rubentito/OCR-IDL huggingface dataset:
8 It is not a chemical interaction which causes an inclusion compound to form, although this may be a factor in the net complexation observed. Van der Waals forces may be an important factor here (25), because of the close- ness of the components. The steric configurations of the molecules are such that the enclosing, or "host", molecule can spatially enclose the included, or "guest", molecule, leaving uneffected the bonding systems of the components. Powell (30) notes that the important factor in inclusion formation is geometry, rather than chemistry. Geometrical rather than chemical charac- teristics of the molecules are limiting factors in the interaction. The most essential feature (1) of the host is its ability to form a rigid structure containing hollow spaces within which the prospective guest can fit. Pauling point out (29) that often the lattice is formed by mole- cules linked to one another by hydrogen bonds, host to host. In view of the interesting properties of the cyclodextrins, it was decided to study their effectiveness in selective filtration of cigarette smoke. During this study, the commercial potential of the cyclodextrin inclusion compounds as flavorants and other uses were recognized (35-38) and investigated. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
9 B. EXPERIMENTAL I. Preparation of Bacillus Macerans Amylase The Bacillus macerans cultures were prepared by Miss Margaret Long of this laboratory (24). Amylase was isolated from the Bacillus macerans culture and purified using a modification of the methods of E. Tilden, et al. (40) and R. Kerr (21). The culture was centrifuged and the supernate filtered through a large Seitz filter (E.K size 14), capacity 2 liters. The amylase was precipitated by adding acetone, 33 percent by volume, and storing overnight at -15°C The mixture was then centrifuged and the precipitate (amylase) dissolved in water. The enzyme (amylase) solution was stored at 5°C. until used for preparation of the cyclodextrin. The activity of the Bacillus macerans amylase was determined initially using the method of D. French and co-workers (12) and later using the method of W. Hale and L. Rawlins (17) as modified by W. Blackwell of this laboratory (4). II. Preparation of Alpha, Beta and Gamma Cyclodextrins a. Introduction The original separation method of Schardinger depended on the ease of crystallization of the beta cyclodextrin from water and its low solubility (about 1.5 percent at room temperature) the alpha cyclodextrin was obtained from the mother liquor by treatment with alcohol (33). A substantial advance in technique was provided by Freudenberg and Jacobi (15), who relied not only on solubility differences of the cyclodextrins themselves, but also on the differences of solubilities and rates of crystallization of the cyclodextrin acetates. Although the method is somwhat tedious, insofar as it required several acetyla- - tions and saponifications, it has the distinct merit of producing an alpha dextrin quite free from other dextrin impurities. In further studies, Freudenberg, Plankenhorn and Knauber (16) modified the previous fractionation scheme, without decreasing its complexity. These authors made use of the observation (14) that bromobenzene does not precipitate alpha cyclodextrin, but the beta and gamma cyclodextrins are readily precipitated. Beta cyclodextrin was separated from gamma cyclodextrin by differential dissolution of the gamma cyclodextrin in warm pyridine. b. The French Method French and co-workers (1949) (12) sought to decrease the complexi- ties of previous fractionation schemes. Fractionation was achieved, Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
10 without acetylation of the cyclodextrins, by taking advantage of the low solubility of beta cyclodextrin in water, the differential precipi- - tating action of bromobenzene on beta and gamma cyclodextrin (leaving alpha cyclodextrin in solution), and the low solubility of gamma cyclo. dextrin in 60 percent n-propanol. These authors also made use of the fact that conditions during the original enzymolysis of the starch can be preadjusted, depending on which cyclodextrin is desired (26) Large yields of beta cyclodextrin are obtained if the enzymolysis is con- ducted in the presence of a precipitant such as toluene or trichloro- ethylene. Maximal yields of alpha cyclodextrin are obtained, in the absence of a precipitant, with a relatively short conversion. For preparation of gamma cyclodextrin, the enzymolysis is very extensive, again in the absence of a precipitant (see figure 1). Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
11 FIGURE 1 FRACTIONATION SCHEME OF FRENCH, LEVINE, PAZUR AND NORBERG extensive enzymolysis enzymolysis in in presence of toluene absence of dextrin mixture precipitant centrifuge dilute to ppt. with 2% solids bromo- benzene discard or ppt. with effluent trichlorethylene concentrate to 5% solids, precipitate with trichloro- ethylene 8-dextrin + y-dextrin Q-dextrin solution remove tol- - concentrate (discard) uene or bro- to 25% sol- mobenzene ids, cool recrystallize from 60% by boiling 1-propanol with water; a-dextrin-1-propanol complex boil with 8-dextrin cool water recrystallize dilute to ppt. with from boiling 3% solids, bromobenzene pure a-dextrin water pure B -dextrin dissolve in boil to remove solution (discard) boiling bromobenzene water add 1.5 vol. of 1-propanol Y-dextrin-l-propano: complex repeated crystallization from 60% 1-propanol pure y-dextrin-1- mother liquors propanol complex (B-dextrin + y-dextrin) remove 1-propanol - and boil with concentrate to return to B-Y crudes water 40-50% solids, cool pure y-dextrin Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
12 c. The Cramer Method Cramer and Henglein (10) found that by using suitable cyclodextrin occlusion compounds they were able to work out a procedure for the separation of alpha, beta and gamma cyclodextrin in which specific precipitation agents were employed. The separation procedure also made possible the isolation of gamma cyclodextrin. In the separation procedure (Figure 2) beta and gamma cyclodextrin are first precipitated with p-cymene and them separated with fluoro- benzene. Gamma cyclodextrin is obtained in pure form by precipitating it as an anthracene derivative from the mother liquor. Alpha cyclo- dextrin is isolated with cyclohexane. The use of specific precipitating agents in this method eliminates the concentration of large liquid volumes. The compounds precipitate in very pure form. FIGURE 2 FRACTIONATION SCHEME OF CRAMER AND HENGLEIN I. Mixture a, p- and y-dextrin 4% solution + p-cymene V. Solution II. Precipitate -dextrin, remaining B. - B - and Y-dextrin-p-cymene adduct, and y-dextrin, double removal of p-cymene, preparation volume of 1% solution + cyclohexane fluorobenzene VII. Remäinder VI. Precipitate IV. Solution III. Precipitate a, B- and y-dextrin -dextrin-cyclo- Solution of y-dextrin B-dextrin-fluoro- is added to the next hexane-adduct; is adjusted to 10%, benzene, removal of separation maximum removal of cyclo- treated with anthra- - fluorobenzene con- 20% of I. hexane, crystal- - cene in ether, re- centrate in vacuo, lizing in water, peated if necessary. and 8-dextrin is pure -dextrin. The mother liquor is obtained in pure added to VII. The form. Y-dextrin-anthracene adduct is decomposed in water and - dextrin crystallize. 1234 Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094
13 d. The Kono Method Kono and Nikuni (22) found that stirring the digestion mixture at a reduced temperature (40°F.) resulted in an increased yield of beta cyclodextrin. Otherwise, this method very closely resembles that of D. French et al. (12). In addition, this method also offers selective preparation of predominately alpha cyclodextrin or of beta cyclodextrin. The fractionation scheme of this method is presented in Figure 3. FIGURE 3 FRACTIONATION SCHEME OF KONO AND NIKUNI Potato starch 200 g. + macerans amylase 4% solution Toluene 100 cc 5 1 40°C. 96 hrs. (24 C.P.) digestion stirred for 15 hrs. in cold room boiled for 1 hr. . impure B-dextrin Tetrachlorethane 100 cc added 70 g stirred for 24 hrs. crystallized twice from water, then treated c carbon impure a-dextrin (30 g.) pure B-dextrin [a D = + 162° (51 g.) treated c carbon crystallized twice from 60% propanol crystallized from water filtrate pure a-dextrin D = + 150.06 (15 g.) Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094
14 e. Effect of Enzyme_Concentrationon the Yield of Beta Cyclodextrin Six different concentrations of Bacillus macerans amylase were added to uniform solutions of soluble starch to determine which concen- tration of enzyme would give the greatest yield of beta cyclodextrin. Six aqueous solutions of soluble starch were prepared (100 gm./2 liters) . The enzyme was added to each flask in different concentration (see Table I). The total volume of each flask was adjusted to 3 liters with distilled water. The flasks were incubated at 40°C for 11 days for static digestion. Beta cyclodextrin was isolated from the digest according to the method of Cramer (10) The results of this study are presented in Table I. TABLE I EFFECT OF THE CONCENTRATION OF B. MACERANS AMYLASE ON THE YIELD OF BETA CYCLODEXTRIN Concentration of Amylase Yield of Beta Sample No. (Hale & Rawlins Units) Cyclodextrin (gms.) 1 250 3.2 2 200 0.3 3 150 7.2 4 100 6.3 5 50 7.1 6 10 5.3 The data in Table I indicate the optimum concentration of B. macerans amylase for production of beta cyclodextrin is between 50 and 150 Hale & Rawlins units of the enzyme per 100 gms. of soluble starch. However, a reasonably high yield of beta cyclodextrin was obtained even at the low enzyme concentration of 10 Hale & Rawlins units per 100 gms. of soluble starch. The low yield of beta cyclo- dextrin obtained at an enzyme concentration of 200 Hale & Rawlins units per 100 gms. of soluble starch may have been caused by a contaminant. f. Photomicrographs of the Cyclodextrins Photomicrographs were made of the alpha, beta and gamma cyclo- dextrins which were prepared in this laboratory. The crystal structures of the three types of cyclodextrins prepared were found to duplicate those described by French and co-workers (12). These photomicrographs are presented in Figures 4, 5 and 6. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
15 FIGURE 4 ALPHA CYCLODEXTRIN FROM 60% N-PROPANOL, HEXAGONAL FORM, APPROXIMATELY 700 MAGNIFICATIONS Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
50096 1238 c a
17 FIGURE 6 GAMMA CYCLODEXTRIN FROM 60% N-PROPANOL, SQUARE CRYSTALS, APPROXIMATELY 700 MAGNIFICATIONS A t Source: :ttps://www.industrydocuments.ucsf.edu/docs/ffbb0094
18 n. Large Batch Preparations Large batches of beta cyclodextrin were prepared using improvised equipment. All three of the methods (French, Cramer, and Kono) men- tioned in Sections B.II.b.,c ,d. of this report were used in these large batch preparations. Soluble starch solution (4-5%) was prepared in 2 liter batches. Usually 150 liters of this starch solution was used as the substrate. This solution was placed in a 55 gallon stain- less steel drum and the Bacillus macerans amylase was added; the mixture was then stirred with a large stirring motor. The temperature (40°C.) was controlled by use of copper coils in the drum and circu- lating water or by locating the entire apparatus in a cubicle (Yerkes Building) at a constant temperature and humidity. After digestion, the mixture was centrifuged. The supernate was concentrated on the "turba-film" evaporator in the pilot plant. The alpha, beta and gamma cyclodextrins were then isolated from this concentrate using one of the methods mentioned above. The following table illustrates the yields of beta cyclodextrin obtained using the "large batch" methods and laboratory scale methods. TABLE II COMPARISON OF THE YIELD OF BETA CYCLODEXTRIN FROM LABORATORY SCALE AND LARGE BATCH PREPARATIONS Wt. Soluble Wt. of Beta Large Batch Ave. Lab. Scale Method Starch (gms.) Cyclodextrin (gms.) % Yield % Yield French 2800 75 2.7 12.9 Cramer 5700 750 13.1 29.5 Kono 5000 No Yield 0 25.0 Cramer (Substituted 244 4.9 29.5 for above) Cramer 7500 304 4.0 29.5 It is felt that with proper fermentation equipment much greater yields of beta cyclodextrin could have been obtained in the large scale preparations. In the case of the Kono method in the above table, no yield was obtained because the proper experimental conditions could not be maintained. Therefore, the Cramer method was substituted to isolate the beta cyclodextrin which had been formed. 0511 Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094
19 The following amounts of the cyclodextrin are presently on hand in a highly pure form: alpha cyclodextrin - 215 g. beta cyclodextrin - 1457 g. gamma cyclodextrin - 239 g. III. Investigation of the Selective Filtration Properties of the Cyclodextrins a. Preparation of Cigarettes and Filter_Tips Approximately ten pounds of C3-C9 strips (flue-cured tobacco fraction of the CAMEL blend) were obtained from the Manufacturing Department for making the cigarettes. The strips were cut on an Imhoff cutter set at 32 cuts per inch. The cut tobacco was stored in a chamber at the proper relative humidity to obtain a 12-12.5 percent moisture content in the tobacco. The tobacco was fabricated into cigarettes (68 mm.) on the Hauni machine in this laboratory. The cigarettes were stored in a chamber at the proper relative humidity to maintain a 12-12.5 percent moisture level. Unplasticized filter plugs (Estron) were obtained from the Manufacturing Department. The "plugs" were wrapped with plastic tape (Scotch Pressure Sensitive. Tape-Water Insoluble) and cut into 25.5 mm. sections using a filter plug cutter. The cyclodextrins were introduced into the filter plug by applying 1.2 ml. of a 2 percent (aqueous) dextrin solution (burette) to the plug and drying it in a forced draft oven for 15 hours at 80°C. After drying, the "treated" plugs were cut into 17 mm. sections by cutting 4.25 mm. off of each. end of the 25.5 mm. plug. These ends were cut from the treated plugs to remove the cyclodextrin which crystallized in these areas during drying. In the case of the con- trols, 17 mm. sections were cut directly from the original plugs. The filter plugs (17 mm.) were attached to the C3-C9 cigarettes using plastic tape cut into sections 1/2 inch wide and 55 mm. long. The final tipped cigarettes which were used for analyses were free of loose ends and soft spots and were selected in a weight range of 1.02-1.06 grams. b. Polycyclics Samples of the cigarettes described in Section III.a. were analyzed for polycyclics in the smoke by the Analytical Division (18) The results of these analyses are presented in Tables III-A and III-B. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
20 TABLE III-A (First Determination) POLYCYCLICS IN THE SMOKE OF CIGARETTES WITH CYCLODEXTRIN-TREATED FILTER TIPS Amount of Draft Benz [b] 1,4-Benzo- Filter Cyclodextrin Resistence Fluorathene Fluorathene % pyrene % Type Mg. % of Filter y/150 cig. y/150 cig. Diff. y/150 cig. Diff. - Control -- -- 2.12 5.75* 1.45 -- 2.23 -- Alpha 8.8 6.2 2.34 18.3 0.72 -56.3 1.50 -32.7 Beta 6.0 4.3 2.22 18.7 0.78 -46.2 1.83 -17.9 Gamma 12.6 8.6 2.33 13.7 0.87 -40.0 2.23 N.D. *This sample was found to have a break in the flask and an undeterminable amount of the sample was lost. TABLE III-B (Second Determination) POLYCYCLICS IN THE SMOKE OF CIGARETTES WITH CYCLODEXTRIN-TREATED FILTER TIPS Amount of Benz [b] 3,4-Benzo- Filter Cyclodextrin Puffs Fluoranthene % Fluoranthene % pyrene % Type Mg. % Cig. y/150 cig. Diff. y/150 cig. Diff. y/150 cig.Diff Control -- -- 9.21 11.95 - - 1.01 -- 1.31 -- Alpha 11.21 7.9 9.88 12.65 +5.9 0.99 N.D. 1.41 +7.6 Beta 10.77 7.7 9.98 12.65 +5.9 0.83 -17.8 1.14 -13.0 Gamma 6.10 4.5 9.33 11.25 -5.9 0.85 -15.8 1.31 N.D. The data in Table III-A suggest a significant removal of 3,4- benzopyrene from the smoke of cigarettes with alpha and beta cyclodextrin- treated filter tips. In addition, there is also a significant removal of benz [b] fluoranthene from the smoke of cigarettes in all three samples treated with cyclodextrins. No definite conclusion could be made in the case of the fluoranthene because of the loss of the control sample. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
21 The data in Table III-B do not substantiate the original con- clusions. Instead, it is found there was only slight removal of 3, 4-benzopyrene by the beta cyclodextrin, slight removal of benz [b] fluoranthene by the beta and gamma cyclodextrin and also a slight removal of fluoranthene by the gamma cyclodextrin. Since there were two sets of conflicting data, analysis of another set of these same samples was requested of the Analytical Division. This set of samples has not yet been analyzed. Therefore, no definite conclusion can be made on the removal of polycyclics from the smoke of cigarettes by cyclodextrin-treated filter tips at this time. C. Phenols Smoke from the cigarettes described in Section III.a. was analyzed for phenols by the Analytical Division (41) . The results of these analyses are presented in Table IV. Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094
TABLE IV PHENOLS IN THE SMOKE OF CIGARETTES WITH CYCLODEXTRIN-TREATED FILTER TIPS (A11 the results are expressed as Mg./Cigarette) Phenol 0-Cresol M-Cresol Total Cresol Guiaciol Xylencl Total Phenol Control No. 1 .0621 .0154 .0248 .0397 .0199 .0275 .1492 Control No. 2 .0627 .0169 .0231 .0396 .0191 .0269 .1483 Control Ave. 06241.5% .0162+4.6% 0240+3.5% .039710.3% .019512.1% .0272+1.1% .1488 Alpha No. 1 .0646 .0162 .0273 .0429 .0211 .0289 .1575 Alpha No. 2 .0664 .0174 .0261 .0431 .0206 .0286 .1587 22 Alpha Ave. 0655+1.4% 016813.5% .026712.2% .043010.2% .0209+1.2% 028810.5% .1581 Beta No. 1 .0628 .0166 .0248 .0408 .0198 .0273 .1507 Beta No. 2 .0677 .0175 .0275 .0444 .0210 .0296 .1627 Beta Ave. 065323.5% 017112.6% 026215.3% .042614.2% .020443.0% 0285+4.0% .1567 Gamma No. 1 .0603 .0155 .0246 .0396 .0189 .0257 .1445 Gamma No. 2 .0597 .0157 .0247 .0399 .0188 .0259 .1443 Gamma Ave. .06001.5% .0156+0.6% .0247+0.2% .0398+0.3% .0189+0.3% .0258*0.4% .1444 9600 9 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
23 The data in Table IV indicate there is no significant difference between the total phenol content of the smoke from the cigarettes with cyclodextrin-treated filter tips and the control cigarettes. d. Fatty Acids Samples of the cigarettes described in Section III.a. were analyzed for fatty acids by Dr. S. A. Bellin (3). The results of these analyses are presented in Table V. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
TABLE V FATTY ACIDS IN THE SMOKE OF CIGARETTES WITH CYCLODEXTRIN-TREATED FILTER TIPS Alpha Beta Gamma Analyses Control Cyclodextrin Cyclodextrin Cyclodextrin Weight of smoke condensates 134 144 145 141 (mg./10 cig.) Hexane solubles retained on filters 63 65 63 64 (mg./10 cig.) Weight of fatty acids in smoke condenser* 10.3 11.6 10.6 10.4 (mg./10 cig.) Unknown acid (As % of Total Fatty Acid) 1.4 0.4 - - -- (Mixture) Unknown acid " -- 0.3 -- -- Unknown acid " 2.1 1.8 0.8 1.9 Myristate " 3.5 2.8 3.6 2.6 Pentadecanoate " 2.1 2.0 2.4 2.2 Pentadecanoate " 2.5 2.0 2.3 2.0 Palmitate " 39.1 36.3 36.3 35.8 Unknown acid " 0.9 0.5 -- 1.0 Stearate " 8.5 7.7 8.6 7.9 Oleate " 8.9 9.6 11.0 10.2 Linoleate " 11.8 13.4 13.0 12.4 Unknown acid " 0.1 1.4 -- 2.6 Linolenate ii 19.1 22.0 22.0 21.4 *Weight of methyl esters of C14-C18 Acids 9721 9600 S Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
25 There is no significant difference between the control and treated cigarette samples in any of the above analyses (3) IV. Investigation of the Selective Filtration Properties of the Cyclodextrin-Acetates a. Preparation of the Cyclodextrin Acetates The cyclodextrin-acetates were prepared by the method of D. French (12). Dry alpha cyclodextrin was added in four equal parts to five parts of boiling acetic anhydride containing one-half part anhydrous sodium acetate. After the final addition, the mixture was refluxed for 30 minutes, allowed to cool to room temperature, and poured with stirring into cracked ice and water. The cyclodextrin- acetate was then suction filtered. Beta and gamma cyclodextrin were acetylated in the same manner. The crude alpha and beta cyclodextrin- acetates were crystallized from 10-15 parts of boiling toluene by cooling to room temperature. Gamma cyclodextrin-acetate was crystal- lized from 2-3 parts of hot butyl acetate. b. Preparation of Cigarettes and Filter_Tips Approximately 1,000 WINSTON cigarettes (untipped) were obtained from the Manufacturing Department. Cigarettes which were free of loose ends and soft spots were selected in a weight range of 0.96-1.02 grams for tipping. Unplasticized filter plugs were obtained from the Manufacturing Department. The plugs were wrapped with plastic tape (Scotch Pressure Sensitive Tape-Water Insoluble) and cut into 25.5 mm. sections using a filter plug cutter. The cyclodextrin-acetates were introduced into the filter plugs by applying 1 ml. of a 1 percent (methanol) cyclodextrin-acetate solution (burette) to the plug and drying at room temperature for 4 hours. After drying, the treated plugs were cut into 17 mm. sections by cutting 4.25 mm. off of each end of the 25.5 mm. plug. These ends were cut from the treated plugs to remove the cyclodextrin-acetate which had crystallized in these areas during drying. In the case of the controls, 17 mm. sections were cut directly from the original plugs. The filter plugs (17 mm.) were attached to the WINSTON ciga- rettes using plastic tape cut into sections 0.5 inch wide and 55 mm. long. The final tipped cigarettes were submitted to Mr. Wade Williard of Quality Control who selected 20 cigarettes of each cyclodextrin- acetate for submission to Dr. A. Laurene of the Analytical Division for analysis. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
26 C. Smoke Analysis for Phenol The final tipped cigarettes were submitted to Dr. A. Laurene (23) of the Analytical Division for analysis of phenols. The results are presented in Table VI. TABLE VI PHENOL IN THE SMOKE OF CIGARETTES WITH CYCLODEXTRIN ACETATE-TREATED FILTER TIPS Phenol (mg./cigarette) Draft Wt. Range Vapor Mass Samples Code Resistence Cigarettes Fractometer Spectrometer alpha-acetate L-340A 1.8-2.4 1.279-1.313 74.5 67.7 beta-acetate L-340C 1.9-2.1 1.275-1.318 68.6 67.4 gamma-acetate L-340D 1.8-2.1 1.265-1.301 67.3 69.1 control L-340B 2.7-3.4 1.260-1.300 63.1 58.0 There is no removal of phenols from the smoke of cigarettes with cyclodextrin acetate-treated filter tips. V. Preparation of Cyclodextrin-Flavorant Inclusion Compounds The method used in the preparation of the cyclodextrin-inclusior compounds was essentially that of H. Schlenk (34). The inclusion component was added to a hot, saturated, aqueous cyclodextrin solution, which was allowed to cool slowly (room temperature) while being shaken for 12-24 hours. The following list of inclusion compounds were successfully prepared using this technique. The ratio of beta cyclodextrin to the inclusion com- ponent was determined by T. G. Harrell (19) of the Analytical Division. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
27 TABLE VII LIST OF BETA CYCLODEXTRIN-FLAVORANT INCLUSION COMPOUNDS PREPARED 1. Imitation prune flavor, No. 50.066/A, Firmenich & Co. 2. Imitation peach flavor, No. 51.976/A, Firmenich & Co. 3. Imitation apple flavor, No. 50.047/A, Firmenich & Co. 4. Imitation grape flavor, No. 59.150/T, Firmenich & Co. 5. Imitation butter flavor, No. 51.561/A, Firmenich & Co. 6. Imitation butter flavor, No. 59.049/A, Firmenich & Co. 7. Imitation lemon juice flavor, No. 51.124/A, Firmenich & Co. 8. Imitation vanilla flavor, No. 59.106/A, Firmenich & Co. 9. Imitation burnt sugar flavor, No. 59.267/AP, Firmenich & Co. 10. Imitation coffee flavor, No. 50.439/AP, Firmenich & Co. 11. Imitation orange juice flavor, No. 59.107/A, Firmenich & Co. 12. Imitation rum flavor, No. 50.860/C, Firmenich & Co. 13. Liquid sunshine, No. 9601, Firmenich & Co. 14. Oil of 1emon, No. 1160, Felton Chemical Co. 15. Oil of lime, No. n.o., Felton Chemical Co. 16. Imitation honey base, No. 27, Felton Chemical Co. 17. Imitation fruit flavor, No. 952, Felton Chemical Co. 18. Cinnamic aldehyde, Fritzsche Bros. 19. Oil of peppermint, USP XIV, Fritzsche Bros. 20. Compounded perfume oil, No. 19434, Fritzsche Bros. 21. Vanillin, No. 167, Dow Chemical Co. 22. Diacetyl, No. 1591, Eastman Organic Chemical 23. Ethyl propionate, Source Unknown 24. Imitation McKinley apple, No. N-602, Dragoco, Inc. 25. 50 Spearmint, 50 Carvone, Norda Co. 26. Imitation peach concentrate, F-1975, Givandan Flavors, Inc. 27. Imitation peach flavor, No. F-110, Givandan Flavors, Inc. 28. Imitation apple concentrate, S-3778, International Flavor & Fragrances, Inc. 29. Arome artif. n. alc. lime, No. 13588, Antoine Chiris Bros., Paris 30. Isovaleric acid, Dr. C. Nystrom, this laboratory 31. 8-methyl valeric acid, Dr. C. Nystrom, this laboratory 32. a-methyl butyric acid, Dr. C. Nystrom, this laboratory 33. n-butyric acid, Dr. C. Nystrom, this laboratory A beta cyclodextrin-coffee headnote flavor inclusion compound was also prepared. For this preparation, 100 grams of coffee (A & P, Red Circle, drip grind, freshly ground) was placed in a one-liter Erlenmeyer flask and 500 ml. of water were added. The mixture was slowly heated to boiling and the flask flushed with nitrogen which was passed through a condenser and then bubbled through a solution of beta cyclodextrin. The beta cyclodextrin solution was in a gas-washing bottle fitted with a sintered glass sparger; the solution was held at approximately 30-40°C. with constant stirring on a magnetic stirrer hot plate combination unit. After boiling for 2 hours, a fresh batch of coffee was placed in the Erlenmeyer flask and the process repeated. After two pounds of coffee had been treated in this way, the Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
28 beta cyclodextrin solution was allowed to cool to room temperature and stirred for approximately 12 hours. The white precipate of the beta cyclodextrin-coffee headnote flavors was separated by filtering with vacuum and dried. VI. Investigation of the Commercial Applications for the Cyclodextrin-Flavorant Inclusion Compounds a. Cakes One of the problems in the Baking Industry today is the use of volatile flavors and their subsequent loss during the baking process. An example of this problem is the butter flavor 51.561/A developed by the Firmenich Company. It was found that a high percentage of this flavor was lost during the baking process (27). With this problem in mind, it was proposed that beta cyclodextrin-inclusior compounds of these flavors might offer a solution. These compounds were prepared and tested in cakes. Cakes were baked using comparable beta cyclodextrin-flavor inclusion compounds and commercially available powdered flavors as flavorants. For each different flavor tested, three boxes of Duncan Hines, White, Deluxe Cake Mix (1 1b. 2-1/2 oz.) were used and the cakes prepared according to the directions on the package. During the mixing step, the beta cyclodextrin-flavo inclusion compound was added to one mix, the comparable powdered flavor was added to another mix, and the third mix was used per se, as the control. All of the flavors (both liquid and powdered) used in this experiment were supplied by Firmenich & Co. The preparation of the beta cyclodextrin-flavor inclusion compounds using the Firmenich & Co. liquid flavors is described in Section B.V. of this report. Equiva- lent amounts of each type of flavor were always added to the cake mixes. Firmenich & Co. recommended that 1 ounce of the powdered flavor be added to 100 pounds of dry cake mix. This recommendation was used as the basis for calculating the equivalent amounts of each flavorant to add to the cake mixes. The ratio of the beta cyclodextrin to the flavor component was determined by T. G. Harrell (19) of the Analytical Division for each of the inclusion compounds used in this experiment. These values were used to calculate equivalent amounts of the beta cyclodextrin-flavor inclusion compounds. The cakes were panel-tested to determine which cake retained the greatest amount of flavor after baking. The cakes were cooled and approximately 1 inch cubes were cut and presented to the panelists. A paper cup of water was offered for rinsing between samples. The question was asked of each panelist, "Which sample has the strongest flavor?" It was emphasized that we were not interested in preference. Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094
29 The results of these panel tests are presented in Table VIII. TABLE VIII COMPARISON OF BETA CYCLODEXTRIN-FLAVOR INCLUSION COMPOUNDS WITH POWDERED FLAVORS IN CAKES Test No. 1 - Imitation butter flavor, 51.561/A, Firmenich & Co. Total panelists 47 Number of panelists selecting the beta cyclodextrin complex 40 Number of panelists selecting the powdered flavor 5 Number of panelists selecting the control 2 Test No. 2 - Imitation butter flavor, 59.049/A, Firmenich & Co. Total panelists 50 Number of panelists selecting the beta cyclodextrin complex 39 Number of panelists selecting the powdered flavor 8 Number of panelists selecting the control 3 Test No. 3 - Imitation burnt sugar flavor, 59.267/AP, Firmenich & Co. Total panelists 50 Number of panelists selecting the beta cyclodextrin complex 38 Number of panelists selecting the powdered flavor 11 Number of panelists selecting the control 1 Test No. 4 - Imitation orange juice flavor, 59.107/A, Firmenich & Co. Total panelists 49 Number of panelists selecting the beta cyclodextrin complex 45 Number of panelists selecting the powdered flavor 3 Number of panelists selecting the control 1 Test No. 5 - Imitation 1emon juice flavor, 51.124/AP, Firmenich & Co. Total panelists 50 Number of panelists selecting the beta cyclodextrin complex 20 Number of panelists selecting the powdered flavor 23 Number of panelists selecting the control 7 1531 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
30 The panel overwhelmingly selected the cakes flavored with the beta cyclodextrin-flavor complex over the powdered flavor cakes in four of the five flavors tested. These results indicate that in four of the flavors tested the flavor is retained during baking temperature in the beta cyclodextrin complex. In the case of the 1 emon juice flavor, it may be rationalized that the powdered form of this flavor is more stable to heat, since the panel could not distinguish between the two types of flavors in the 1emon cakes. b. Chewing Tobacco Beta cyclodextrin-flavor inclusion compounds were tested as flavorants on chewing tobacco. The flavor inclusion compounds were prepared as described in Section B.V. of this report and were submitted to Mr. E. H. Harwood of this laboratory for panel testing. The fol- lowing inclusion compounds were prepared with beta cyclodextrin: 1. Imitation McKinley apple, N-602, Dragoco, Inc. 2. Diacetyl, Eastman Organic Chemicals 3. Imitation honey base, Felton Chemical Co. 4. Imitation apple concentrate, S-3778, International Flavors & Fragrances, Inc. 5. Imitation peach concentrate, F-1975, Givandan Flavors, Inc. 6. Imitation peach flavor, F-110, Givandan Flavors, Inc. 7. Imitation prune flavor, 50.066/A, Firmenich & Co. 8. Imitation peach flavor, 51.976/A, Firmenich & Co. 9. Imitation apple flavor, 50.047/A, Firmenich & Co. 10. Imitation grape flavor, 59.150/T, Firmenich & Co. According to Mr. E. H. Harwood (20), two of the above flavor complexes were successfully panel-tested on chewing tobacco. The samples containing the imitation peach flavor, F-110, Givandan Flavors, Inc., inclusion compound was overwhelmingly selected over the control in the panel test. In another panel test, the samples containing the Imitation McKinley apple flavor, N-602, Dragoco, Inc., inclusion compound were also overwhelmingly selected over the control samples. It was also observed that there was a slow release effect of the flavor in the case of the beta cyclodextrin inclusion compound. Many of the panelists remarked that this flavor was "longer lasting". None of the other flavor inclusion compounds were panel tested on a. large scale. 2341 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
31 c. Smoking Tobacco Beta cyclodextrin-flavor inclusion compounds became of interest for use as flavors in smoking tobacco from the standpoint of stabilizing volatile flavors. It was also proposed that flavors which reacted with the tobacco itself (e.g. oil of 1 emon) could also be utilized when applied to tobacco in the form of a cyclodextrin inclusion compound. It was also considered that these inclusion compounds could be used to mask the external odor (if objectionable) of some flavors. 1. BETA CYCLODEXTRIN SMOKING PANEL TEST Since the cyclodextrins were an unknown entity from the standpoint of smoking quality in cigarettes, a smoking panel test was made on cigarettes which were treated with beta cyclodextrin. CAMEL blend tobacco (700 g.) was sprayed with an aqueous solution of beta cyclodextrin (7 gn./200 ml.). Another portion of the tobacco (700 g.) was sprayed with 200 ml. of distilled water. The two samples were fabricated into 70 mm. cigarettes. Cigarettes were selected for panel testing which were free of soft spots and loose ends in a weight range of 1.00-1.08 gm. The selected cigarettes were then submitted to Dr. S. A. Bellin for panel testing (2). Two triangle tests were made, one with the test cigarette odd and the other with the control cigarette odd. In all cases the code numbers, 1, 2, 3, were randomized within the panel. Dr. Bellin concluded from this test that the 30-member panel was unable to distinguish the cigarettes treated with beta cyclo- dextrin from the control cigarettes (2). 2. VOLATILE FLAVORANTS Ethyl propionate was suggested as a good example of a volatile flavorant by Mr. E. H. Harwood. A beta cyclodextrin- ethyl propionate inclusion compound was prepared as described in Section B.V. of this report. The ethyl propionate inclusion compound was applied to CAMEL blend tobacco by sprinkling on the dry powder. Cigarettes were then fabricated using a small hand- operated machine and stored for 30 days at room temperature. When the cigarettes were smoked, the odor and flavor of the ethyl pro- pionate were readily apparent in both the mainstream and side- stream of the smoke. Control cigarettes were prepared in the same way except that a solution of ethyl propionate was sprayed on the tobacco. After 30 days storage, these cigarettes were also smoked and had no detectable flavor of ethyl propionate. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
32 3. FLAVORANTS WHICH REACT WITH TOBACCO It was reported by Mr. E. H. Harwood (20) that when oil of lemon or oil of lime flavors are added to tobacco there is an apparent reaction which after several days completely neutralizes these flavors so that they are not detectable by smell or smoking. Beta cyclodextrin-inclusion compounds of oil of 1 emon and oil of lime were prepared as described in Section B.V. of this report. Aqueous slurrys of these compounds were sprayed on CAMEL blend tobacco and, after drying, the tobacco was fabricated into cigarettes (treated) using a hand machine. Control samples were prepared by spraying an equivalent amount of the liquid flavors on equivalent amounts of CAMEL blend tobacco and fabricating cigarettes. Both the "control" cigarettes and "treated" cigarettes were stored in glass jars at room temperature for approximately 30 days. At this time, the cigarettes were smoked and evaluated. It was found that in both bases (lemon and lime flavors) the cigarettes which had been treated with the beta cyclodextrin- inclusion compounds retained these flavors, while neither of the control cigarettes yielded these flavors, upon smoking. 4. FLAVORANTS WITH "LOUD" OR OBJECTIONABLE EXTERNAL ODORS For this study, vanillin was selected as an example of a "loud" flavorant (i.e. one which masks the effect of a group of other.flavorants), while isovaleric acid was selected as a flavorant with an objectionable external odor. The beta cyclodextrin-inclusion compounds of vanillin and isovaleric acid were prepared as described in Section B.V. of this report. The beta cyclodextrin-vanillin inclusion compounds had no external odor. When a small amount was placed on a burning cigarette, the odor of vanillin was readily apparent in both the mainstream and sidestream of the smoke. In the case of the beta cyclodextrin-isovaleri acid inclusion compound, the external odor of the isovaleric acid was never com- pletely removed from the inclusion compound. Various techniques including drying under high vacuum and repeated washings with ether did not completely remove the external odor of the isovaleric acid. When the compound was tested on a burning cigarette, there was a substantial release of isovaleric acid in the mainstream and side- stream of the smoke. d. Laundry Fragrance The object of this experiment was to determine whether a cyclo- dextrin-fragrance inclusion compound could withstand the washing, Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
33 drying and ironing of the laundry cycle. Mr. C. H. Milton suggested two fragrances for use in this experiment: Liquid sunshine No. 9601, Firmenich & Co. and Compound oil No. 19434, Fritzsche Bros. The beta cyclodextrin-inclusion compounds of these fragrances were prepared as described in Section B.V. of this report. The experiment was conducted at a local launderette (Thruway Shopping Center). Twelve cotton diapers and one bottle of "Downy", concentrated, fabric softener, Procter and Gamble Co. were obtained. for the experiment. For the "control", six diapers were washed in a conventional automatic washer through. the 30 minute wash cycle. One capful of "Downy" and 0.526 grams of Liquid Sunshine No. 9601 were added at the beginning of the wash cycle. For the treated samples, six diapers were washed exactly as described above except that in addition to one capful of "Downy" 1.35708 grams of beta cyclodextrin Liquid Sunshine No. 9601 inclusion. compound were added at the beginning of the wash cycle. Both sets of diapers were dried in separate dryers for ten minutes at. 160°F After drying, the diapers were steam-ironed by Mrs. C. H. Milton. The diapers were then evaluated for fragrance by Mr. C. H. Milton (28) It was found that the diapers which were washed with the beta cyclodextrin Liquid Sunshine No. 9601 inclusion compound retained a perceptable amount of fragrance even after steam-ironing. The control diapers had no odor at the end of this cycle. This experiment was repeated twice with the same results. e. Chewing Gum Beta cyclodextrin-flavor inclusion compounds were panel-tested in chewing gum. Five flavor inclusion compounds were prepared as described in Section B.V. of this report and are listed below. 1. 50 Spearmint, 50 carvone, Norda, Inc. 2. Oil of peppermint, USP x IV, Fritzsche Bros. 3. Imitation fruit flavor No. 952, Felton Chemical Co. 4. Cinnamic aldehyde, Fritzsche Bros. 5. Imitation licorice flavor, No. 50.691/A, Firmenich & Co. The method used for making the chewing gum was obtained from Firmenich & Co. and is presented in Appendix I. Chewing gum samples were prepared using beta cyclodextrin-flavor inclusion compounds and panel-tested with chewing gum made using com- parable liquid flavors in equivalent amounts. Some difficulty was encountered in establishing the most acceptable flavor level (i.e. the amount of flavor added to the gum) for panel testing. It became apparent that smaller amounts of the flavor inclusion compounds could Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
34 be used since none of the flavor was lost because of the high tempera- ture of the gum when the flavor was added. In the preliminary gum samples, the gum containing the flavor inclusion compound invariably had a stronger flavor than the control gum sample which was prepared using a liquid flavor. Therefore, an acceptable flavor level for the cyclodextrin-flavor chewing gum had to be established by panel testing. For panel testing, the chewing gum was weighed in 3-gram portions and wrapped with aluminum foil. The wrapped gum was then placed in glass tubes fitted with air-tight screw caps. The samples were sent to the panel in this form for testing. The results of panel testing chewing gum are presented in Table IX. TABLE IX COMPARISON OF BETA CYCLODEXTRIN-FLAVOR INCLUSION COMPOUNDS WITH LIQUID FLAVORS IN CHEWING GUM Test No. 1 - Oil of peppermint, USP XIV, Fritzsche Bros. Total panelists 50 Number of panelists selecting the beta cyclodextrin complex 4 Number of panelists selecting the control (liquid flavor) 46 Test No. 2 - 50 Spearmint 50 Carvone, Norda, Inc. Total panelists 50 Number of panelists selecting beta cyclodextrin complex 14 Number of panelists selecting control (liquid flavor) 36 Test No. 3 - Imitation fruit flavor, No. 952, Felton Chemical Co. Total panelists 49 Number of panelists selecting beta cyclodextrin complex 15 Number of panelists selecting control (liquid flavor) 34 Test No. 4 - Cinnamic aldehyde, Fritzsche Bros. Total panelists 48 Number of panelists selecting beta cyclodextrin complex 6 Number of panelists selecting control (liquid flavor) 42 In all four panel tests, the panelists overwhelmingly preferred the control (liquid flavor) chewing gum over the chewing gum made with the beta cyclodextrin complex. However, it is felt that the beta cyclo- dextrin flavor complexes still may have a potential as chewing gum flavors because of the smaller amount of the flavorant which can be used in preparing the chewing gum. Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
35 f. Coffee An apparatus was devised for using a solution of beta cyclodextrin to complex the "headnote" flavors of fresh ground coffee (see Section B.V. of this report). The object of this preparation was to add the "headnote" flavor complex to instant coffee in an attempt to improve the flavor of this product. For the panel test, 0.050 gm. of Maxwell House Instant Coffee was weighed on a Mettler balance and dissolved in 10 ml. of an aqueous sucrose solution (1 1b./1 qt.) in a paper cup. This solution was termed the "control". The "test" sample was prepared exactly as described above except that 0.050 gm. of the beta cyclodextrin-coffee headnote flavor complex were also added to the solution. Each panelist was presented with two unlabeled cups (1 control and 1 test sample) and asked which liquid had the best coffee flavor. The panel consisted of 30 people who were avowed coffee drinkers. Twenty-one out of thirty panelists preferred the test sample (beta cyclodextrin-coffee complex), while nine out of thirty panelists preferred the control sample of instant coffee. VII. Thin Layer Chromatography of the Cyclodextrins The object of this work was to develop a method for separating alpha, beta and gamma cyclodextrin using thin layer chromatography. Thin layer glass plates were prepared using the Brinkman apparatus. Kieselguhr G was used as the absorbent. The glass plates were coated with Kieselguhr G and dried for 2 hours at 100°C and stored in a desiccator. Solutions of the cyclodextrins (0.1 percent) were prepared by dissolving 25 mg. of each in 25 ml. of water. These solutions were spotted (10 m1.) on the plates for chromatography. The plates were irrigated in various solvents, dried and developed by spraying with 0.1N iodine in ethanol. It was found that the most promising solvents for separation of the cyclodextrins were: methanol, ethanol, n-propanol, n-butanol and water. The best separation was obtained with methanol and ethanol. It is pro- posed that a combination of these solvents with water in the proper pro- portions would be the best solvent system for separating the alpha, beta and gamma cyclodextrins. The cyclodextrin project was suspended shortly after this phase of the work was begun. 4531 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0094
36 C. DISCUSSION No appreciable time was spent on the development of better techniques for the preparation of the cyclodextrins. Instead, three published methods were investigated and the method of F. Cramer (10) was found to be superior. When attempting to prepare large scale batches of beta cyclodextrin, the laboratory method was scaled up and the apparatus was improvised. It is felt that with proper fermentation equipment much greater yields of beta cyclodextrin could have been obtained. During the investigation of the selective filtration properties of the cyclodextrins, some difficulty was encountered in obtaining a uniform dis- tribution of the cyclodextrin throughout the filter plug. Unplasticized Estron filter plugs had to be used, since the aqueous solutions of the cyclodextrins could not be introduced into the plasticized plugs. The filter plugs had to be wrapped with plastic tape (water insoluble) previous to the introduction of the aqueous solution in order to maintain a uniform plug cylinder. Aqueous solutions of the cyclodextrin were introduced into the filter plug, dropwise, using a burette to measure the volume. The filter plug was then dried in a forced draft oven. It was found that during the drying period, the cyclodextrins crystallized in greater amounts at the ends of the filter plugs. It was therefore necessary to cut 4.25 mm. sections off of each end of a 25.5 mm. plug to obtain a 17 mm. filter plug with a uniform distribution of cyclodextrin. The weight of cyclodextrin present in the final filter plug was estimated by the weight difference between treated and untreated plugs of the same exact length. The treated plugs were attached to the cigarettes with plastic tape. Some of the treated plugs had small wrinkles from contact with aqueous solutions and had to be carefully wrapped to avoid air leaks when the cigarettes were smoked on the smoking machine. Beta cyclodextrin-flavorant inclusion compounds were prepared using a number of commercially available flavors. It is known that some of these flavors contained many individual compounds which comprised the total flavor. When inclusion compounds were prepared with these flavors, it was not established whether all of the compounds comprising the total flavor were included in the final inclusion compound. However, this information could have been obtained using gas chromatographic techniques. D. CONCLUSIONS 1. Of the three published methods for the preparations of the cyclo- dextrins, the method of F. Cramer (10) was found to be superior. 2. The optimum concentration of B. macerans amylase for production of beta cyclodextrin is between 50 and 150 Hale & Rawlins units of enzyme per 100 gms. of substrate. Source: :https://www.industrydocuments.ucsf.edu/docs/ffbb0094