Appendix E Total Protein Assay of D-Ribose Total Protein Assay of Bioenergy RiboseTM The level of protein in Bioenergy RiboseTM was assayed using the Bradford method. The test method is based on the interaction between protein and a dye, Brilliant Blue G. When dissolved in an acid-alcoholic medium, the Brilliant Blue G dye reacts almost immediately with proteins to form a blue-colored protein- dye complex. The protein-dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorption at 595 nm is proportional to the protein present. This method is simple, rapid, and highly sensitive. The detection limit is 0.1 μg/mL with an advertised linear range to 5mg/mL. Our assay was performed using a kit (Product # TP0100) from Sigma-Aldrich. The kit contains all the necessary reagents and a standard protein as reference. Before assaying the test samples, the method/kit was first calibrated using the reference protein standard included in the kit. Standard protein solutions of different concentrations (from 0.0 to 6.0 μg/mL) were prepared and their absorptions at 595 nm were measured on a Shimadz UV-Vis spectrometer following the manufacturer’s suggested procedures. The results are presented in Table 1 and Figure 1. The absorption was proportional to the protein concentration. It is clear that the method works very well and the assay is linear in the test range. Six lots of Bioenergy RiboseTM (three from year 2006 and three from 2005) were randomly selected for the protein content assay. Two grams of each Bioenergy RiboseTM were dissolved in 0.85% NaCl solution to make a 20% D-ribose solution. The 0.85% NaCl solution used to dissolve D-ribose is recommended by Sigma–Aldrich since both the positive and negative controls employ the solution. The positive control consisted of the standard protein solution and the negative control contained 0.85% NaCl solution only. The D-ribose solutions were assayed following the same procedure as the reference protein in the calibration. The results are summarized in Table 2. It is clear that the assay works very well because both the positive and negative controls gave repeatable result as in the calibration. However, no detectable level of protein was detected in any lot of Bioenergy RiboseTM. The absorption at 595 nm showed some random small numbers indicating the assay is beyond the detectable limit. Therefore, it is concluded that no protein is detectable in the Bioenergy RiboseTM products. Table 1. Calibration of the Total Protein Assay Method Protein Absorption concentration (595nm) (μg/mL) 0.0 0.000 1.2 0.031 2.4 0.074 3.6 0.101 4.8 0.135 6.0 0.159 Figure 1. Calibration Curve for the Total Protein Assay Method Bradford Assay 0.200 0.150 0.100 0.050 Absorption (595 nm) 0.000 0.0 2.0 4.0 6.0 8.0 Protein Concentation (µg/mL) Table 2. Protein Assay for Bioenergy RiboseTM Test Sample /Lot Absorption Protein content number (595nm) (μg/mL) Negative control 0.000 No protein 50126501 0.001 Not detectable 50546501 0.002 Not detectable 50856501 -0.006 Not detectable 60126501 -0.001 Not detectable 60546501 0.004 Not detectable 60956501 0.000 Not detectable Positive control 0.158 6 μg/mL Appendix G Estimated Daily Intake of D-Ribose by the U.K. Population from Proposed Food- Uses in the E.U. ESTIMATED DAILY INTAKE OF D-RIBOSE BY THE U.K. POPULATION FROM PROPOSED FOOD-USES IN THE E.U. Prepared for: Bioenergy, Inc. 13840 Johnson St. NE Minneapolis, MN 55304 USA Prepared by: Cantox Health Sciences International 2233 Argentia Road, Suite 308 Mississauga, Ontario, Canada L5N 2X7 December 13, 2007 Mississauga, ON, Bridgewater, NJ, Fleet, Hampshire, Shinjuku, Tokyo CANADA USA UK JAPAN 905-542-2900 908-429-9202 +44 (0) 1252 400 505 81-3-5287-3522 ESTIMATED DAILY INTAKE OF D-RIBOSE BY THE U.K. POPULATION FROM PROPOSED FOOD-USES IN THE E.U. Table of Contents Page 1.0 INTRODUCTION 1 2.0 FOOD CONSUMPTION SURVEY DATA 1 2.1 Survey Description 1 2.2 Statistical Methods 3 3.0 FOOD USAGE DATA 3 4.0 FOOD SURVEY RESULTS 4 4.1 Estimated Daily Intake of D-Ribose from All Proposed Food-Uses in the E.U. 5 4.2 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses in the E.U. 6 4.2.1 All-Person Intakes 6 4.2.2 All-User Intakes 7 5.0 CONCLUSIONS 8 6.0 REFERENCES 8 List of Appendices APPENDIX A Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Different Population Groups Within the U.K. APPENDIX B Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Different Population Groups Within the U.K. APPENDIX C Representative MAFF Food Codes for All Proposed Food-Uses of D-Ribose in the E.U. Bioenergy, Inc. i December 13, 2007 List of Tables Table 3-1 Summary of the Individual Proposed Food Uses and Use-Levels for D-Ribose in the U.K. 4 Table 4.1-1 Summary of the Estimated Daily Intake of D-Ribose from All Proposed Food Categories in the U.K. by Population Group (NDNS Data) 5 Table 4.1-2 Summary of the Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from All Proposed Food Categories in the U.K. by Population Group (NDNS Data) 6 Table A-1 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Children Aged 1½ to 4½ Years Within the U.K. (NDNS Data, 1992- 1993) A-1 Table A-2 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Young People Aged 4 to 10 Years Within the U.K. (NDNS Data, 1997) A-3 Table A-3 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Female Teenagers Aged 11 to 18 Years Within the U.K. (NDNS Data, 1997) A-5 Table A-4 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Male Teenagers Aged 11 to 18 Years Within the U.K. (NDNS Data, 1997) A-7 Table A-5 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Female Adults Aged 16 to 64 Years Within the U.K. (NDNS Data, 2000-2001) A-9 Table A-6 Estimated Daily Intake of D-Ribose from Individual Proposed Food-Uses by Male Adults Aged 16 to 64 Years Within the U.K. (NDNS Data, 2000- 2001) A-11 Table B-1 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Children Aged 1½ to 4½ Years Within the U.K. (NDNS Data, 1992-1993) B-1 Table B-2 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Young People Aged 4 to 10 Years Within the U.K. (NDNS Data, 1997) B-3 Table B-3 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Female Teenagers Aged 11 to 18 Years Within the U.K. (NDNS Data, 1997) B-5 Table B-4 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Male Teenagers Aged 11 to 18 Years Within the U.K. (NDNS Data, 1997) B-7 Table B-5 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Female Adults Aged 16 to 64 Years Within the U.K. (NDNS Data, 2000-2001) B-9 Table B-6 Estimated Daily Per Kilogram Body Weight Intake of D-Ribose from Individual Proposed Food-Uses by Male Adults Aged 16 to 64 Years Within the U.K. (NDNS Data, 2000-2001) B-11 Bioenergy, Inc. ii December 13, 2007 ESTIMATED DAILY INTAKE OF D-RIBOSE BY THE U.K. POPULATION FROM PROPOSED FOOD-USES IN THE E.U. 1.0 INTRODUCTION D-Ribose is proposed for use in the European Union (E.U.) in foods, such as chocolate confectionary (soft candies (excluding chocolate bars)), dietary supplements (meal replacement beverages and meal replacement bars), ice cream (ice cream excluding frozen novelties), juices and ades (calorie reduced fruit drinks and ades, fruit juice, fruit nectars, and vegetable juice), miscellaneous (milk drinks excluding malts and shakes), low calorie soft drinks (low calorie carbonated soft drinks (excluding cola-type soft drinks), and low calorie ready-to-drink soft drinks), soft drinks (carbonated soft drinks, not low calorie (excluding cola-type beverages), ready-to-drink soft drinks, not low calorie, and sport and energy drinks), sugar confectionary (hard candy), tea, coffee, and water (instant and herbal tea), and yogurt, fromage frais, and other dairy desserts [yogurt (including frozen yogurt, excluding yogurt drinks)]. Estimates for the intake of D-ribose in the E.U. were based on the proposed use-levels and food consumption data collected as part of the United Kingdom (U.K.) Food Standards Agency’s, Dietary Survey Programme (DSP). Calculations for the mean and high-level (97.5th percentile) all-person and all-user intakes, and percent consuming were performed for each of the individual proposed food-uses for D-ribose. Similar calculations were used to determine the estimated total intake of D-ribose from all proposed food-uses combined. In both cases, the per person and per kilogram body weight intakes were reported for the following population groups: children, ages 1½ to 4½ ; young people, ages 4 to 10; female teenagers, ages 11 to 18; male teenagers, ages 11 to 18; female adults, ages 16 to 64; male adults, ages 16 to 64.