could further reduce the amount of digestible starch that would delay the digestion and absorption of digestible starches.

Cost considerations:

The estimated price of Hi-maize is $125/100wt. Replacing up to 20% flour in the Baked goods formulations would be considerable requiring higher premium for the products. Generally, flour constitutes about 2/3 of the ingredients in most baked goods. The estimated price is $20-25/100wt.

Recommendations:

In order to establish potential benefits and a competitive advantage of Phase 2 over Hi-maize resistant starch, two approaches are recommended. Results of these studies could provide a marketing edge to Pharmachem.

Clinical evaluation:

To determine the relative benefits of Phase 2 and Hi-maize, food products containing Phase 2 and Hi-maize should be evaluated individually for the reduction in glycemic index in a condensed clinical study.

In addition, another study on the reduction in glycemic index with products containing both Phase 2 and Hi-maize together would determine if their impact on glycemic index is cumulative or synergistic.

Product evaluation:

Identical food products containing Phase 2 and Hi-maize for should be tested in sensory evaluation for their relative consumer acceptance. Suggested food products are wheat bread and cheese pizza.

Depending upon the outcome of clinical studies recommended above, sensory evaluation of food products with both Phase 2 and Hi-maize should also be evaluated.

Summary:

Hi-maize is being marketed as a dietary fiber. It claims benefits of fiber as well as reduction in glycemic response. While Phase 2 is not a fiber source, its benefits are similar to those of Hi-maize.

Incorporating Hi-maize in food products requires major reformulation and sensory properties, particularly taste are expected to be affected.

Cost of Hi-maize to reduce digestible starch in foods could be an important factor.

Since health concerns for healthy living and obesity are major public issues, products such as Phase 2 and Hi-maize that could provide nutritional benefits would be perceived as useful products and would provide business opportunities.

Studies and Applications Data for Phase 2 Carb Controller/Starchlite

For Trade Distribution, only.

Phase 2 Carb Controller® incorporation into baked goods was researched in a joint effort with the Geffen Center for Human Nutrition at UCLA.

Amylase enzyme content of flours must be considered, as it will affect Phase 2 Carb Controller® performance in the product (showing that it works).

Extensive tests have been conducted to check the effects of standard processing, such as homogenization, UHT treatment, pasteurization, sterilization, baking, extrusion, molding, fermentation and acidification. Alpha-amylase inhibition remains unaffected (See Figure 1 and Figure 2).

Figure 1. Inhibition of Alpha-Amylase Activity by Phase 2 Carb Controller® in Mashed Potatoes

Activity (Optical Density) (Optical Activity

Amylase - Alpha

Inhibition of Inhibition Samples

Control No Phase 2 Carb Controller® - No mashed potatoes P2 Post Prep Phase 2 Carb Controller® added to mashed potatoes post preparation MP Mashed potatoes - No Phase 2 Carb Controller® P2 Prep Mashed potatoes prepared with Phase 2 Carb Controller®

Figure 2. Inhibition of salivary alpha-amylase activity by Phase 2 Carb Controller® in Chewing Gum

3 Tests Each: 1. Uninhibited Salivary Alpha-Amylase 2. Control Chewing Gum

Activity (Optical Density) Activity (Optical 3. Phase 2 Carb Controller®

Chewing Gum Amylase - Alpha

Inhibition of

Phase 2 Carb Controller® in Liquid Products Phase 2 Carb Controller® can simply be mixed with liquids (e.g. milk) just before processing, while retaining alpha-amylase inhibiting activity (see Figure 3 and 4).

Figure 3. Inhibition of Alpha-Amylase Activity by Phase 2 Carb Controller® in a Pasteurized Orange Drink

Activity (Ex 485/Em (Ex 538 Activity

Amylase - Alpha

Enzyme Control reaction without any inhibitor Control Untreated drink (No Phase 2 Carb Controller®) P2 Pre Pre-treated drink (Phase 2 Carb Controller® added during processing/pasteurization @ 100 mg/24 mL) P2 Post Untreated drink with added Phase 2 Carb Controller® in the laboratory for assay purpose

Figure 4. Alpha-amylase activity in untreated and Phase 2 Carb Controller® treated pasteurized milk

Activity

Relative

Untreated Milk P2 Treated Milk

Stability and Shelf Life:

pH Stability Phase 2 Carb Controller® shows very good stability for acid pH, so it is fully suitable for applications such as acidic drinks. At very basic pH, the activity decreases, probably because of a lower solubility of the powder.

Thermal Stability Phase 2 Carb Controller® shows good stability from 20°C to 120°C. Activity decreases with long-time heat treatment, but retains over 40% inhibition of alpha-amylase. Therefore, Phase 2 Carb Controller® is fully suitable for industrial processes that use heat treatment over 70°C, such as pasteurization or sterilization.

Microwave Stability Phase 2 Carb Controller® was treated with a microwave oven (500 W) for 1-5 minutes. Alpha- amylase inhibitory activity was not decreased by microwave treatment.

Source Material The source material used for Phase 2 Carb Controller® (Phaseolus vulgaris) is 100% grown in the U.S.A. and supplied by Archer Daniels Midland Company. The extract is manufactured under (c)GMP conditions in a Pharmachem Laboratories, Inc. owned and operated U.S. facility. There is full traceability from farm to manufacture.

Regulatory Aspects • Self-affirmed GRAS (Generally Recognized as Safe) for use as an ingredient in foods and bever- ages, thanks to the assistance of CANTOX. • Non-GMO • Prop 65 compliant • Solvent-free

Phase 2 Carb Controller® Outdoes the Competition • Very low, efficacious dose • 18 clinical and safety studies, and clinical citations support claims • Applications range from supplements, to foods, to unique food applications (such as seasonings) • Source material is fully traceable, non-GMO, and grown in the U.S.A. • More versatile for food applications than resistant starch • New forms being developed for improved formulations and applications

“Enjoy a little more, absorb a lot less!”™ with Phase 2 Carb Controller®

StarchLiteStarchLite® stabilitystability andand activityactivity inin freshfresh pastapasta

July 2011 Introduction

TRIALSTRIALS AIMAIM

Aim of this study

To observe the impact of StarchLite® on the quality attributes of fresh pastas (aspect, flavour and taste)

To quantify and validate StarchLite® activity in the finished product

To measure the influence of process on StarchLite® stability.

2 RecipeRecipe

Fresh pasta Control with 2% Raw materials Fresh pasta StarchLite®

Durum wheat semolina 67.03 % 65.03 %

Water (50C) 30.33 % 30.33 %

Milk Protein 2.64% 2.64%

StarchLite® X 2 %

Fresh pastas were made without egg

3 ProcessProcess

Mixing all ingredients

Kneading 10 minutes

Leaving the dough as a ball 30 minutes

Dividing the dough in small parts

Rolling the dough in a pasta machine

Dividing in long and thin parts

Drying the pastas 24h at 55C

4 ResultsResults

Visual appearance: No significant difference between the fresh pastas with StarchLite and the reference (no browning, no discoloration).

Physical stability: No significant difference between the fresh pastas with StarchLite and the reference (no hardening).

Sensory properties (triangle test): No significant difference between the fresh pastas with StarchLite and the reference.

5 AnalyticalAnalytical methodmethod

Extraction method : Starchlite® decreases the Glycemic Index by inhibiting alpha- amylase, an enzyme present in saliva and also produced by the pancreas. Extraction method :

Control : Fresh pastas Test : Fresh pastas without StarchLite with StarchLite

100g of Test 100g of control + 100g of control + product + phosphate buffer phosphate buffer phosphate buffer up to 100mL up to 100mL + 4g StarchLite up to 100 mL

Mixing by vortex

Centrifugation (9000rpm / 20C / 30 min)

Supernatant

Centrifugation (9000rpm / 1 min) 6 EnzymaticEnzymatic inhibitioninhibition method

Principle:

Measurement of alpha-amylase enzymatic activity during the hydrolysis of its specific substrate (Amylose, Sigma A0512)in presence of Control extract (to observe matrix effect), by absorbance analysis Measurement of alpha-amylase enzymatic activity when StarchLite® is added. Comparison of both results to express alpha-amylase inhibitory activity of StarchLite®.

Products:

Pasta Control without StarchLite Pasta with 2% of StarchLite StarchLite in powder U08502

Sample preparation:

Pasta control without StarchLite:  100 g of product are mixed and dissolved in 100mL of Buffer phosphate 20mM pH 6.9.  Mixing and centrifuged at 9 000 rpm 20C during 30min.  Taking the supernatant and before analysis do a new spin 9 000rpm 1min.  Analysis this final solution.

Pasta control + StarchLite in Powder (U08502) :  100g of product are mixed and dissolved in 100mL of Buffer phosphate 20mM pH 6.9  Addition of 4g of StarchLite® U08502 (in order to get approx 20mg of StarchLite®/mL of Buffer Phosphate 20mM pH 6.9)  Mixing and centrifuged at 9 000rpm 20C during 30min.  Taking the supernatant and before analysis do a new spin 9 000rpm 1min.  Analysis this final solution

Pasta sample with 2% of StarchLite :  100g of product are mixed and dissolved in 100mL of Buffer phosphate 20mM pH6.9 (on the basis that the product contains about 2% of StarchLite®).  Mixing and centrifuged at 9 000rpm 20C during 30min.  Taking the supernatant and before analysis do a new spin 9 000rpm 1min.  Analysis of the final solution.

Analytical method:

Enzymatic Method (M.RD.AA.012) – 450nm Used of 0.1mL of each supernatant

7 EnzymaticEnzymatic inhibitioninhibition Results

Determination of Inhibition rate after extraction

Inhibition Recovery Rate (%)

Pasta control + Starch Lite U08502 100% Pasta sample (2% of Starch Lite) 99%

 Enzymatic activity was determined from the analysis of Pasta control in order to overcome the matrix effect.

 An inhibitory activity was determined in Pasta control + StarchLite (Comparison between enzymatic activity in Pasta control and enzymatic activity in Pasta control when StarchLite is added) to obtain a reference.

 The recovery rate is obtained by comparing Inhibitory activity in Pasta control + StarchLite and inhibitory activity in Pasta extract sample.

Evidences

Compared to Pasta Control + Starchlite Powder extract:

 99% of the inhibitory activity normally find in Pasta samples have been dosed.

 Very few part of StarchLite and his activity must have been lost during sample extraction.

8 ConclusionsConclusions

The addition of StarchLite® has no significant impact on the quality attributes of fresh pastas.

The dosage of StarchLite® in pastas samples with the enzymatic method showed a recovery rate of 99%.

With an addition of 2% StarchLite® in pastas, the alpha-amylase inhibitor activity is totally preserved.

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9 ISSI LABORATORIES, INC. 515 BLUE RIDGE AVENUE PISCATAWAY, NEW JERSEY 08854-5013 Telephone: 732-246-3930 (Voice), 732-247-4977 (Facsimile) Email:

CONFIDENTIAL

April 12, 2007

Mr. Mitch Skop Pharmachem Laboratories, Inc. 265 Harrison Avenue Kearny, NJ 07032 201-246-1000 (Fax 8105)

Dear Mr. Skop:

Phase2®/StarchLite™

This is to report to you on the analytical results obtained at our laboratory on Phase2®/StarchLite™.

Introduction

Ingested starch (amylose and amylopectin) is hydrolyzed into simple sugars (such as glucose, maltose and maltotriose) by an enzyme (-amylase), resulting in increased availability of the monosaccharides. If this process can be minimized, it would limit the availability of sugars in the blood stream. A glycoprotein from white kidney bean was known to have some inhibitory effect on -amylase (up to 50% inhibition). In human subjects, the glycemic index was considerably reduced upon ingestion of white kidney bean powder. It would, therefore, be a beneficial situation if the white kidney bean powder is incorporated into certain regular food items, especially to the benefit of obese and diabetic individuals.

Analytical Problem

When the white kidney bean powder (Phase2®/StarchLite™) was incorporated into a prepared food, ingestion of it yielded the desired glycemic effect in vivo. However, the available laboratory assay techniques were unable to detect the expected inhibitory effect in vitro. This apparent anomaly had to be resolved, in order to unequivocally establish the inhibitory activity of Phase2®/StarchLite™ in prepared foods.

Objective of Investigation

The objective of the proposed investigation is to determine the fate of Phase2®/StarchLite™ in the processed foods and to develop a method for monitoring its inhibitory activity. At the outset, the investigation rested on the hypothesis that (1) Phase2®/StarchLite™ was active in the prepared food, but was inaccessible for measurement in vitro; (2) the source of a-Amylase was not that of human; and (3) the assay reaction mixture was inappropriate for the enzyme-inhibitor complex.

Page 1 of 7

Metabolism -- Biochemistry -- Analytical Chemistry Research -- Testing -- Consultation ISSI No. P25036 Pharmachem / StarchLite® Page 2

Materials and Methods

Equipment and Materials

1. Stove, domestic style (natural gas) 2. Cooking pan, stainless steel (4 quart size) 3. Mixing bowl, stainless steel (4 quart size) 4. Fork, stainless steel, domestic 5. Balance (Scale), capable of 100 g at 0.01 g increments 6. Water (city tap) 7. Butter, unsweetened, unsalted 8. Reagent grade water 9. Mashed Potatoes (Potato Buds®, Betty Crocker® Brand by General Mills) 10. Phase2®/StarchLite™ 11. Plastic storage bags 12. Refrigerator (~5 oC) 13. Microcentrifuge (Centronics Model C0240; Spintron Inc., Metuchen, NJ or equivalent unit) 14. Microcentrifuge plastic tubes for above, 1.5 mL capacity 15. Refrigerated Centrifuge (2000 rpm at 10 oC) 16. 50-mL Capacity plastic centrifuge tubes for above 17. Multiwell plate (96-well) 18. Pipettes (assorted) 19. -Amylase, human salivary (from volunteers at our laboratory) 20. Substrate (2-Chloro-4-nitrophenyl--D-maltotrioside; C24H34ClNO18, FW 660) 21. Phosphate Buffered Saline, pH 7.4, 10 mM. 22. Microplate Reader (Bio-Rad Model 680 or equivalent unit) 23. Hot surface (~30 oC) 24. Multi-channel pipette, adjustable volume

Preparation of Instant Mashed Potatoes with Phase2®/StarchLite™ (“MP”)

Pharmachem’s Stove-Top Method:

Water-- 300 mL (73%) Butter-- 27.12 g (6.6%) Potato Buds-- 71.51 g (17.4%) StarchLite® -- 12.33 g (3.0%)

“Premix Potato Buds and Phase2®/StarchLite™ until Phase2®/StarchLite™ is completely blended into the mixture. Combine water and butter in a pot. Heat until boiling. Remove pot from heat. Stir in Potato Buds/ StarchLite® mixture until moistened. Let stand for approximately 2 minutes or until liquid is absorbed and whip up with a fork.”

A control preparation without Phase2®/StarchLite™ was prepared in a similar manner.

The preparations was allowed to cool at room temperature for about an hour, then transferred into plastic bags and kept overnight at ca. 5 oC in a refrigerator.

Extraction of -Amylase Inhibitor of Phase2®/StarchLite™ from Instant Mashed Potatoes (MP)

Water-soluble constituents in MP were extracted with Phosphate Buffered Saline (PBS) by sonication, shaking and centrifugation, as follows:

1. Weigh out 10 g of Instant Mashed Potatoes with Phase2®/StarchLite™ (“SMP”) into a 50-mL plastic centrifuge tube. 2. Weigh out 10 g of Instant Mashed Potatoes without Phase2®/StarchLite™ (“CMP”) into a 50-mL plastic centrifuge tube. ISSI No. P25036 Pharmachem / StarchLite® Page 3

3. Weigh out 10 g of Instant Mashed Potatoes without Phase2®/StarchLite™ (“CMP”) into a 50-mL plastic centrifuge tube, and add 306 mg of StarchLite. 4. Weigh out 306 mg of StarchLite into a 50-mL plastic centrifuge tube. 5. To all 4 tubes above, add 10 mL of PBS. 6. Mix with a Votex® mixer for 30 sec. 7. Sonicate in an ultrasonic water bath for 5 min. 8. Centrifuge for 1 hr at 2,000 rpm and 10 oC. 9. Pipette out aliquots of the supernatant from each tube into 4 microcentrifuge tubes. 10. Centrifuge with the microcentrifuge for 10 min at room temperature. 11. Use the clear supernatant for assays.

Assay Method

The assay method is based on the principle that the hydrolysis of 2-Chloro-4-nitrophenyl--D- maltotrioside, catalyzed by -Amylase, yields 2-Chloro-4-nirophenol that is quantitatively measured by its absorbance at 415 nm. Its formation is directly proportional to the -Amylase activity. Hitherto, the measurements were mostly made with spectrophotometers that usually require milliliter volumes of reaction mixture. However, with the advent of multi-well plate reader technology, it is now more convenient, efficient, and economical to conduct these tests, up to 96 reactions at a time.

In the present study, assays were carried out with a total volume of 150 L of reaction mixture per well, as follows:

1. -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1); equivalent of 10 L human saliva 2. 100 L of sample extract (100 L PBS for control) (-Amylase inhibitor was equivalent of 3 mg of Phase2®/StarchLite™) 3. Incubate for the assigned time over a warm plate (~30 oC), covering the multiwell plate loosely with a plastic lid 4. Add 40 L of the substrate (2-Chloro-4-nitrophenyl--D-maltotrioside) solution 5. Measure the Optical Density at 415 nm. If time course values are planned, continue measurements at the selected time intervals.

Results and Discussion

The following 5 reaction conditions were assayed in 2 separate assays, and in duplicate within each assay:

1. Control-- No Phase2®/StarchLite™ and Mashed Potatoes. This is a reaction that would provide the extent of enzymatic hydrolysis of the substrate, free of any influence by the ingredients in the food preparation. 2. SL-- Phase2®/StarchLite™ Only (No Mashed Potatoes). This is a reaction that would provide the extent of enzymatic hydrolysis of the substrate after the enzyme was exposed to Phase2®/StarchLite™, but free of any influence by the ingredients in the food preparation. 3. SL+UT-- Phase2®/StarchLite™ added to Mashed Potatoes prepared without Phase2®/StarchLite™. This is a reaction that would provide the extent of enzymatic hydrolysis of the substrate after the enzyme was exposed to (“uncooked”) Phase2®/StarchLite™, but not free of any influence by the other ingredients in the food preparation. (This is a post-preparation addition of Phase2®/StarchLite™.) 4. UT-- Mashed Potatoes Prepared Without Phase2®/StarchLite™. This is a reaction that would provide the extent of enzymatic hydrolysis of the substrate in the total absence of Phase2®/StarchLite™, but not free of any influence by the ingredients in the food preparation. ISSI No. P25036 Pharmachem / StarchLite® Page 4

5. T-- Mashed Potatoes Prepared With Phase2®/StarchLite™. This is a reaction that would provide the extent of enzymatic hydrolysis of the substrate after the enzyme was exposed to Phase2®/StarchLite™, but not free of any influence by the other ingredients in the food preparation.

The observed Optical density values, which are directly related to the -Amylase activity, are summarized in the following Figure 1.

Optical Density (415 nm) Sample Assay 1 Assay 2 Mean

Control 2.033 2.244 2.139 SL 0.088 0.096 0.092 SL+UT 0.161 0.149 0.155 UT 2.094 2.694 2.394 T0.1520.1600.156

StarchLite Assays

3

2.5

2

1.5

1 Optical Density (OD)

0.5

0 Control SL SL+UT UT T Sample

Figure 1. Inhibition of -Amylase Activity by Phase2®/StarchLite™ in Instant Mashed Potatoes.

Control = No Phase2®/StarchLite™ - No Mashed Potatoes SL = Phase2®/StarchLite™ Only-No Mashed Potatoes SL+UT = Phase2®/StarchLite™ Added to Mashed Potatoes Prepared Without Phase2®/StarchLite™ UT = Mashed Potatoes Prepared Without Phase2®/StarchLite™ T = Mashed Potatoes Prepared With Phase2®/StarchLite™

Vertical bars represent Assay 1, Assay 2, and their Mean values, respectively. ISSI No. P25036 Pharmachem / StarchLite® Page 5

Inhibition of -Amylase by Phase2®/StarchLite™ in Instant Mashed Potatoes (MP)

By a direct comparison of the -Amylase activity between the Instant Mashed Potatoes prepared with and without Phase2®/StarchLite™ (samples T and UT, respectively), the extent of inhibition was calculated as:

100–([0.156/2.394] x 100) = 100–(0.06516 x 100) = 100–6.516 = 93.5%

Interference of Other Ingredients in Instant Mashed Potatoes (MP)

There was a slight increase in the -Amylase activity when the reaction mixture contained MP (see SL 0.92 versus SL+UT 0.155 or T 0.156). It appears that the potato starch was responsible for this situation. Since potato starch is a substrate by itself, it is not surprising that it could add to the overall reaction velocity, as could be expected under the first order kinetics. Nonetheless, the observed effect was negligible and, with the employment of appropriate control, had no negative impact on the assay outcome.

Effect of Incubation Time on Inhibition

An independent assay was carried out to determine the optimum time of exposure (incubation) to achieve a desired level of inhibition. Incubation times of 0, 30, 60 and 120 min were studied. Following the incubation, the substrate was added and activity read (OD415). Following the initial reading, the readings were continued through 2 min, 5 min and 10 min.

The results of these observations are summarized below in Figure 2:

Incubation Optical Density Reading Time (min) Time (min) Initial 2 min 5 min 10 min 00.8901.4351.8442.011 30 0.184 0.195 0.217 0.247 60 0.173 0.175 0.184 0.199 120 0.164 0.164 0.170 0.184 ISSI No. P25036 Pharmachem / StarchLite® Page 6

StarchLite Incubation Time (min)

2.500

2.000

1. 5 0 0 Init ial 2 min 5 min 10 m i n 1. 0 0 0 Optical Density (OD) Density Optical

0.500

0.000 03060120 Incubation Time (min)

Figure 2. Effect of Time of Incubation on -Amylase Activity by Phase2®/StarchLite™ in Instant Mashed Potatoes.

Vertical bars at each Incubation Time represent 4 readings taken initially, and after 2 min, 5 min and 10 min, respectively.

As could be seen above, most of the inhibition (nearly 88%) occurred by 30-min incubation. Additional incubations (up to 2 hours) did not contribute significantly. It appears, therefore, that a 30-min incubation would be adequate, although an hour of incubation was employed in present study.

With regards to the time-course reaction of the substrate hydrolysis, the readings taken at the 4 time intervals showed a clear linear rate, from 0.890 to 2.011 over the 10-min period in the uninhibited reaction (see 0-min incubation data above). For comparative purposes, it appears adequate to make readings at 5 min following the addition of substrate. In the present study, readings were taken at 4 min for routine evaluation of the activity levels.

It is also interesting to note that the reading time intervals did not matter in case of inhibited reactions (see 30-min, 60-min and 120-min incubation data above). This situation suggests that the substrate per se had no influence on the integrity of the enzyme-inhibitor complex. ISSI No. P25036 Pharmachem / StarchLite® Page 7

Conclusions

1. The -Amylase of human saliva was significantly inhibited by the inhibitor in Phase2®/StarchLite™. 2. The inhibitor in Phase2®/StarchLite™ was unaffected during the preparation of Instant Mashed Potatoes. 3. Exposure of -Amylase to Phase2®/StarchLite™ for 30 min resulted in significant inhibition. 4. The assay method has been adapted for a multiwell plate reader.

Respectfully Submitted

Yesu T. Das, Ph.D. ISSI Laboratories, Inc. ISSI LABORATORIES, INC. 515 BLUE RIDGE AVENUE PISCATAWAY, NEW JERSEY 08854-5013 Telephone: 732-246-3930 (Voice), 732-247-4977 (Facsimile) Email:

CONFIDENTIAL

July 19, 2007

Mr. Gregory Drew Pharmachem Laboratories, Inc. 265 Harrison Avenue Kearny, NJ 07032 201-246-1000 (Fax 8105)

Dear Mr. Drew:

Phase2®/StarchLite™ in Chewing Gum

This has reference to your samples of chewing gum that were processed with- and without Phase2®/StarchLite™.

Objective of Investigation

The objective of the analytical efforts is to assess the efficacy of Phase2®/StarchLite™ in the gum towards inhibiting the human salivary -Amylase.

When Phase2®/StarchLite™ was incorporated into the chewing gum, there appears to be an intimate physical association of it with the gum components. This situation prevented the measurement of inhibitory activity, since Phase2®/StarchLite™ was not present as an aqueous solution. It was, therefore, necessary to first release Phase2®/StarchLite™ from the gum before an assay could be conducted. The following were the practical issued to be resolved:

(1) The assay methodology requires that all reagents must be in aqueous solutions. (2) The chewing gum per se is insoluble in water, although there may be some minor constituents that may be water-soluble. (3) Phase2®/StarchLite™ is soluble in water, but only after it is released from the gum. (4) Blending or squeezing in aqueous medium did not release Phase2®/StarchLite™ from the gum.

Experimental Strategy

It became evident that a valid assay for Phase2®/StarchLite™ could not be conducted without releasing it from the gum.

The following aspects were, therefore, included in the present investigation.

Page 1 of 4

Metabolism -- Biochemistry -- Analytical Chemistry Research -- Testing -- Consultation ISSI No. P25036-B Pharmachem / StarchLite® Page 2

(1) Apply a suitable method to “break up” the gum. (2) Extract Phase2®/StarchLite™ into an aqueous buffer. (3) Assay the aqueous buffer extract for the -Amylase inhibition.

Materials and Methods

Sample Preparation

The Chewing Gum had an average weight of 1 g/piece. Using a kitchen cheese grater, the pieces were individually grated into particles of ca. 1-3 mm.

Extraction Method

1. Place 1 g of chewing gum particles into a 50-mL capacity plastic vial (centrifuge tube). 2. Add ca. 10 g of clean sand (ca. 1-mm size). 3. Add 10 stainless steel balls (4-mm diameter). 4. Add 5 mL of Potassium Phosphate Buffer Saline (PBS) (pH 7.4, 10 mM). 5. Shake in an Orbit Shaker for 140 min at 300 rpm. 6. Pipette out 1 mL-aliquots of the supernatant into 4 microcentrifuge tubes. 7. Centrifuge with a microcentrifuge for 20 min at room temperature (at ca. 14,000 g). 8. Use the clear supernatant for assays.

Assay Method

The assay method is based on the principle that the hydrolysis of 2-Chloro-4-nitrophenyl- -D- maltotrioside, catalyzed by -Amylase, yields 2-Chloro-4-nirophenol that is quantitatively measured by its absorbance at 405 nm. Its formation is directly proportional to the -Amylase activity.

In the present study, assays were carried out with a total volume of 150 L of reaction mixture per a microplate well, and measuring the Optical Density with a Microplate Reader (Bio-Rad Model 680), as follows:

1. -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1); equivalent of 1 L human saliva. 2. 100 L of sample extract (-Amylase inhibitor was equivalent of 2.5 mg of Phase2®/StarchLite™). 3. Incubate for 30 min over a warm plate (~30 oC), covering the microplate loosely with a plastic lid. 4. Add 160 L of the substrate (2-Chloro-4-nitrophenyl--D-maltotrioside) solution. 5. Measure the Optical Density at 405 nm at 5 min.

Measurements were made with 3 replications of each of the following:

Table 1

Reaction Enzyme (L) Buffer (PBS) (L) Gum Extract (L) Substrate (L)

1. Blank (Background) None 110 None 160 2. Untreated Gum None 10 100 160 3. Uninhibited Enzyme 10 100 None 160 4. Treated Gum 10 None 100 160 5. Phase2/StarchLite 10 None 100 160 6. Untreated Gum plus 10 None 100 160 Phase2/StarchLite ISSI No. P25036-B Pharmachem / StarchLite® Page 3

Results

The assays were conducted in triplicate for the Untreated (Chewing Gum without Phase2®/StarchLite™) and Treated (Chewing Gum with Phase2®/StarchLite™, along with the experimental controls.

The Optical density values, which are directly related to the -Amylase activity, are summarized in the following Table 2 and Figure 1.

Table 2

Optical Density (405 nm)

Replication Enzyme Untreated Chewing Treated Chewing (Uninhibited) Gum Gum

1 1.915 1.809 0.246 2 1.862 1.915 0.218 3 1.925 1.951 0.289

Mean 1.901 1.892 0.251

Activity Remaining 99.53% 13.20%

Phase2/StarchLite in Chewing Gum

2

1.8

1.6

1.4

1.2

1

0.8

0.6 Optical Density nm)(405 0.4

0.2

0 123 Uninhibited Enzyme (1), Untreated Chewing Gum (2), and Treated Chewing Gum (3), Each with 3 Replications

Figure 1. Inhibition of -Amylase Activity by Phase2®/StarchLite™ in Chewing Gum. ISSI No. P25036-B Pharmachem / StarchLite® Page 4

The data obtained with the recovery of Phase2®/StarchLite that was spiked (mixed) with Untreated Gum ( see Reactions 5 and 6 in Table 1) are summarized below in Table 3.

Table 3

Optical Density (405 nm)

Replication Phase2®/ Chewing Gum Spiked with ® StarchLite Phase2 /StarchLite

1 0.156 0.146 2 0.166 0.129 3 0.156 0.170

Mean 0.159 0.148

Recovery 93.08%

As could be seen from the above data, about 7% of Phase2®/StarchLite was left behind after the extraction.

Conclusions

(1) The -Amylase of human saliva was significantly inhibited by the inhibitor in Phase2®/StarchLite™. The mean inhibition of -Amylase activity was 86.80%, calculated as follows:

[100-13.20] = 86.80%

(2) The remainder of 12.88% is most likely the amount of Phase2®/StarchLite™ that was still unextractable from the Chewing Gum. At least, about 7% of it could be attributed to the unextractability, as was seen in the spiked experiment. Since the spiking of the Untreated Chewing Gum does not mimic the actual formulation of the product, it is reasonable to expect that an additional 5% of the activity could be still resident in the Treated Gum.

(3) The inhibitor in Phase2®/StarchLite™ was unaffected during the processing/manufacturing of the Chewing Gum.

Respectfully submitted:

Yesu T. Das, Ph.D. Pasteurized Orange Drink

Inhibition of -Amylase Activity by Phase2/StarchLite in Pasteurized Orange Drink

Author

Yesu T. Das, Ph.D.

Completion Date

April 16, 2008

Performing Laboratory

ISSI Laboratories, Inc. (ISSI) 515 Blue Ridge Avenue Piscataway, NJ 08854 Telephone (732) 246-3930

ISSI Number

P28031

Sponsor

Pharmachem Laboratories, Inc. 265 Harrison Avenue Kearny, New Jersey 07032 Telephone 201-719-7405

Sponsor Representative

Gregory Drew

Page 1 of 7 ISSI 28031 Pharmachem / Orange Drinks Page 2

Objective

The objective of the analytical efforts is to assess the efficacy of Phase2®/StarchLite™ that was incorporated into the pasteurized orange drink towards inhibiting the human salivary -Amylase.

Materials and Methods

Materials

Substrate

DQ-BODIPYFL Conjugate (Invitrogen Detection Technologies/Molecular Probes Inc., Eugene, Oregon). This is a derivatized corn starch (DQ) labeled with a fluorescent dye (BODIPYFL).

Enzyme

Human salivary -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1), clarified by freezing and centrifugation, and used undiluted.

Buffers

Potassium Phosphate Buffer Saline (PBS) (pH 7.4, 10 mM). Sodium Acetate Buffer (pH 4.0, 50 mM). MOPS Buffer (pH 6.9, 1 M).

Fluorescence Microplate Reader

Zynaxis Zymmune Auto-Reader F, Labsystems Model 372, Thermo Electron Corporation, Finland.

Sample Preparation

The pasteurized orange drink bottles (Untreated and Treated) were gently shaken with hand to obtain a uniform consistency. For preparing the spiked sample, 24 mL of the untreated drink was mixed with 100 mg of Phase2®/StarchLite™, and gently shaken with hand until a uniform consistency was obtained. Aliquots of 100 L were used for assay.

Assay Method

The assay method is based on the principle that the hydrolysis of a derivatized starch substrate, catalyzed by -Amylase, yields a fluorescent product that is quantitatively measured by its fluorescence (Excitement485/Emission538). The intensity of fluorescence is directly proportional to the -Amylase activity. ISSI 28031 Pharmachem / Orange Drinks Page 3

Assays were carried out with a total volume of 200 L of reaction mixture per a microplate well, and by measuring the fluorescence with a Microplate Reader, as follows:

1. -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1); equivalent of 50 L human saliva. 2. 100 L of sample extract (-Amylase inhibitor was equivalent of 0.417 mg of Phase2®/StarchLite™). 3. Incubate for 30 min over a warm plate (~30 oC), covering the microplate loosely with a plastic lid. 4. Add 50 L of substrate solution (containing 10 g of DQ-BODIPYFL Conjugate). 5. Monitor fluorescence (Excitement485/Emission538) at various time intervals.

Results

The fluorescence values for the triplicate analysis conducted on each reaction, which are directly related to the -Amylase activity, are summarized in Table 1 and Figure 1.

The fluorescence values increased from 518 to 787 in Enzyme, from 251 to 463 in Untreated, from 234 to 349 in Treated, and from 199 to 300 in Spiked, over a period of 20 min. As could be expected, the highest activity was seen with uninhibited enzyme control, and lowest activity was seen with spiked inhibitor control (Figure 1). However, the focus of attention was on the Untreated and Treated drinks, which provided significant difference between them. Their rates of increase in fluorescence (Reaction Velocities), along with the spiked control, were statistically derived by subjecting the data to Correlation-Regression analysis (Figure 2). The resulting velocity values were 9.50 and 5.52 for the Untreated and Treated drinks, respectively. A normalized comparative picture of these values is shown in Figure 3.

Conclusions

(1) The -Amylase of human saliva was significantly inhibited by the inhibitor in Phase2®/StarchLite™. Based on the reaction velocities, the mean inhibition of -Amylase activity in the Treated drink was 41.90%, calculated as follows:

[100-58.10] = 41.90%

(2) The spectrophotometric assays that are routinely employed for monitoring -Amylase activity met with some problems by the interference of orange pigments in the drinks. For this reason, a fluorescence technique was employed in the present investigation. Further optimization of certain parameters of this assay system, such as substrate/enzyme concentration and reaction time, offer scope for better data acquisition.

(3) The inhibitor of Phase2®/StarchLite™ appeared to be unaffected by the processing/pasteurization stress during the manufacturing of the drinks.

Submitted By:

Yesu T. Das, Ph.D. ISSI Laboratories, Inc. ISSI 28031 Pharmachem / Orange Drinks Page 4

Table 1. Hydrolysis of Starch Substrate (DQ) by Human Salivary -Amylase Showing Observed Fluorescence Values (Excitement485/Emission538) Under Various Reaction Conditions.

Time Intervals Reaction Replication 0 min 3 min 13 min 20 min

Enzyme 1 512 706 806 841 2502656726749 3541697752772 Mean 518 686 761 787

Control 1 231 348 422 450 2288397478510 3234333400428 Mean 251 359 433 463

Treated 1 236 275 334 363 2248294347374 3219245290309 Mean 234 271 324 349

Spiked 1 199 233 276 291 2198226274297 3199240290311 Mean 199 233 280 300

Enzyme: Control reaction without any inhibitor. Control: Untreated drink (No Phase2/StarchLite). Treated: Treated drink (Incorporated with Phase2/StarchLite during processing/pasteurization @ 100 mg/24 mL). Spiked: Untreated drink with added Phase2/StarchLite in the laboratory for assay purpose. ISSI 28031 Pharmachem / Orange Drinks Page 5

alpha-Amylase Activity

900

800

700

600

500 538 Value

Em 400

300 485 / Ex 200

100

0 0510152025 Time (min) Enzyme Control Spiked Treated

Figure 1. Hydrolysis of Starch Substrate (DQ) by Human Salivary -Amylase Monitored By Fluorescence Microplate Reader (Excitement485/Emission538) Under Various Reaction Conditions.

Enzyme: Control reaction without any inhibitor. Control: Untreated drink (No Phase2/StarchLite). Treated: Treated drink (Incorporated with Phase2/StarchLite during processing/pasteurization @ 100 mg/24 mL). Spiked: Untreated drink with added Phase2/StarchLite in the laboratory for assay purpose. ISSI 28031 Pharmachem / Orange Drinks Page 6

Reaction Velocities of alpha-Amylase Activity

500 Untreated (v = 9.50) 450

400 Treated (v = 5.52) 350 538 538 Value 300 Spiked (v = 4.85) Em

250 485 / 485 200 Ex

15 0

10 0 0510152025 Time (min)

Figure 2. Reaction Rates (v) of Starch Substrate (DQ) Hydrolysis by Human Salivary -Amylase Monitored By Fluorescence Microplate Reader (Excitement485/Emission538) Under Various Reaction Conditions.

Untreated (Control): Untreated drink (No Phase2/StarchLite). Treated: Treated drink (Incorporated with Phase2/StarchLite during processing/pasteurization @ 100 mg/24 mL). Spiked: Untreated drink with added Phase2/StarchLite in the laboratory for assay purpose.

See Figure 3 for a comparison of reaction rates of untreated and treated drinks. ISSI 28031 Pharmachem / Orange Drinks Page 7

Activity of alpha-Amylase in Pasteurized Orange Drinks

120% 100% 100% e 80% 58.10% 60%

Relative % Valu % Relative 40%

20%

0% Untreated (Velocity = 9.50) Treated (Velocity = 5.52)

Figure 3. Comparative Reaction Rates (Velocities, v) of Starch Substrate (DQ) Hydrolysis by Human Salivary -Amylase Monitored by Fluorescence Microplate Reader (Excitement485/Emission538) For Untreated and Treated Drinks.

Untreated (Control): Untreated drink (No Phase2/StarchLite). Normalized to 100%. Treated: Treated drink (Incorporated with Phase2/StarchLite during processing/pasteurization @ 100 mg/24 mL). Pasteurized Milk

Inhibition of -Amylase Activity by Phase2 in Pasteurized Milk

Author

Yesu T. Das, Ph.D.

Completion Date

May 22, 2008

Performing Laboratory

ISSI Laboratories, Inc. (ISSI) 515 Blue Ridge Avenue Piscataway, NJ 08854 Telephone (732) 246-3930

ISSI Number

P28042

Sponsor

Pharmachem Laboratories, Inc. 265 Harrison Avenue Kearny, New Jersey 07032 Telephone 201-719-7405

Sponsor Representative

Mitch Skop

Page 1 of 4 ISSI P28042 Pharmachem / Milk Page 2

Objective

The objective of the analytical efforts is to assess the efficacy of Phase2® that was incorporated into the pasteurized milk towards inhibiting the human salivary -Amylase.

Materials and Methods

Materials

Test Substances

Untreated (Control) Milk (No Phase2®)(Toong Yeuan Blank) Treated Milk (Phase2® Milk)(Toong Yeuan Phase2 Milk, 400 mg Phase2®/200 mL)

Substrate

2-Chloro-4-nitrophenyl- -D-maltotrioside (CNPG3)

Enzyme

Human salivary -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1), clarified by freezing and centrifugation, and used undiluted.

Buffers and Reagents

Potassium Phosphate Buffer Saline (PBS) (pH 7.4, 10 mM). Sodium hydroxide (1 N aqueous solution).

Microplate Reader

Bio-Rad Model 680.

Assay Principle

The assay method is based on the principle that the hydrolysis of 2-Chloro-4-nitrophenyl- -D- maltotrioside, catalyzed by -Amylase, yields 2-Chloro-4-nirophenol that is quantitatively measured by its absorbance at 405 nm. Its formation is directly proportional to the -Amylase activity.

The pasteurized milk samples were treated with a-Amylase and allowed to interact at 37 oC for 60 min. The milk proteins were then precipitated with Trichloroacetic acid (TCA), removed by centrifugation, and the clear supernatant was used for evaluating the residual enzymatic activity, after neutralizing with alkali (1N NaOH). ISSI P28042 Pharmachem / Milk Page 3

Assay Method

In the present study, assays were carried out with a total volume of 300 L of reaction mixture per a microplate well, and measuring the Optical Density with a Microplate Reader (Bio-Rad Model 680), as follows:

1. -Amylase (1,4--D-Glucan-glucanohydrolase; E.C. 3.2.1.1); equivalent of 3 L human saliva. 2. 100 L of sample (-Amylase inhibitor was equivalent of 100 g of Phase2®). 3. 100 L of sodium hydroxide solution (1 N) 4. 100 L of the substrate (2-Chloro-4-nitrophenyl--D-maltotrioside) solution. 5. Optical Density measurement at 405 nm.

Results

The Optical Density (OD) values of the duplicate assays, which are directly related to the -Amylase activity, are summarized in the following Table 1 and Figure 1.

Table 1

Optical Density (405 nm)

Replication Untreated Milk Treated Milk

137.0 31.0 239.5 30.0

Mean 38.3 30.5

Relative Activity 79.6%

Conclusions

(1) The -Amylase of human saliva was significantly inhibited by the inhibitor in Phase2®. The mean inhibition of -Amylase activity in the Treated Milk was 20.4%, calculated as follows:

[100-79.6] = 20.4%

(2) The spectrophotometric assay that is routinely employed for monitoring -Amylase activity met with some problems by the interference of the milk proteins. For this reason, a special step was introduced to precipitate out the proteins to minimize the opacity.

(3) The inhibitor in Phase2® appeared to be unaffected by the processing/pasteurization stress during the manufacturing of the milk.

(4) Further optimization of the assay parameters, such as substrate/enzyme/inhibitor concentrations and reaction time, offer scope for better data acquisition and better visibility of the inhibitory level. ISSI P28042 Pharmachem / Milk Page 4

Phase2 in Pasteurized Milk

120.0% 100.0% 100.0% 79.6% 80.0%

60.0%

40.0% Relative Activity Relative

20.0%

0.0% Untreated Milk Treated Milk

Figure 1. Relative Activity of Human Salivary -Amylase in Untreated and Treated Milk.

Untreated (Control) Milk (No Phase2). Normalized to 100%. Treated Milk (Incorporated with Phase2).

Submitted By:

Yesu T. Das, Ph.D. ISSI Laboratories,Inc. STABILITYSTABILITY REPORTREPORT pH and thermal stability of StarchLite

February 2009 Introduction

TRIAL AIM : To measure the influence of pH and heating conditions on the stability of StarchLite in water.

Product : StarchLite powder dissolved in distilled water (1% w/w).

Experimental conditions : Stability of StarchLite was assessed by using experimental design (Doelhert matrix), which studies the variation of 3 factors : pH : from 2 to 10 Temperature : from 20C to 120C Duration : from 0 to 20 minutes

Process : Samples were prepared as followed :

1% Starchlite solution

pH adjustment (HCl 2N or NaOH 2N)

heat treatment

cooling analysis 2 Method

ANALYTICALANALYTICAL METHODMETHOD

Analytical method : Principle : α-amylase enzymatic activity is measured during the hydrolysis of its specific substrate (2-chloro-4-nitrophenyl- alpha-D-maltotrioside) by absorbance analysis.

Enzymatic Test : Preparation of reactional mix : [StarchLite solution 2g/L + PBS buffer pH 5.2 + enzyme (porcine pancreatic α-amylase 150mM)] Incubation (1 hour / 38C).

Addition of substrate C24H34Cl N O18 =2-chloro-4-nitrophenyl- alpha-D-maltotrioside (1mM)

Addition of Na2CO3 after 5 min to stop reaction (t0). Measurement of Absorbance at 404nm.

Results expression :

The stability of StarchLite is determined from the comparison of StarchLite α-amylase inhibition activity before and after heat/pH treatment.

3 Results

RESULTSRESULTS && COMMENTSCOMMENTS

α -amylase inhibition activity of StarchLite at pH 3

120

y ivit 100 act 95% 60-70% activity 70- 80

60

Temperature (°C) Temperature 95-100% activity 40

20 05101520 Time (min)

Heat stability of StarchLite at pH = 3 At pH 3, whatever the heat treatment applied on StarchLite, the activity remains > 60%. In general, at acidic pH, StarchLite is more stable at temperature under 60C. In case of a long heating time treatment, StarchLite activity is preserved. However, the lower α -amylase inhibition rate is observed for short heating time treatment.

4 Results

RESULTSRESULTS && COMMENTSCOMMENTS

α -amylase inhibition activity of StarchLite at pH 5

120

100 50-70% activity

80 70-90% activity

60 90-100% activity Temperature (°C) Temperature 40

20 05101520 Time (min)

Heat stability of StarchLite at pH 5 At pH 5, no side effect on the α-amylase inhibition rate was observed for heat treatment until 80-90C. Higher temperatures have consequences by decreasing the α-amylase inhibition rate. When temperature decreases, StarchLite activity is preserved.

5 Results

RESULTSRESULTS && COMMENTSCOMMENTS

α -amylase inhibition activity of StarchLite at pH 7

120 60-65% activity

100

65-75% 80 activity 90-100% activity 60 y Temperature (°C) Temperature vit 90% acti 40 75-

20 05101520 Time (min)

Heat stability of StarchLite at pH 7 At pH 7, whatever the heat treatment applied on StarchLite, the activity remains > 60%. There is no degradation of inhibiting activity for short time heat treatment until 110C. When heating time decreases, StarchLite activity is preserved.

6 Results

RESULTSRESULTS && COMMENTSCOMMENTS

Stability of StarchLite in industrial processes Thermisation : 75C / 30 sec ; at pH 3 Pasteurization : 85C / 3 min ; at pH 5 Sterilization : 120C / 10min ; at pH 7

Activity of StarchLite in industrial processes 80-100% 

60-80% Thermisation Sterilization

Pasteurization 40-60% % activity% StarchLite of 20-40% 0- 20%

7 Conclusions

GENERALGENERAL CONCLUSIONSCONCLUSIONS

pH stability StarchLite shows very good stability for acid pH. At very basic pH, the activity decreases probably because of a lower solubility of the powder. Application : StarchLite is fully suitable for applications such as acid drinks.

Thermal stability StarchLite shows a good stability from 20C to 120C. Starchlite activity decreases at long time heat treatment but remains >40% inhibition of α-amylase Application : StarchLite is fully suitable for industrial process with heat treatment over 70C such as pasteurization or sterilization.

General conclusion StarchLite is fully suitable for a large spectrum of applications.

8

MATERIALS AND METHODS

Microwave treatment

Powder of Phase 2 was treated with microwave oven for 1, 2, 3, 4 or 5 min.

Alpha-amylase inhibitory assay

Determination of α-amylase inhibitory activity was analyzed by iodine-starch assay. The test compounds suspended in 50 µl of distilled water were incubated with 10 µl of 125 U/ml α-amylase (Type -B from Porcine Pancreas; SIGMA)

prepared in 20 mM phosphate buffer (pH 7.4) containing 20 mM CaCl2, at room temperature. After 5 min of incubation, 125 µl of 4% soluble starch was added to the mixture, and further incubated for 7.5 min at room temperature. After the incubation, 125 µl of 0.002 N iodine solution and 695 µl of distilled water were added, and the absorbance at 620 nm was measured using a microplate reder. Inhibitory activity was expressed the absorbance difference (%) of the test samples relative to the change in absorbance of the control (100%) that used distilled water instead of the test solution.

4% soluble starch 0.002 N iodine absorbance at 620 nm solution was measured RESULT

% control % inhibition (control) Non-treatment (control) Non-treatment 1 min23 min 45 1 min2345 Time ofmicrowavetreatment Time of microwavetreatment Enzyme +:0.0632 Enzyme -:0.5733 OD value RESULT

The stability of Phase 2 is determined from the comparison of α-amylase inhibition activity before and after microwave treatment. Alpha-amylase inhibitory activity of Phase 2 was not decreased by microwave treatment. Inhibitory activity was found to have been sustained for 1-5 min microwave treatment.

CONCLUSION

Phase 2 shows a good stability for microwave treatment. Protein - alpha amylase inhibitor-1 precursor [Phaseolus vul... Page 1 of 1

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Showing 223 aa region from aa 22. Change Region Shown GenBank: AAT35809.1 Whole sequence alpha amylase inhibitor-1 precursor [Phaseolus vulgaris] Selected Region from: 22 to: end Features Sequence Update View LOCUS AAT35809 223 aa linear PLN 28-JUL- 2008 Customize View DEFINITION alpha amylase inhibitor-1 precursor [Phaseolus vulgaris]. ACCESSION AAT35809 REGION: 22..244 VERSION AAT35809.1 GI:47571317 Analyze This Sequence DBSOURCE accession AY603476.1 KEYWORDS . Run BLAST SOURCE Phaseolus vulgaris Identify Conserved Domains ORGANISM Phaseolus vulgaris Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Recent activity Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; Turn Off Clear rosids; fabids; Fabales; Fabaceae; Papilionoideae; Phaseoleae; Phaseolus. REFERENCE 1 (residues 1 to 223) alpha amylase inhibitor-1 AUTHORS Prescott,V.E., Campbell,P.M., Moore,A., Mattes,J., precursor [Phaseolus vulgaris] Rothenberg,M.E., Foster,P.S., Higgins,T.J. and Hogan,S.P. AAT35809.1 (1) TITLE Transgenic expression of bean alpha-amylase inhibitor in peas results in altered structure and immunogenicity alpha-amylase inhibitor-2 JOURNAL J. Agric. Food Chem. 53 (23), 9023-9030 (2005) [Phaseolus vulgaris] PUBMED 16277398 REFERENCE 2 (residues 1 to 223) BAA05105.1 (1) AUTHORS Moore,A.E. and Higgins,T.J. TITLE Direct Submission Towards biochemical reaction JOURNAL Submitted (21-APR-2004) CSIRO Plant Industry, G.P.O. Box 1600, monitoring using FT-IR Canberra, ACT 2601, Australia COMMENT Method: conceptual translation. » See more... FEATURES Location/Qualifiers source 1..223 /organism="Phaseolus vulgaris" All links from this record /cultivar="Pinto" /db_xref="taxon:3885" Protein <1..223 BLink /product="alpha amylase inhibitor-1 precursor" mat_peptide 1..223 Related sequences /product="alpha amylase inhibitor-1" Conserved domains Region 2..201 /region_name="Lectin_legB" Domain relatives /note="Legume lectin domain; pfam00139" Nucleotide /db_xref="CDD:143910" CDS <1..223 PubMed /coded_by="AY603476.1:1..735" Related structure ORIGIN 1 atetsfnidg fnktnlilqg daivssngnl qlsynsydsm srafysapiq irdsttgnva Taxonomy 61 sfdtnftmni rthrqansav gldfvlvpvq peskgdtvtv efdtflsris idvnnndiks 121 vpwdvhdydg qnaevrityn sstkvfavsl lnpstgksnd vsttveleke vydwvrvgfs LinkOut 181 atsgayqwsy ethdvlswsf sskfinhkdq ksersnivln kil //

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http://www.ncbi.nlm.nih.gov/protein/47571317?from=22&to=244&report=gpwithparts 2010/04/07 ExPASy ProtParam tool Page 1 of 2

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ProtParam

User-provided sequence:

10 20 30 40 50 60 ATETSFNIDG FNKTNLILQG DAIVSSNGNL QLSYNSYDSM SRAFYSAPIQ IRDSTTGNVA 70 80 90 100 110 120 SFDTNFTMNI RTHRQANSAV GLDFVLVPVQ PESKGDTVTV EFDTFLSRIS IDVNNNDIKS 130 140 150 160 170 180 VPWDVHDYDG QNAEVRITYN SSTKVFAVSL LNPSTGKSND VSTTVELEKE VYDWVRVGFS 190 200 210 220 ATSGAYQWSY ETHDVLSWSF SSKFINHKDQ KSERSNIVLN KIL References and documentation are available.

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 223

Molecular weight: 24896.3

Theoretical pI: 4.92

Amino acid composition: CSV format Ala (A) 11 4.9% Arg (R) 8 3.6% Asn (N) 20 9.0% Asp (D) 17 7.6% Cys (C) 0 0.0% Gln (Q) 8 3.6% Glu (E) 9 4.0% Gly (G) 10 4.5% His (H) 4 1.8% Ile (I) 13 5.8% Leu (L) 13 5.8% Lys (K) 10 4.5% Met (M) 2 0.9% Phe (F) 12 5.4% Pro (P) 5 2.2% Ser (S) 30 13.5% Thr (T) 18 8.1% Trp (W) 4 1.8% Tyr (Y) 8 3.6% Val (V) 21 9.4% Pyl (O) 0 0.0% Sec (U) 0 0.0%

(B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 26 Total number of positively charged residues (Arg + Lys): 18

Atomic composition:

Carbon C 1100 Hydrogen H 1687 Nitrogen N 297 Oxygen O 360 Sulfur S 2

http://www.expasy.ch/cgi-bin/protparam 2010/04/07 ExPASy ProtParam tool Page 2 of 2

Formula: C1100H1687N297O360S2 Total number of atoms: 3446

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 33920 Abs 0.1% (=1 g/l) 1.362

Estimated half-life:

The N-terminal of the sequence considered is A (Ala).

The estimated half-life is: 4.4 hours (mammalian reticulocytes, in vitro). >20 hours (yeast, in vivo). >10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 37.33 This classifies the protein as stable.

Aliphatic index: 77.71

Grand average of hydropathicity (GRAVY): -0.386

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Showing 198 aa region from aa 23 to 220. Change Region Shown GenBank: BAA05105.1 Whole sequence alpha-amylase inhibitor-2 [Phaseolus vulgaris] Selected Region from: 23 to: 220 Features Sequence Update View LOCUS BAA05105 198 aa linear PLN 01-FEB-2000 DEFINITION alpha-amylase inhibitor-2 [Phaseolus vulgaris]. Customize View ACCESSION BAA05105 REGION: 23..220 VERSION BAA05105.1 GI:529075 DBSOURCE locus PHVAA2 accession D26109.1 Analyze This Sequence KEYWORDS . SOURCE Phaseolus vulgaris Run BLAST ORGANISM Phaseolus vulgaris Identify Conserved Domains Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; fabids; Fabales; Fabaceae; Papilionoideae; Phaseoleae; Identical Proteins for BAA05105.1 Phaseolus. RecName: Full=Alpha-amylase inhibitor[Q41114] REFERENCE 1 2; Short=Alpha-AI-2; Short=Alpha-AI2; AUTHORS Suzuki,K., Ishimoto,M. and Kitamura,K. TITLE cDNA sequence and deduced primary structure of an alpha-amylase » See all... inhibitor from a bruchid-resistant wild common bean JOURNAL Biochim. Biophys. Acta 1206 (2), 289-291 (1994) PUBMED 8003534 Recent activity REFERENCE 2 (residues 1 to 198) AUTHORS Suzuki,K. Turn Off Clear TITLE Direct Submission JOURNAL Submitted (07-DEC-1993) Kazunori Suzuki, National Agriculture Research Center, Legume Breeding Laboratory; 3-1-1 Kannondai, alpha-amylase inhibitor-2 Tsukuba, Ibaraki 305, Japan (Tel:0298-38-8503, Fax:0298-38-8515) [Phaseolus vulgaris] FEATURES Location/Qualifiers source 1..198 BAA05105.1 (1) /organism="Phaseolus vulgaris" /isolate="breeding line OAr4" Towards biochemical reaction /db_xref="taxon:3885" monitoring using FT-IR /clone="pOAr4-1" hdii /tissue_type="cotyledon" Purification and partial /clone_lib="lambda gt11" characterization of an elastolytic /dev_stage="midmature seed" Protein <1..>198 Inhibition of human leucocyte /product="alpha-amylase inhibitor-2" elastase by ursolic acid. Evidence Region 1..198 /region_name="Lectin_legB" » See more... /note="Legume lectin domain; pfam00139" /db_xref="CDD:143910" CDS <1..>198 All links from this record /gene="alpha-ai2" /coded_by="D26109.1:1..723" /experiment="experimental evidence, no additional details BLink recorded" ORIGIN Related sequences 1 sdtsfnfysf netnlilqgd atvsskgylq lhtvdsmcsa fysapiqird sttgnvasfd Identical proteins 61 tnftmnittq reansvigld falvpvqpks kghtvtvqfd tfrsrisidv nnndiksvpw 121 deqdydgqna kvritynsst kvlavslsnp stgksnevsa rmevekeldd wvrvgfsais Conserved domains 181 gvheysfetr dvlswsfs Domain relatives // Nucleotide PubMed Related structure Taxonomy LinkOut

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http://www.ncbi.nlm.nih.gov/protein/529075?from=23&to=220&report=gpwithparts 2010/04/07 ExPASy ProtParam tool Page 1 of 2

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You are here: ExPASy CH > Tools > Primary structure analysis > ProtParam

ProtParam

User-provided sequence:

10 20 30 40 50 60 SDTSFNFYSF NETNLILQGD ATVSSKGYLQ LHTVDSMCSA FYSAPIQIRD STTGNVASFD 70 80 90 100 110 120 TNFTMNITTQ REANSVIGLD FALVPVQPKS KGHTVTVQFD TFRSRISIDV NNNDIKSVPW 130 140 150 160 170 180 DEQDYDGQNA KVRITYNSST KVLAVSLSNP STGKSNEVSA RMEVEKELDD WVRVGFSAIS 190 GVHEYSFETR DVLSWSFS References and documentation are available.

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 198

Molecular weight: 22029.2

Theoretical pI: 4.79

Amino acid composition: CSV format Ala (A) 10 5.1% Arg (R) 8 4.0% Asn (N) 14 7.1% Asp (D) 15 7.6% Cys (C) 1 0.5% Gln (Q) 8 4.0% Glu (E) 9 4.5% Gly (G) 9 4.5% His (H) 3 1.5% Ile (I) 10 5.1% Leu (L) 10 5.1% Lys (K) 8 4.0% Met (M) 3 1.5% Phe (F) 12 6.1% Pro (P) 5 2.5% Ser (S) 28 14.1% Thr (T) 17 8.6% Trp (W) 3 1.5% Tyr (Y) 6 3.0% Val (V) 19 9.6% Pyl (O) 0 0.0% Sec (U) 0 0.0%

(B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 24 Total number of positively charged residues (Arg + Lys): 16

Atomic composition:

Carbon C 968 Hydrogen H 1487 Nitrogen N 261 Oxygen O 320 Sulfur S 4

http://www.expasy.ch/cgi-bin/protparam 2010/04/07 ExPASy ProtParam tool Page 2 of 2

Formula: C968H1487N261O320S4 Total number of atoms: 3040

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 25440 Abs 0.1% (=1 g/l) 1.155, assuming all pairs of Cys residues form cystines

Ext. coefficient 25440 Abs 0.1% (=1 g/l) 1.155, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is S (Ser).

The estimated half-life is: 1.9 hours (mammalian reticulocytes, in vitro). >20 hours (yeast, in vivo). >10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 23.82 This classifies the protein as stable.

Aliphatic index: 72.27

Grand average of hydropathicity (GRAVY): -0.362

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http://www.expasy.ch/cgi-bin/protparam 2010/04/07 DisEMBL 1.5 - Predictors of intrinsic protein disorder Page 1 of 2

DisEMBL™

Prediction results for AI1

Download PostScript file

Disordered by Loops/coils definition

>AI1_LOOPS 1-65, 74-99, 110-133, 151-162, 196-217 ATETSFNIDG FNKTNLILQG DAIVSSNGNL QLSYNSYDSM SRAFYSAPIQ IRDSTTGNVA SFDTNftmni rthRQANSAV GLDFVLVPVQ PESKGDTVTv efdtflsriS IDVNNNDIKS VPWDVHDYDG QNAevrityn sstkvfavsl LNPSTGKSND VSttveleke vydwvrvgfs atsgayqwsy ethdvLSWSF SSKFINHKDQ KSERSNIvln kil

Disordered by Hot-loops definition

>AI1_HOTLOOPS 1-14, 90-99, 107-122, 149-162, 203-223 ATETSFNIDG FNKTnlilqg daivssngnl qlsynsydsm srafysapiq irdsttgnva sfdtnftmni rthrqansav gldfvlvpvQ PESKGDTVTv efdtflSRIS IDVNNNDIKS VPwdvhdydg qnaevrityn sstkvfavSL LNPSTGKSND VSttveleke vydwvrvgfs atsgayqwsy ethdvlswsf ssKFINHKDQ

http://dis.embl.de/cgiDict.py 2010/04/07 DisEMBL 1.5 - Predictors of intrinsic protein disorder Page 2 of 2

KSERSNIVLN KIL

Disordered by Remark-465 definition

>AI1_REM465 none atetsfnidg fnktnlilqg daivssngnl qlsynsydsm srafysapiq irdsttgnva sfdtnftmni rthrqansav gldfvlvpvq peskgdtvtv efdtflsris idvnnndiks vpwdvhdydg qnaevrityn sstkvfavsl lnpstgksnd vsttveleke vydwvrvgfs atsgayqwsy ethdvlswsf sskfinhkdq ksersnivln kil

Low-complexity predicted by CAST

>AI1_LOW-COMPLEXITY none atetsfnidg fnktnlilqg daivssngnl qlsynsydsm srafysapiq irdsttgnva sfdtnftmni rthrqansav gldfvlvpvq peskgdtvtv efdtflsris idvnnndiks vpwdvhdydg qnaevrityn sstkvfavsl lnpstgksnd vsttveleke vydwvrvgfs atsgayqwsy ethdvlswsf sskfinhkdq ksersnivln kil

Beta aggregation predicted by Tango

>AI1_TANGO none atetsfnidg fnktnlilqg daivssngnl qlsynsydsm srafysapiq irdsttgnva sfdtnftmni rthrqansav gldfvlvpvq peskgdtvtv efdtflsris idvnnndiks vpwdvhdydg qnaevrityn sstkvfavsl lnpstgksnd vsttveleke vydwvrvgfs atsgayqwsy ethdvlswsf sskfinhkdq ksersnivln kil

JOB-ID AI1_0304470yJQJAoBAY8AAGwCJvsAAAAA Frames used smooth=8 peak=8 join=4 Thresholds used coils=0.516 rem465=0.6 hot loops=0.1204 Name AI1 Description none Title/ID AI1 Sequence length 223 Download predictions smoothed scores raw scores

DisEMBL™ is Copyright © 2003-2006 by Rune Linding & Lars Juhl Jensen - EMBL

http://dis.embl.de/cgiDict.py 2010/04/07 DisEMBL 1.5 - Predictors of intrinsic protein disorder Page 1 of 2

DisEMBL™

Prediction results for AI2

Download PostScript file

Disordered by Loops/coils definition

>AI2_LOOPS 1-31, 39-65, 72-97, 103-130, 147-157 SDTSFNFYSF NETNLILQGD ATVSSKGYLQ LhtvdsmcSA FYSAPIQIRD STTGNVASFD TNFTMnittq rEANSVIGLD FALVPVQPKS KGHTVTVqfd tfRSRISIDV NNNDIKSVPW DEQDYDGQNA kvritynsst kvlavsLSNP STGKSNEvsa rmevekeldd wvrvgfsais gvheysfetr dvlswsfs

Disordered by Hot-loops definition

>AI2_HOTLOOPS 85-127, 144-158, 178-198 sdtsfnfysf netnlilqgd atvsskgylq lhtvdsmcsa fysapiqird sttgnvasfd tnftmnittq reansvigld falvPVQPKS KGHTVTVQFD TFRSRISIDV NNNDIKSVPW DEQDYDGqna kvritynsst kvlAVSLSNP STGKSNEVsa rmevekeldd wvrvgfsAIS GVHEYSFETR DVLSWSFS

http://dis.embl.de/cgiDict.py 2010/04/07 DisEMBL 1.5 - Predictors of intrinsic protein disorder Page 2 of 2

Disordered by Remark-465 definition

>AI2_REM465 148-161 sdtsfnfysf netnlilqgd atvsskgylq lhtvdsmcsa fysapiqird sttgnvasfd tnftmnittq reansvigld falvpvqpks kghtvtvqfd tfrsrisidv nnndiksvpw deqdydgqna kvritynsst kvlavslSNP STGKSNEVSA Rmevekeldd wvrvgfsais gvheysfetr dvlswsfs

Low-complexity predicted by CAST

>AI2_LOW-COMPLEXITY none sdtsfnfysf netnlilqgd atvsskgylq lhtvdsmcsa fysapiqird sttgnvasfd tnftmnittq reansvigld falvpvqpks kghtvtvqfd tfrsrisidv nnndiksvpw deqdydgqna kvritynsst kvlavslsnp stgksnevsa rmevekeldd wvrvgfsais gvheysfetr dvlswsfs

Beta aggregation predicted by Tango

>AI2_TANGO none sdtsfnfysf netnlilqgd atvsskgylq lhtvdsmcsa fysapiqird sttgnvasfd tnftmnittq reansvigld falvpvqpks kghtvtvqfd tfrsrisidv nnndiksvpw deqdydgqna kvritynsst kvlavslsnp stgksnevsa rmevekeldd wvrvgfsais gvheysfetr dvlswsfs

JOB-ID AI2_0305481sPgawoBAY8AAGNHSCgAAAAD Frames used smooth=8 peak=8 join=4 Thresholds used coils=0.516 rem465=0.6 hot loops=0.1204 Name AI2 Description none Title/ID AI2 Sequence length 198 Download predictions smoothed scores raw scores

DisEMBL™ is Copyright © 2003-2006 by Rune Linding & Lars Juhl Jensen - EMBL

http://dis.embl.de/cgiDict.py 2010/04/07 AI1 1 ATETSFNIDGFNKTNLILQGDAIVSSNGNLQLSYNSYDSMSRAFYSAPIQIRDSTTGNVASFDTNFTMNIRTHRQA AI2 1 -SDTSFNFYSFNETNLILQGDATVSSKGYLQL--HTVDSMCSAFYSAPIQIRDSTTGNVASFDTNFTMNITTQREA consensus 1 ...**** ** ********* *** * ***.. . *** **************************** * * *

AI1 81 GLDFVLVPVQPESKGDTVTVEFDTFLSRISIDVNNNDIKSVPWDVHDYDGQNAEVRITYNSSTKVFAVSLLNPSTG AI2 78 GLDFALVPVQPKSKGHTVTVQFDTFRSRISIDVNNNDIKSVPWDEQDYDGQNAKVRITYNSSTKVLAVSLSNPSTG consensus 81 **** ****** *** **** **** ****************** ******* *********** **** *****

AI1 161 VSTTVELEKEVYDWVRVGFSATSGAYQWSYETHDVLSWSFSSKFINHKDQKSERSNIVLNKIL AI2 158 VSARMEVEKELDDWVRVGFSAISGVHEYSFETRDVLSWSFS------consensus 161 ** .*.***. ********* ** .*.**.********...... Functional Seasoning Application

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In four delicious flavors! Add it to your favorite comfort foods! NUTRITIONAL DATA PRODUCT: PHASE 2 ITALIAN HERB SEASONING NUTRIENT PER 100 GRAM

TOTAL CALORIES (Kcal) 275 CALORIES FROM FAT (Kcal) 14.30 TOTAL FAT 1.60 GM - SATURATED FAT 0.25 GM - TRANS FAT 0 GM - CHOLESTEROL 0 GM - SODIUM 6720 MG - TOTAL CARBOHYDRATE 60 GM - SUGAR 12.3 GM - DIETARY FIBER 2.70 GM - PROTEIN 12.50 GM - VITAMIN A 4100 IU - CALCIUM 220 MG - VITAMIN C 14 MG - IRON 5.8 MG - BETA CAROTENE 1460 MCG - WATER/MOISTURE 5.8 GM -

THIS CALCULATED VALUE BASED ON INFORMATION RECEIVED FROM SUPPLIER. DATE: AUGUST 30, 2011

PHARMACHEM LABORATORIES, INC.

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phar-ma-foodsÆ... efficacious food supplements standardized for specific potency, solubility, direct compression and disintegration characteristics... NUTRITIONAL DATA PRODUCT: PHASE 2 BUTTER GARLIC NUTRIENT PER 100 GRAM

TOTAL CALORIES (Kcal) 265 CALORIES FROM FAT (Kcal) 34 TOTAL FAT 3.8 GM - SATURATED FAT 2.1 GM - TRANS FAT 0.1 GM - CHOLESTEROL 0 GM - SODIUM 8510 MG - TOTAL CARBOHYDRATE 51.5 GM -- SUGARS 16 GM - DIETARY FIBER 2.70 GM - PROTEIN 11.2 GM - VITAMIN A 135 IU - CALCIUM 115 MG - VITAMIN C 1.7 MG - IRON 2.5 MG - BETA CAROTENE 50 MCG - WATER/MOISTURE 5.4 GM -

THIS CALCULATED VALUE BASED ON INFORMATION RECEIVED FROM SUPPLIER. DATE: AUGUST 30, 2011

PHARMACHEM LABORATORIES, INC.

265 Harrison Ave. ï Kearny, NJ 07032-4315 ï (201) 246-1000 ï Fax (201) 246-8105 ï 1-800-526-0609 ï www.pharmachemlabs.com

phar-ma-foodsÆ... efficacious food supplements standardized for specific potency, solubility, direct compression and disintegration characteristics... NUTRITIONAL DATA PRODUCT: PHASE 2 CINNAMON SUGAR NUTRIENT PER 100 GRAM

TOTAL CALORIES (Kcal) 350 CALORIES FROM FAT (Kcal) 4.5 TOTAL FAT 0.5 GM - SATURATED FAT 0.1 GM - TRANS FAT 0 GM - CHOLESTEROL 0 GM - SODIUM 675 MG - TOTAL CARBOHYDRATE 84.5 GM - SUGAR 62 GM - DIETARY FIBER 3 GM - PROTEIN 8.5 GM - VITAMIN A 40 IU - CALCIUM 220 MG - VITAMIN C 14 MG - IRON 5.8 MG - BETA CAROTENE 1460 MCG - WATER/MOISTURE 5.8 GM -

THIS CALCULATED VALUE BASED ON INFORMATION RECEIVED FROM SUPPLIER. DATE: AUGUST 30, 2011

PHARMACHEM LABORATORIES, INC.

265 Harrison Ave. ï Kearny, NJ 07032-4315 ï (201) 246-1000 ï Fax (201) 246-8105 ï 1-800-526-0609 ï www.pharmachemlabs.com

phar-ma-foodsÆ... efficacious food supplements standardized for specific potency, solubility, direct compression and disintegration characteristics... NUTRITIONAL DATA PRODUCT: PHASE 2 ASIAN RUB NUTRIENT PER 100 GRAM

TOTAL CALORIES (Kcal) 275 CALORIES FROM FAT (Kcal) 19 TOTAL FAT 2.1 GM - SATURATED FAT 0.3 GM - TRANS FAT 0.01 GM - CHOLESTEROL 0.2 GM - SODIUM 7780 MG - TOTAL CARBOHYDRATE 59 GM - SUGAR 27 GM - DIETARY FIBER 4.2 GM - PROTEIN 12.30 GM - VITAMIN A 465 IU - CALCIUM 185 MG - VITAMIN C 2.4 MG - IRON 3 MG - BETA CAROTENE 220 MCG - WATER/MOISTURE 4.8 GM -

THIS CALCULATED VALUE BASED ON INFORMATION RECEIVED FROM SUPPLIER. DATE: AUGUST 30, 2011

PHARMACHEM LABORATORIES, INC.

265 Harrison Ave. ï Kearny, NJ 07032-4315 ï (201) 246-1000 ï Fax (201) 246-8105 ï 1-800-526-0609 ï www.pharmachemlabs.com

phar-ma-foodsÆ... efficacious food supplements standardized for specific potency, solubility, direct compression and disintegration characteristics...

Sensory Evaluation and Product Recipes

For Trade Distribution, only. Product development of baked goods with a proprietary Fractionated White Bean Extract KANAK UDANI, Ph.D. P. O. box 1031 Agoura Hills, CA 91376

INTRODUCTION

With the growing consumer demand for low carbohydrate and low glycemic foods, several methods have been evaluated to lower the effective glycemic index of existing foods. These include the addition of soluble fiber, psyllium, blackgram fiber, and barley, or the substitution of simple starches with resistant starches.

One novel approach is the addition of the Phase 2 Carb Controller™/ StarchLite™ brand of proprietary fractionated white bean extract (FWBE) prepared from the White Kidney Bean (Phaseoulus vulgaris) which has α-amylase inhibitory properties. The purpose of this paper is to describe the results of the incorporation of the FWBE into baked goods and consumer acceptance of such products.

The proposed mechanism of action for this FWBE is its ability to partially inhibit or delay the breakdown of complex carbohydrates into their more absorbable sub-constituents (mono and disaccharides). The delay in digestion may delay the gut absorption rate of glucose, and may also blunt the subsequent insulin rise.

The White Kidney Bean (Phaseolus vulgaris) inhibits salivary and pancreatic α-amylase (1). Phaseolus vulgaris binds to α-amylase in non-competitive fashion, optimally at a pH of 5.5, forming a 1:1 molar complex (2). Similar binding can be seen at neutral pH as well. White bean extracts are not expected to have any activity on the brush border enzymes and an vitro study (3) confirmed that it does not inhibit the brush-border enzyme maltose/glucoamylase.

The activity of Phaseolus vulgaris is dose dependent and extract dependent. Threshold levels of greater than 90% inhibition of amylase are required before any carbohydrate malabsorption is seen. Below this threshold, incomplete inhibition of amylase may not have any significant effect on carbohydrate malabsorption (4).

Animal toxicity studies (5) (both acute and chronic) were performed on the proprietary fractionated white bean extract (from Pharmachem).

Human clinical trials have yielded mixed results regarding weight loss, but pilot data shows promise for the ability of this ingredient to lower the glycemic index of carbohydrate containing foods.

FWBE is currently available in tablet, capsule or a powder form and consumers take this product before or with meals. This requires consumers to have easy access to FWBE during their meal time as well as to remember to take it before or with meals. This can become a cumbersome regimen and an inconvenience for the consumers.

Since convenience is one of the most important attribute desired by consumers in their lifestyle in general and in selection of food products in particular, incorporating FWBE into the high carbohydrate foods would provide consumers the desired convenience of not having to remember to take FWBE before or with their meals. Thus high carbohydrate foods with FWBE would become value added food products.

REQUIREMENTS

Incorporating FWBE into food products, particularly baked goods made with yeast raised dough such as breads and pizzas without affecting their product quality is challenging. When FWBE are to be incorporated in an existing baked goods product, the resulting product must be comparable in key attributes such as appearance, texture and taste to the existing product and liked by consumers as well as the existing product. The product must also be formulated to take into consideration the serving size and allow appropriate amount of FWBE per serving size of the product as consumed. In addition it should have a comparable shelf life.

A key factor to be taken into consideration in the development of baked goods would be the effect of FWBE on the performance of yeast during the preparation of the dough for the baked goods. FWBE should not be added during dough fermentation as they would reduce the available assailable carbohydrates and prevents yeast to act on assailable carbohydrates to convert them into carbon dioxide and alcohol.

Another important factor affecting the product performance would be the composition of flours, particularly the α-amylase enzyme content of the flours as it would affect the FWBE performance in the product. For example, rye flour contains high levels of α-amylase and makes it particularly difficult to formulate with FWBE.

A study to find ways to incorporate FWBE in baked goods using the product requirements described above was recently done by the author in a joint effort with the manufacturer of FWBE and the Center for Human Nutrition at UCLA. Among the products developed were three varieties of breads, cheese pizza, coffee cake and muffins.

Basic formulations for breads and pizza were developed broadly based on information available in the literature (6).

The wheat bread formulation consisted of wheat flour, flour, water, honey, vegetable oil, gluten, yeast and salt.

The multigrain bread formulation consisted of water, wheat flour, flour, multigrain mix, honey, vegetable oil, yeast, gluten and salt.

The rye bread formulation consisted of flour, water, rye flour, sugar, yeast, vegetable oil, caraway seeds and salt.

The cheese pizza formulation had three parts: Crust (flour, water, shortening, yeast, sugar, salt and gluten), pizza sauce and mozzarella cheese. Both pizza sauce and mozzarella cheese used in our work were retail products purchased from a local supermarket.

The coffee cake formulation was an adaptation of a basic coffee cake recipe from a retail cake mix product.

The blueberry muffin mix was a commercial product available locally.

Several trials and iterations in the method of preparation and processing conditions were required to develop successful prototypes of control and test products with white bean extract. Key factors in the preparation that influenced the product performance were the order and method of ingredient incorporation, the time and temperature requirements for the dough development and the baking of the product.

Prototype samples of both control products and test products with white bean extract were than evaluated by an outside consumer research company (Tragon Corp.) to assess consumer acceptance of the prototype test products made with white bean extract relative to a control product.

Products were tested using a sequential monadic, balanced block design by qualified consumers (Heads of household, 50% female & 50% male, age 25-65, category users) in the San Francisco area.

RESULTS

Wheat bread: Number of tasters -34 Hedonic (9-point liking) Results

 Control and Test were liked similarly by consumers. – The two products scored at statistical parity at the 95% and 90% confidence levels for all hedonic means.  Control and Test scored the same (6.1) for Overall Appearance.  Test was numerically better liked for Overall Texture (6.0 vs. 5.7).  Control was numerically better liked for Overall Taste (6.4 vs. 6.1).  Control (6.0) received a slightly better Overall Opinion score than Test (5.9).

Multigrain bread: Number of tasters -35 Hedonic (9-point liking) Results

 Control and Test were liked similarly by consumers. – The two products scored at statistical parity at the 95% and 90% confidence levels for all hedonic means.  Test was liked numerically higher than Control across all hedonic means. - Overall Appearance 6.8 vs. 6.5 - Overall Texture 6.4 vs. 6.1 - Overall Taste 6.5 vs. 6.3 - Overall Opinion 6.6 vs. 6.1

Rye Bread: Number of tasters -34 Hedonic (9-point liking) Results

 Consumers scored Control and Test similarly for Overall Texture, Overall Taste and Overall Opinion. – The two products scored at statistical parity at the 95% and 90% confidence levels for all hedonic means.  Consumers liked Control significantly more than Test for Overall Appearance (7.1 vs. 6.1)  Test was liked numerically higher than Control for Overall Taste (6.8 vs. 6.6)  Control was liked numerically higher than Test for Overall Texture (6.8 vs. 6.6)  Consumers numerically rated both products equally in Overall Opinion (6.6 vs. 6.6)

Cheese pizza: Number of tasters - 29 Hedonic (9-point liking) Results

 Control and Test were liked similarly by consumers. – The two products scored at statistical parity at the 95% and 90% confidence levels for all hedonic means.  Test was numerically better liked than Control for Overall Appearance (6.07 vs. 6.03)  Test was numerically better liked than Control for Overall Texture (6.28 vs. 6.07)  Test was numerically better liked than Control for Overall Taste (6.48 vs. 6.31)  Test was numerically better liked than Control for Overall Opinion (6.45 vs. 6.17)

Coffee Cake: Number of tasters -32 Hedonic (9-point liking) Results

 Control and Test were liked similarly by consumers.  The two products rated at statistical parity across all hedonic measures at the 90% and 95% Confidence Levels.  Test rated numerically higher in liking for Overall Appearance (6.2 vs. 6.1), Overall Texture (6.2 vs. 5.9) and Overall Taste (6.1 vs. 6.0)  Control and Test were rated equal numerically (6.0 vs. 6.0) in overall opinion.

Blueberry Muffins: Number of tasters -32 Hedonic (9-point liking) Results

 Control and Test were liked similarly by consumers.  The two products rated at statistical parity across all hedonic measures at the 90% and 95% Confidence Levels.  Control rated slightly higher numerically in liking for Overall Appearance (6.3 vs. 6.1) and Overall Opinion (6.7 vs. 6.5).  Test rated slightly higher numerically in liking for Overall Texture ( 6.7 vs. 6.5)  Control and Test were numerically rated equally in liking for Overall Taste ( 6.7 vs. 6.7)

SUMMARY

The StarchLite brand of proprietary fractionated white bean extract can be successfully incorporated in baked food products such as breads and pizzas without affecting the product quality and consumer acceptance while providing consumers a convenient way to reap the benefits of proprietary fractionated white bean extract.

DISCLOSURES AND REFERENCES

Dr. Udani provides consulting services for Pharmachem Laboratories, manufacturer of Phase2®

1. B.H. Meyer et al, Inhibition of starch absorption by alpha-amylase inactivator given with food. Lancet, 1(8330) p.934 (1983)

2. P. Layer et al, Partially purified white bean amylase inhibitor reduces starch digestion in vitro and inactivates intraduodenal amylase in humans. Gastroenterology, 88(6) pp.1895-1902 (1985)

3. C.B. Hollenbeck et al, Effects of a commercial starch blocker preparation on carbohydrate digestion and absorption: in vivo and in vitro studies. Am J Clin Nutr, 38(4) pp.498-503 (1983)

4. M. Boivin et al, Effect of a purified amylase inhibitor on carbohydrate metabolism after a mixed meal in healthy humans. Mayo Clin Proc, 62(4) pp.249-255 (1987)

5. R. Maheshwari et al, Acute and Chronic Toxicity Studies of Phaseolamin2250. Report to Sponsor. 2002

6. E. J. Tyler, Baking Science and Technology, I, II

KANAK UDANI, Ph.D. 5630 Silver Valley Avenue, Telephone: (818) 991 6590 Agoura Hills, CA 91301 Email: [email protected]

December 24, 2005

Pharmachem – Phase 2 project:

Wheat bread formulation with Phase 2:

All-purpose flour 550 grams Whole wheat flour 516 grams Water (100 degrees F) 504 grams Honey 192 grams Olive oil 55 grams Phase 2 34 grams Gluten 30 grams Yeast (active dry) 18.8 grams Salt 13.8 grams

Procedure: 1. Yeast proofing: Mix water, yeast and 12 grams of honey in a small bowl. Set aside for 10-15 minutes to activate yeast. Mixture should be bubbly and have a rounded crown. 2. Mixing: Place dry ingredients in a Hobart mixer bowl. Add oil, remaining honey and proofed yeast mixture. Mix with a dough hook at speed one for four minutes. Change to speed two and knead for five minutes, until the dough is smooth and elastic. 3. Place the dough in a greased bowl. Turn over to coat both sides. Cover with a plastic film and place in a warm place (90-100°F) to rise until the dough volume is doubled; approximately 1.5 hours. 4. Punch down dough. Divide dough into two 750 gram portions. Shape into loaves and place in greased baking loaf pans. (There will be excess dough and can be discarded). 5. Cover loaves loosely with plastic film; allow to rise in a warm place, until doubled in volume; approximately 1.0 hour. 6. Bake at 350°F for 25 minutes in a convection oven: until the bread loaves turn golden brown. Temperature at the center of the loaf should be 185-190°F. 7. Allow to rest in pans for 5 minutes. Remove loaves from the pans and put them on a cooling rack. 8. Allow the loaves to cool completely. 1-3 hours. KANAK UDANI, Ph.D. 5630 Silver Valley Avenue, Telephone: (818) 991 6590 Agoura Hills, CA 91301 Email: [email protected]

December 24, 2005 Pharmachem – Phase 2 project:

Wild Blueberry Muffins formulation with Phase 2:

Betty Crocker Wild Blueberry Muffin mix 423 grams (1package minus 9 grams) Large Eggs 2 eggs Whole Milk 71 grams Safflower Oil 56 grams Water 15 grams Phase 2 9 grams

Procedure: 1. Preheat oven to 425 degrees F. 2. Place paper baking cups in12 regular size muffin cups. 3. Drain blueberries and set aside. 4. Add dry muffin mix into a medium sized bowl. 5. Mix eggs, oil, water and milk together in a separate bowl. Stir in gently Phase 2 powder using a fork or a spatula until evenly suspended. 6. Add the mixture in step #5 to the muffin mix and stir until combined. 7. Gently stir in drained blueberries. 8. Divide the batter among 12 muffin cups, filling each cup about 2/3 full. 9. Bake 18 minutes or until golden brown and top of the muffin spring back when touched. 10. Place on a cooling rack to cool completely. KANAK UDANI, Ph.D. 5630 Silver Valley Avenue, Telephone: (818) 991 6590 Agoura Hills, CA 91301 Email: [email protected]

December 24, 2005 Pharmachem – Phase 2 project:

Cheese Pizza formulation with Phase 2:

Pizza dough: All-purpose flour 996 grams Water (110 degrees F) 550 grams Shortening 48 grams Yeast (active dry) 25 grams Sugar 12 grams Salt 10.5 grams Gluten 10 grams Phase 2 4 grams Ragu Thick & Zesty Pizza sauce: 200 grams/360 grams pizza dough Kraft Mozzarella cheese: 100 grams/360 grams pizza dough

Procedure: 1. Yeast proofing: Mix 200 grams of water, yeast and sugar in a small bowl. Set aside for 15 minutes to activate yeast. Mixture should be bubbly and have a rounded crown. 2. Dry blend 96 grams of all-purpose flour and 4 grams of Phase 2 in a small bowl and set aside. 3. Mixing: Add remaining water, yeast mixture, shortening and remaining flour, gluten and salt into a Hobart mixer bowl. Mix with a dough hook at speed one for four minutes until the dough structure is formed. Scrape the sides of the bowl. Change to speed two and knead for five minutes, until the dough is smooth and elastic. 4. Place the dough in a large bowl coated with cooking spray. Turn over to coat both sides. Cover with a plastic film and place in a warm place (90-100°F) to rise until the dough volume is doubled; approximately 45 minutes. 5. Punch down dough. Cover and let rest for five minutes. Knead in the remaining flour/Phase 2 mixture by hand until completely incorporated. 6. Divide dough into four 360 gram pieces. (There will be excess dough and can be discarded). 7. Roll each piece into a 14-inch circle between two sheets of plastic wrap. Make the whole round sheet of dough of the same thickness, including the edges. Shape and form each pizza at staggered rate to allow for staggered rising and baking times, e.g., approximately 10 minutes between each formed pizza. 8. Coat four 12-inch round pizza pans with cooking spray. Place dough in prepared pans; rotate pan, pressing dough until edges of pan are covered. Cover lightly with plastic wrap and let rise 20 minutes or until slightly puffy. Pierce dough with a fork 20 times to prevent excess puffing during baking. 9. Top each pierced and unbaked crust with 200 grams of pizza sauce and 100 grams of mozzarella cheese. Cover with sauce and cheese right to the edges, distributing the cheese as evenly as possible over the top of the pizza. 10. Bake one pizza at a time at 450°F for 7 minutes or until the crust is golden brown, rotating pan 180 degrees halfway through baking. (The crust and cheese should only be very slightly browned if the pizza is going to be reheated later. Reheating would than finish cooking). 11. Allow to rest in pans for 5 minutes. Remove the pizza on a on a cooling rack. 12. Weigh the finished pizza: Target weight 600-620 grams.

KANAK UDANI, Ph.D. 5630 Silver Valley Avenue, Telephone: (818) 991 6590 Agoura Hills, CA 91301 Email: [email protected]

December 24, 2005 Pharmachem – Phase 2 project:

Coffee cake formulation with Phase 2:

Part I Cake Batter:

Original Bisquick mix 242 grams Whole Milk 162.5 grams Sugar 38 grams Large Eggs 1 egg Phase 2 8 grams

Part II Cinnamon Streusel Mix:

Brown Sugar 73 grams Original Bisquick mix 40 grams Parkay soft margarine 28 grams Ground Cinnamon 3 grams

Procedure:

1. Prepare Cinnamon Streusel by mixing all ingredients in Part II in a small bowl until crumbly. Set aside. 2. Preheat oven to 375°F. 3. Spray an 8-inch square pan with corn oil PAM spray. 4. Beat together the milk and egg in a small bowl. In another bowl, stir together the Phase 2 powder and sugar. 5. Add the Phase 2 powder/sugar mixture to the milk and egg mixture, and beat with a rotary beater for two minutes, or until all clumps of Phase 2 are broken and dissolved. 6. Add this mixture to the dry Bisquick mix, and mix until blended to form the batter. 7. Spread the batter into the 8-inch square pan. 8. Sprinkle the Cinnamon Streusel made in step 1 over the batter. 9. Bake for 27 minutes or until golden brown. 10. Cool completely on a wire cooling rack. 11. Cut to make 10 servings. Product Documents and Technical Specifications

For Trade Distribution, only.