This thesis, having been approved by the special Faculty Committee, is accepted by the Committee on Graduate Study o f the University o f Wyoming, in partial fu lfillm en t o f the requirements fo r the degree .. ......... Chairman o f the Committee on Graduate Study. Secretary. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SOME PHYSICAL CONSTANTS OF THE OIL OF FUSARIUM LINI by Charles Francis Kinahan Thesis submitted to the Department of Chemistry and the Committee on Grad uate Study at the University of Wyoming, in partial fulfillment of the require ments for the degree of Master of Arts Laramie, Wyoming 1940 UNIV. WY9. LIBRARY Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP19855 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignm ent can adversely affect reproduction. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI ® UMI Microform EP19855 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page List of Ta ble s .............................................. iii Introduction................................................ 1 3 Ex p eri me nta l................................ Growth of F u n g u s ...................................... 3 Preparation of Fungus for Extraction................. 5 Extraction of Oil from F u n g u s ................ 6 Determination of Some Physical Constants of the O i l ................................................ 1. •Specific Gravity at 25°/25°C............. 8 9 2. Refractive Index at 20°C ................ 10 3. Iodine Number (Hanus Method)............... 11 4. Saponification N u m b e r ........... 5. Per Cent Soluble Acids Calculated as 13 Butyric A c i d ............ ............ ....... 15 6. Hehner N u m b e r ................................ 16 7. Acid V a l u e ................................. ..17 8. Per Cent of Free Acid Calculated as Oleic A c i d ...................................18 Discussion of R e s u l t s .......................................19 Sum mary. .............................................. Bibliography................................................ 22 ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 LIST OF TABLES Page Table 1 ............................ 3 Table I I ........................... 7 Table I I I .......................... 10 Table I V ............................ 11 Table V ............................. 12 Table V I ............................ 14 Table V I I ...........................18 Table V I I I ..........................20 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SOME PHYSICAL CONSTANTS OF THE OIL OF FUSARIUM LINI Introduction Some work has been done on the products of metabolism of Fusarium l i n i . the fungus of flax wilt. Anderson, M or row and Willaman^ found after extensive investigation that the principal products of metabolism were carbon dioxide and ethyl alcohol, with smaller amounts of organic acids. When the fungus was grown on a carbohydrate medium, these pro ducts accounted for 90# of the carbon contained in the car bohydrate. In 1926, Willaman and Letcher^ reported that al though alcohol was produced there were also traces of ace tone and acetaldehyde formed as intermediates during the growth of fungus. Dammann found that Fusarium lini grown on a 2# solution of dextrin produced carbon dioxide and ethyl alcohol. She reported no other products. During an investigation of the specificity of the in tercellular globulin of Fusarium l i n i , Nelson^ found that when the fungus was grown on a culture medium containing carbohydrate in excess, the growth yielded very little pr o tein and rapidly reached maturity and converted the excess 1. 2. 3. 4. Anderson, A. K . , Morrow, C. A., and Willaman, J. J . , Minnesota Agr. Expt. S t a . , Ann. Rept. 1922, p. 35. Willaman, J. J. and Letcher, Houston. Phytopathology 16, 941-49 (1926). Dammann, Else. B e r . 7 1 B , 1865-68 (1938). Nelson, Casper I., J. Agr. Research 4 6 , No. 2, 183-187 (1933). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 carbohydrate into storage fats. Such synthesis of fat was followed by a considerable lag in the rate of growth. When the protein was extracted by grinding in a ball mill for from 36 to 40 hours, there was also extracted an oil which when exposed to the atmosphere over a period of time became a dark solid mass. Solheim5 , on seeing this oil and the h a r dened mass resulting from it, became interested in the syn thesis of the oil by the fungus and suggested this investiga tion in order to determine whether the oil produced by the fungus, in the conversion of the excess carbohydrate to a fat, was a drying oil. In view of this suggestion, this preliminary study was undertaken to (l) find a method for extraction of the oil, in a pure form, from the fungus and (2) to determine some of the physical constants of the oil which might serve in establishing the nature of the oil. At this time, the author wishes to acknowledge grat e fully: the advice and assistance of Dr. E. R. Schierz, under whose direction this work was completed; the assistance of Dr. W. G. Solheim in culturing and growing the fungus; and the grant-in-aid from the Research Committee of the U n i versity with which some necessary apparatus was purchased. 5. Solheim, W. G . , Head of Botany D e p t . , Univ. (Private communication). of Wyoming. Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. Experimental Growth of fungus: The fungus was grown on a synthetic culture medium of which the important consideration was the per cent carbo hydrate. Several types of culture media were tried before one was obtained that would give a satisfactory growth at room temperature in a reasonable length of time, in from 30 to 35 days. that is, Nelson^ found that if too little carbohydrate was present the growth was feeble, however, that when there was an excess of carbohydrate the fungus rapidly reached maturity and converted the excess carbohydrate into reserve stores of fat. Rawlin's solution 7 Q » was found to give a good growth of fungus in about four weeks when the carbohydrate concentration was £0 per cent of the solution. The Rawlin solution prepared for these cultures contained the following ingredients in the quanti ties indicated in Table I. Table I Sucrose, C i g K g g O ^ Tartaric acid, (CHOHCOgH)g Ammonium nitrate, (NH^NO-j......... Ammonium phosphate, 6. 7. 8. (NH^)gHPO^..... 300 grams 4 grams 4 grams .60 grams Nelson, op. c i t . , pp. 183-187. Harshberger, John W . , A Tex t-Bo ok of Mycology and Plant Pa th ol og y, p. 59£. P. Bl a k i s t o n ’s Son & Co., Philadelphia (1917). Smith, Erwin F . , Bacteria in Relation to Plant Diseases Vol. I. p. 197. Carnegie Institution of Washington, Wash ington, D. C . , (1905). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 Potassium carbonate, K C O .60 grams 2 3 Magnesium carbonate, M g C O g . . ................ 40 grams Ammonium sulfate, Zinc sulfate, ( N H ^ ) g S O ^ H g O ) .......... 25 grams Z n S O ^ H g O ) ^ ................... 07 grams Ferrous sulfate, F e S O ^ H g O ) ^ ................ 07 grams Potassium silicate, K _ S i O „ ............ 2 6 07 grams Distilled wa t e r .......................... 1500 cc. The cultures were grown in Erlenmeyer flasks ranging in size from 250 ml. to 2000 ml., the size of the flask having little effect on the amount of growth obtained. The cultures were prepared by filling the flasks to a depth of about three quarters of an inch with the culture medium, and then pl ac ing them in an autoclave for treatment at a steam pressure of 22 pounds for one-half hour. After autoclaving the cul ture medium was inoculated with Fusarium lini transfered from the stock by means of a sterile pipette* Following the inoc ulation the flasks were loosely stoppered with sterile cotton and placed on tables in a room in which the temperature varied from 18 to 30 degrees centigrade, the usual temperature being 25°C. During the period of incubation no attempt was made to control the room temperature and the cultures remained und is turbed except for an occasional shaking which brought the mycelium to the surface of the liquid medium. After an incu bation period of from 30 to 35 days there was a considerable lag in the rate of growth and it was considered complete. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 Preparation of fungus for extraction: In the early experimental part of this work attempts were made to extract the oil from the fungus immediately after removing the growth from the culture medium and while it was still moist. In these trials, using either hot al cohol or ether as the solvent, much water and solid mater ial were extracted and it was found impossible to free the oil from this mixture in a pure form or in a good yield. Further experiments showed that it was necessary to dry the fungus before extraction and if extracted when dry the oil could be obtained free from water. To dry the fungus, the growth was removed from the culture flasks and pressed as dry as possible in a cheese cloth towel. The fungus was then placed in a large beaker and washed several times with cold 95^ alcohol and then pressed dry in a cheese cloth towel. This process removed most of the surface water. complete the drying operation, vacuum desiccator, To the fungus was placed in a containing anhydrous calcium chloride, * and carbon dioxide passed over it to replace the air in the desiccator. The desiccator was attached to a water aspir ator and evacuated to a pressure of 20 mm. Hg. The drying process required about one week and during this time it was necessary to supply the desiccator with fresh calcium chloride; when this was done the air was replaced with carbon dioxide and the desiccator was evacuated from this atmosphere. The fungus was dried until crisp and then Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 ground in a mortar to break it up into small pieces. Extraction of the oil from the funcus: Of the solvents tried for extracting the oil, anhydrous ether that had been distilled over sodium was found to be the most satisfactory. Using ether as the solvent the oil was obtained in good yield and almost free of solid mater ials. The extraction of the oil from the dried fungus was carried out in a Soxhlet extractor eauioped with glass .joints and a flask of 500 ml. capacity for the solvent. fungus was carefully weighed, The dried to the second decimal place, into the porous paper extraction cup and placed in the ex tractor. For the extraction, ether was used. about 400 ml. of the anhydrous The apparatus was heated on an electric hot plate. Experiments were made to determine the extraction time that would give the greatest percentage of oil. These exper iments showed that the optimum extraction time was one hour. This time was determined by discontinuing the extraction at the end of an hour and removing the extract and adding a fresh supply of solvent to the extraction flask. was then continued another hour. The extraction When the solvent was evap orated from this second extraction it was found there had been very little oil extracted and that the extract contained a greater percentage of solid materials. The effect of the extraction time on the percentage by weight of oil extracted is shown in Table II. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 Table of Dried Fungus II Extraction Time Wt. of Oil Extracted Per Cent yield 206.63 g. 1/2 hour 11.66 g. 5. 64$ 164.71 g. 3/4 hour 8.13 g. 4.93$ 157.70 g. 1 hour 11.52 g. 7.30 $ 199.45 g. li? hours 15.73 g. 7.89 $ 147.92 g. lir hours 15.85 g. 10.72$ 259.91 g. 1 hour 29.91 g. 11.50$ It was found that the solvent could be successfully re moved by distillation at diminished pressure. The solution was transfered from the extraction flask to a Claisen dis tillation flask of 250 ml. capacity. A boiling tube, drawn out to a fine capillary, was inserted through the rubber stopper in the neck of the flask. This tube was then con nected with a tank of carbon dioxide by means of a rubber tubing equipped with a pinch clamp. The side arm of the flask was closed with a rubber stopper carrying a glass stop-cock. The outlet tube of the flask was connected to a water aspirator through a manometer. of the solvent, During the evaporation a fine stream of carbon dioxide was allowed to flow through the boiling tube thereby preventing any oxi dation of the oil and also the bumping of the solution as it became more concentrated. When the pressure became constant at about 20 mm. H g . , as shown b y the manometer, indicating that the ether had been removed, heat was applied to the dis tillation flask, by means of an electric hot plate. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The heat 8 melted the oil which had become solid during the evaporation, and also removed the last traces of the solvent. red, The orange- slightly viscous oil was transfered to a small weighed glass-stoppered bottle. It was preserved under an atmos phere of carbon dioxide and stored in a cool place, protected from light. All samples of oil were obtained and preserved in the same manner. The per cent yield of oil was calculated as the ratio of the weight of oil extracted to the weight of the dried fungus. Determination of Some Physical Constants of the Oil: In the determination of the constants, the official methods of analysis for fats and oils as followed by the A s sociation of Official Agricultural Chemists^ were adopted. These methods will be referred to as the A.O.A.C. Methods in the following pages. In some cases it was found n e ce s sary to change the method slightly, particularly as to the quantity of oil used in any one determination, tity of oil available for analysis was limited. starting the analyses, as the quan Before the six samples of oil were combined in a flask and filtered through a hot water funnel, accord ing to the A.O.A.C. Method'*'0 ’^ , to remove any suspended 9. Official and Tentative Methods of Analysis of the Assoc iation of Official Agricultural Chemists, Fourth Edition, 1935, George Bant a Publishing Company (1936). 10. Ibid. , p. 404 11. Leach, Albert E., and Winton, Andrew L . , Food Inspection and A n a l y s i s . Fourth Edition, p. 489. John Wil ey and Sons (1932). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 solid materials and to make certain that the oil was homo geneous. The oil was collected in a previously weighed glass-stoppered bottle and stored in a cool place protected from light and air, to prevent the oil from becoming rancid. In determining the physical constants all samples were trans- fered directly from the stock flask. The total weight of the six combined samples was 79.9140 g. 1. Determination of Specific Gravity at 25°/25°C. The specific gravity of a substance is defined as the ratio of the weight of a certain volume of a substance to the weight of an equal volume of water at the same temper ature. Following the A.O.A.C. Method^-2, the specific grav ity of the oil was determined at 25°C as related to water at the same temperature. A cleaned pycnometer, filled with recently boiled distilled water previously cooled to 20°C, was placed in a constant temperature water bath at 25°C for a period of thirty minutes. At the end of this time, the level of the water was adjusted to the proper mark and the pycnometer stoppered and removed from the water bath and wiped dry. After standing for thirty minutes the pycnometer and water were weighed. The weight of the water contained at 25°C was ascertained by subtracting the weight of the empty pycnometer from its weight when full. For the determination of the specific gravity, the pyc- 12. Official and Tentative Methods of Analysis of the Associa tion of Official Agricultural Ch em is ts , op. c i t . , p. 404 Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. 10 nometer was filled with oil and treated in the same manner. The weight of the oil at 25°C was found by subtracting the weight of the empty pycnometer from its weight when filled with oil. Then the ratio of the weight of the oil to the weight of the water gave the specific gravity at 25°C. The results from three trials gave an average value of 0.9118, as shown in Table III. Table III Trial 2. Wt. of Water Wt. of Oil Specific Gravity 25°/25°C I 9.4998 g. 8.6428 g. 0.9098 II 9.4954 g. 8.6734 g. 0.9134 III 9.5077 g. 8.6733 g. 0.9122 Refractive Index at 20°C. For determining the refractive index of the oil a Spencer Refractometer of the Abbe Type was used according to the A.O.A.C. M e t h o d ^ . The accuracy of the instrument was checked against redistilled water, which has a refract ive index of 1.2998 at 2 0 ° C . , and against a calibrated glass plate supplied with the instrument. The temperature of the instrument was maintained constant by a stream of water circulated through the prisms. reading, Before taking the the instrument and the sample were allowed to stand for a few minutes in order that both would be at the temper ature indicated by the thermometer of the instrument. The 13. Official and Tentative Methods of Analysis of the As soc iation of Official Agricultural Chemists, op. c i t . . pp. 405-406. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 refractive index was found to be n^° 1.4680. Table IV Trial I 3. Refractive Index n ^ D 1.4679 II 1.4680 III 1.4681 IV 1.4680 Determination of Iodine Number (Hanus Method). The iodine absorption number is the number of centi grams of iodine absorbed by 1 gram of a fat or oil. This constant is most valuable in identifying and differentiat ing oils and is based on the property of unsaturated fatty acids to absorb a fixed amount of iodine. In the determin ation of the iodine number by the Hanus method the procedure as outlined in the A.O.A.C. M e t h o d ^ w a s followed. The re agents were prepared and carefully standardized according to the approved methods. The determinations were run in quadruplicate in the fo l lowing manner: a sample of oil, about 0.4 g . , was transfered from a weighing pipette to a glass-stoppered 500 ml. bottle. The exact weight of the transfered oil was found b y differ ence. To the bottle was added 10 ml. of chloroform, to d i s solve the oil, followed by 25 ml. of Hanus iodine solution accurately measured from a burette. This solution was al 14. Official and Tentative Methods of Analysis of the As s o c iation of Official Agricultural Che mi s ts . op. c i t . , pp. 410-412. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 lowed to stand for 30 minutes, a uniform temperature. by stop-watch, in the dark at At the end of this time, which must be closely adhered to in order to obtain comparable results, the stopper was carefully removed from the bottle and while shaking the bottle 10 ml. of 15$ potassium iodide solution was added and then 100 ml. of freshly boiled and cooled water. The solution was titrated with standard sodium thiosulfate solution using starch indicator. Along with each determina tion a blank was prepared in the same manner except that the oil was omitted. From the data collected during the ti tra tions of the determination and the blank, the iodine number of the oil could be calculated from the following formula: Iodine Number = Q. In this formula A represents the difference between the number of milliliters of sodium thiosolfate used to titrate the blank and that used for the determination; B represents the number of grams of iodine equivalent to 1 ml. of the sodium thiosulfate solution; C represents the weight of the oil in the sample. and The aver age iodine number of the oil, as calculated from the four determinations, was 82.72. The weights of the samples and iodine numbers determined from them are shown in Table V. Table V Sample Weight of Oil Iodine Number I 0.4878 g. 82.24 II 0.3549 g. 82.81 III 0.4654 g. 82.94 IV 0.3520 g. 82.88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 4. Determination of Saponification Number (Koettstorfer Number). When treated with basic hydroxides, glycerol and the salts of fatty acids. soaps, fats and oils yield The latter are termed and the process by which they are formed is called saponification. The amount of potassium or sodium hydroxide that will react with a given amount of fat or oil in the pr o cess of saponification will depend on the average length of the constituent fatty-acid chains, acid molecules, for the smaller the fatty- the greater could be their number in a given amount of fat or oil. Upon this principle is based a method for determining the character of different fats and oils. The saponification number is defined as the number of milligrams of potassium hydroxide required to saponify one gram of fat. In determining the saponification number of the oil, the reagents were prepared and carefully standardized as directed in the A.O.A.C. Method 15 that was followed. The determinations were run in quadruplicate in the following manner: a sample of oil (between 1 and 3 grams) was accurately weighed into an Erlenmeyer flask of 250 ml. capacity. Twenty- five milliliters of alcoholic potassium hydroxide were added, by pipette, the flask connected to a water cooled condenser and the solution boiled to saponify the oil. two determinations, In the first the solutions were boiled for 30 minutes 15. Official and Tentative Methods of Analysis of the Assoc iation of Official Agricultural Chemists. op. c i t . , pp. 412-413. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 and in the last two the saponifying time was 45 minutes. With each determination, a blank was prepared in exactly the same manner except that the oil was omitted. After the saponification was complete and the flask had cooled to room temperature, the determination and the blank were titrated with standard hydrochloric acid using phenolphthalein for an indicator. From the data collected during the two titrations it was possible to calculate the saponifica tion number of the oil by the following formula: Saponification Number = In formula A represents the difference between the number of milli liters of alcoholic potassium hydroxide used to titrate the blank and that used for the determination; B represents the normality of the hydrochloric acid used in titrating; C re presents the molecular weight of potassium hydroxide; and D represents the weight of the oil in the sample. The ave rage saponification number of the oil, as calculated from four determinations, was 192.7, The weights of the sample and the saponification number determined from them are shown in Table VI. Table VI Sample Weight of Oil Saponification Number I 1.3181 g 192.9 II 1.4352 g. 192.1 III 2.7277 g. 192.7 IV 3.0310 g 193.0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 5. Per Cent Soluble Acids Calculated as Butyric Acid. The per cents of soluble and insoluble acid were de termined on the same samples of oil as were used in the third and fourth determinations of the saponification n u m ber. The per cent soluble acids was determined by the A.O. A.C. Method X6 as follows: the flasks from the saponification number determinations were placed on a water bath and the alcohol evaporated. Then to each flask was added a Qua n tity of hydrochloric acid equivalent to the quantity of potassium hydroxide used for the saponification of the sample and 1 ml.- more (quantity of acid added I titration for blank titration for sample plus 1 ml.) and the flask placed on a steam bath until the separated fatty acids formed a clear layer on the upper surface of the liquid. The flask was then filled to the neck with boiling distilled water and cooled in an ice bath until the cake of fatty acids was thor oughly hardened. The liquid contents of the flask, contain ing the solid cake, was poured through a filter into a liter flask and the flask refilled with boiling water and placed on the steam bath until the fatty acids had again collected at the surface. three times, This treatment with hot water was repeated cooling and collecting the washings in the liter flask after each treatment. The combined washings were titrated with standard potassium hydroxide using 16. Official and Tentative Methods of Analysis of the Associa tion of Off icial Agricultural Ch emists, op. c i t . , p. 413. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 phenolphthalein as an indicator. A correction was made for the additional 1 ml. of acid added and the milliequivalents of potassium hydroxide neutralized by the soluble acid were calculated. Knowing the number of milliequivalents neutra lized it was possible.to calculate the equivalent weight of the soluble acids as butyric acid. By the ratio of the weight of butyric acid to the weight of the sample was cal culated the per cent of soluble acid. The results from two determinations were 0.143$ and 0.206$. 6. Hehner Number. By the Hehner Number is meant, uble acid in a fat or oil. A.O.A.C, Method 17 the per cent of insol This value was determined by the as a continuation of the preceding deter mination of the per cent soluble acids. The flasks contain ing the cakes of insoluble fatty acids and the filter papers through which the soluble acid had been filtered were al lowed to dry for 12 hours at room temperature. of this time, At the end the cakes were transfered to weighed beakers of 50 ml. capacity. The solid acids that remained on the filter papers were dissolved by pouring hot absolute alcohol through the funnels. The filtrates were collected in the beaker containing the solid cake from the same sample. The beakers were placed on a steam bath to evaporate the alcohol and then dried in an oven at 100°C. to constant weight. The 17. Official and Tentative Methods of Analysis of the Associa tion of Official Agricultural Chemists, o p . c i t . , p. 413. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 weights of the insoluble fatty acids in the beakers were found by difference. The Hehner Numbers for the samples were calculated as the ratio of these weights to the weights of the original samples. The results for the two determinations were 91,35 and 92.12. 7. Acid Value. The acid value is a measure of the amount of free fatty acids in a fat or oil. It is defined as the number of mill i grams of potassium hydroxide required to neutralize the free fatty acids in one gram of fat or oil. For this determina tion it was necessary to reduce the amount of oil used to about one third the amount specified in the A . O . A . C , For the determination, Method*^. an accurately weighed sample of oil (about 6 grams) was transfered to an Erlenmeyer fla.sk of 250 ml. capacity. To the flask was added 50 ml. of neu tra l ized 95^ ethyl alcohol and the flask shaken vigorously to dissolve the oil. Final solution was accomplished by the addition of heat, applied carefully to prevent esterifica- tion of the fatty acids. When the oil was completely dis solved, the solution was allowed to cool to room temperature and titrated with standard potassium hydroxide solution u s ing ohenolphthalein as an indicator. The change in color at the end point was gradual and indefinite, therefore, ough shaking during the titration was essential. thor From the data 18. Official and Tentative Methods of Analysis of the A s so ci a tion of Official Agricultural Ch emi st s. op. c i t . , p. 417. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 collected during the titration and knowing the concentration of the standard potassium hydroxide it was possible to cal culate the number of milligrams of potassium hydroxide r e quired to neutralize the free fatty acids in the sample. The ratio of the number of milligrams of potassium hydroxide to the weight of the sample gave the acid value of the oil. The average acid value, as calculated from two determinations, was 2.810. The weights of the oil in two samples, and the acid values determined from them are shown in Table VII. Table VII Sample 8. Weight of Oil Acid Value I 6.6320 g. 2.818 II 6.4975 g. 2.803 Per Cent of Free Fatty Acid Calculated as Oleic Acid. From the titration values obtained in the determination of the acid value, it was possible to calculate the per cent of free fatty acid as oleic acid. The number of milliequiv alents of potassium hydroxide required to neutralize the free fatty acid was calculated and converted into the equiv alent weight calculated as oleic acid. weight of oleic acid to the weight calculated the per cent By the ratio of the of oil of free fatty acid. values previously obtained, in the sample was For thetwo acid these percentages were 1.416 and 1.408 respectively. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion of Results From the character of the oil and the constants deter mined, it was apparent that the oil extracted from Fusarium lini was not the same oil as found by Nelson"^. followed the normal The oil behavior of a non-drying o i l ^ , that is, when exposed to the air and light for several days it became colorless with little change in fluidity. There was also a change in the odor of the oil during exposure, however, it could not be said that the oil had become rancid during its oxidation. B y a comparison of the physical constants deter mined for the oil with those given in the International Crit ical T a b l e s ^ f o r fats and oils, the placing of the oil as a non-drying oil was substantiated. It was found that the physical constants determined for the oil compared most fav orably with the constants listed in the International Critical Tables type. 22 for non-drying vegetable oils of the olive oil A table showing some of the average physical c o n s t a n t ^ for non-drying vegetable oils and drying vegetable oils as compared to the same constants determined for the oil extrac ted from Fusarium lini is given below. 19. Nelson, ojo. cit. , pp. 183-187. 20. A l l e n 1s Commercial Organic Analysis, Fourth Edition, Vol. 2, p. 3. P. Blakiston's Son and Co. Philadelphia. 21. International Critical T a b l e s . I I , 196-217. McGrawHill Book Company, Inc., New York (1927). 22. Ibid. , p. 201. 23. Ibid. , pp. 201-203. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 Table VIII Classification of oil Iodine Number Non-drying vegetable oil 50-100 Oil from Fusarium lini 82.72 Drying vegetable oil 140-200 Saponification Number Hehner Number Acid Value 180-210 91-96 1-3 192>-7 91.75 2.81 190-210 91-98 2-12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Summary (1) A method has been developed for the extraction of the oil produced by Fusarium l i n i . (2) The following physical constants have been determined for the oil: 1. Specific Gravity at 350C / 2 5 ° C ...... . ........0.9118 2. 3. Refractive Index n 2 0 .......................... 1.4680 D Iodine N u m b e r .................................. 82.72 4. Saponification N u m b e r ........................ 5. Per Cent Soluble Acids Calculated 192.7 as Butyric A c i d ........................ 0.143# to 0.206# 6. Hehner N u m b e r 91.35 and 92.12 7. Acid V a l u e 2.818 and 2.803 8. Per Cent Free Fatty Acid Calculated as Oleic A c i d ....................... 1.416# and (3) 1.4 0 The oil has been classified as a non-drying vegetable oil of the olive oil type. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLIOGRAPHY (20) A l l e n 1s Commercial Organic An aly si s, Fourth Edition, Vol. II, P. Blakiston's Son & Co., Philadelphia (1910). (1) Anderson, A. K . , Morrow, A. C . , and Willaman, J. J. , Minnesota A g r . Expt. S t a . , Ann. R e p t . 1 9 2 2 , p. 35. (3) Dammann, Else, "Fermentation of Dextrin with Fusarium Lini Bolley", Ber. 71B 1865-68 (1938). (7) Harshberger, John ff., A Text-Bo ok of Mycology and Plant Pathology, P. Blakiston's Son & Co., Philadelphia (1917). (21) (22) (23) International Critical Tables of Numerical Data, P h y s i c s . Chemistry and Technology. Vol. II, M c Graw-Hill Book Company, New York (1927), (11) Leach, Albert E . , and Winton, Andrew L . , Food Inspec tion and A n a l y s i s , Fourth Edition, John Wiley and Sons, Inc., Ne w York (1932). Morrow, Clarence A , , Biochemical Laboratory Methods, John Wiley and Sons, Inc., New York (1927). (4) (6) (19) Nelson, Casper I., "A Method of Determining the Specificity of the Intercellular Globulin of F u s arium Lini", Jj_ Agr. Research 46 No. 2 183-87 (1933). (9) (10) (12) (13) (14) (15) (16) (17) (18) Official and Tentative Methods of Analysis of the Association of Official Agricultural C h e m i s t s . Fourth Edition, 1935, George Banta Publishing Company, Menasha, Wis. (1936). (8) Smith, Edwin F . , Bacteria in Relation to Plant D i s eases . Vol. I, Carnegie Institution of Washington, Washington, D. C. (1905). (2) Willaman, J. J . , and Letcher, Houston, "Biochemistry of Plant Diseases: VIII Alcoholic Fermentation of Fusarium Lini", Phytopathology 16 941-49 (1926). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.