968 INDUSTRIAL AND ENGINEERING CHEMISTRY Vol. 20, No. 9

The time required for freezing having been previously determined, the four samples of each mixture were made up a t intervals of 90 minutes in order that the aging of each should be of equal duration.

In preparing the first mixture, the milk was heated to the scalding point. Then it was poured into a sterile bottle and the sugar added. After cooling, the cream was introduced; then the mixture was placed in an ice box and allowed to stand until the following day. In preparing second and third mixtures the gelatin was softened in a little cold milk and dis- solved in the rest of the milk heated to the scalding point. Sugar and cream were added as in the check. In the fourth a custard was made of the thoroughly beaten raw egg and scalded milk. Sugar and cream were added as in the check. The dry egg was combined in the same way as the gelatin. In the sixth the bottled milk was heated to the scalding point and cooled; then the evaporated milk and cream were added.

Just before freezing, the temperature was taken, and the viscosity de- termined with a Mojonniet’s viscometer. In every case the temperature was 12” C. The viscosities were: (1) 16; (2) 17; (3 ) 18; (4) 17; (5) 17; (6) 17. The freezer used was an attachment of a Hobart mixer. The ice used for freezing was finely chopped and mixed with ice-cream salt in the pro- portion of 6:l by weight. The freezer was turned a t medium speed. Each sample was frozen 20 minutes, which produced in all cases ice cream that was quite solid.

Cream from each freezer was removed to a pint card- board carton and then placed in a special container packed with ice. The frozen creams were transferred to cold storage where they stood for 3 days a t a temperature of -14” F. (-26” C.). The photomicrographs were made in this cold- storage room to keep the ice cream solid or in crystalline form. Gauze-thin slices were cut from the center of each ice-cream package with a safety razor blade. Specimens were placed on an ordinary microscopic glass slide with a cover glass on top. They were then examined to see if they were alike in texture. A representative slide from each group was photo-

All mixtures were frozen the following day.

graphed using an ordinary photographer’s camera and a Bausch and Lomb microscope.

Results and Discussion

The accompanying photomicrographs illustrate the in- fluence of the various additions on the texture of ice cream. Formula 1 is seen to produce large, irregular crystals. Finer crystal construction was obtained by formula 2, which con- tains 0.3 per cent gelatin. Formula 3, 0.5 per cent gelatin, gave the finest texture of all ice creams made-so fine that it was almost gummy and for this reason objectionable. The flavor was also poor. The micrograph for formula 4 points out the effectiveness of raw egg which has been cooked with milk as a custard. A similar improvement is shown in the case of dry egg yolk, formula 5. Formula 6 is the ice cream containing evaporated milk and no solids other than those in the first ice cream but there is a much finer texture. In fact, the improvement is quite similar to ice cream containing 0.3 per cent gelatin.

Viscosities of all the mixtures showed little variation when observations were made on the viscometer. The differ- ences, however, were very apparent when the mixtures were poured from one vessel to another-formulas 2, 4, 5, and 6 appearing to be about the same in viscosity but much more viscous than formula 1, which contained milk and cream. Formula 3 was the most viscous of all. The increased vis- cosity of the evaporated milk mixture is due entirely to the evaporated milk itself. Its own viscosity is a result of the finely divided state of the fat globules in the milk that has been brought about in the homogenizing process,6 and the action of heat in sterilization on the casein. Photomicro- graphs showing the effect of homogenization on fat globules are also shown.

Acknowledgment

The author is indebted to the Home Economics Depart- ment of the University of Chicago for the use of a research laboratory, and to Armour and Company for the privilege of using a cold-storage room.

Vitamin B Content of Avocados’ LeRoy S. Weatherby and Eugene W. Waterman

CHEMICM. LABORATORY, UXIVERSITY OF SOUTFIERN CALIPORXIA, Los ANGELES, CALIF.

ORK by Santos2 The vitamin B content of avocados was determined of its long season. It averages showed vitamin B through feeding experiments with albino rats. The about 25 per cent of oil con- to be present in pulp of fresh food was used and was compared with tent. Thefruitusedwassuch

avocados in relatively high as through exterior blemish quantity. His work was the fresh pulp contained approximately one-twelfth failed to meet the require- done on the residue of dried the vitamin value of the dry yeast. ments of first-grade fruit, fruit after the oil had been but which met the standard extracted nith ether. In this investigation it was thought requirements in every other n-ay. advisable to determine the vitamin content of the fresh ripe The vitamin B content was determined in the customary fruit of standard variety as actually consumed. way through feeding experiments with albino rats, using yeast

The fruit tested goes under the trade name of “Calavo.” as a standard of comparison.

Fleischmann’s dry yeast as standard. It was found that

which is the copyrighuted name of the highest grade of avocados put out by the Calavo growers of California, in which the fruit is required to meet certain high specification standards of quality and of maturity. The Fuerte variety was used becquse it is one of the most popular varieties and because

* Received March 16. 1928. Presented before the Intersectional Meet- ing of the Amencan Chemical Society with the Pac:fic Division of the American Association for the Advancement of Science, Pomona, Calif., June 13 to 16, 1928.

2 Am. J . Physiol . , 69, 310 (1922).

Preliminary Experiment

In the first preliminary experiment, rats from three litters, of ages varying from 28 to 33 days, were distributed among three cages. The basal diet common to all the animals was a modification of the Osborne and Mendel vitamin-B-free diet, in which meat residue was substituted for the extracted casein.

The meat residue was prepared by extracting finely ground, lean round steak with hot water three times and then drying

September, 1928 IiVD UXTRIAL .4ND ENGI.VEERIiTG CHEMISTRY 969

in the open air a t a moderate temperature. was Fleischmann’s dry yeast.

The yeast used

Basal Diet

Meat residue Starch Larda Butter fat Salt mixture (hkCollum’s S o . 1 G . t

Per cent 20 52 15 9 4

a Crisco was later substituted for the lard b J . Bid. Chem., 33, 63 11918).

The diet of the animals was as follows: Cage 1-Normal control, basal diet + 1 gram-yeast per rat

per day

day

Cage 2--Vitamin-B-free, basal diet alone Cage 3-Avocado, basal diet + 5 grams avocado per rat per

250

. - T h e in weeks-

Graph I-Cage Average (Preliminary Data)

Record was kept of the food consumption in each cage, and the weight of each animal was recorded weekly. The food consumption of the rats on the vitamin-B-free diet reached a maximum a t the end of the second wetlk, after which it decreased to approximately one-half this amount. The food consumption of the rats on the control diet in- creased throughout, while that of the rats on the avocado diet was stationary or was decreasing slightly toward the end of the experiment, which may be interpreted that these rats were not receiving sufficient vitamin B a t this stage.

The growth curves for the cage average are shown i n Graph I. The growth curve of the control rats shows a normal rate of growth. There were no signs of polyneuritis or other deficiency diseases, indicating that the diet was adequate in all respects.

The growth curve of the rats on the vitamin-B-free diet shows an increase of weight for the first 2 or 3 weeks and than a gradual decline. The old style of cage with the screen bottom resting directly on a tray was used in this experiment, thus enabling the rats to eat their feces. A good deal of vitamin B is excreted in the feces, and this enabled them to keep from any greater loss in weight. This defect was remedied in the second set of experiments by using cages with raised bottoms which permitted the feces to drop I hrough. The rats on this diet were abnormal in appearance and action, their hair was rough, and they appeared to be on the verge of collapse.

From the ciirve showing the growth of the rats on the avocado diet, it can be seen that there is a decided gain in weight over the rats on the vitamin-B-free diet, though their weight did not rise so rapidly as that of the normal rats.

These rats had sleek fur and appeared healthy and normal in every way.

Though this one experiment was not sufficient to deter- mine the exact amount of vitamin B in avocados. it showed decidedly that vitamin B is present and warranted further investigation.

Quantitative Experiments

A second series of experiments was conducted for the purpose of obtaining more nearly quantitative results. From the previous series it was evident that 1 gram of yeast per rat per day was more than adequate to promote normal growth throughout the full time of the experiment. It was therefore determined to use smaller quantities of yeast, and to use varied quantities of avocado for comparison. The same basal diet was used as in the previous experiment.

The diet was as follows: Cage 1-Control (low), basal diet + 0.25 gram yeast per rat

Cage 2-Control (medium), basal diet + 0.5 gram yeast per

Cage 3-Iritamin-B-free, basal diet alone Cage 4-Avocado (low), basal diet + 1.5 grams avocado per

per day

rat per day

rat per day

per rat per day Cage &-Avocado (medium), basal diet + 3.5 grams avocado

Cage 6-Avocado (high), basal diet + 5 grams avocado per rat per day

In this series a smaller number of rats was used in each cage, since in the previous experiment it was demonstrated that growth was uniform among all the animals in each cage, and that an average might justly be deduced from as lo^ a number as three or four animals.

i 2 3 4 s 6 i a --

Time m Weeks - Graph 11-Cage Average (Quantitative Data)

The relative rate of growth of the animals under these conditions is shown in Graph 11.

Curve 1 , showing the growth rate of the rats on 0.25 gram of yeast daily, indicates that this quantity was sufficient for normal growth for a short while only, and that the rats soon dropped in weight due to a shortage of vitamin B.

Curve 2, showing the growth rate of the rats on 0.5 gram of yeast daily, shows that this amount of yeast was adequate for normal growth a t least for the 8 weeks of the experiment. There was a decided increase in growth over those on 0.28 gram daily.

There were three rats on the vitamin-B-free diet. They all began to lose weight after the first week and developed poly- neuritis in from 3 to 4 weeks. Rat number D17B died after jl/, weeks on this diet, and number D160 a t 7 weeks. An au- topsy of rat number D176 found it to be quite normal except that

970 INDUSTRIAL A N D ENGINEERING CHEMIXTRY Vol. 20, No, 9

the intestines were badly disintegrated and the stomach was con- tracted. These findings showed that death was due to a de- ficiency of vitamin B and not to other causes. The other rat, D163, was badly paralyzed and was so close to death that after the sixth week it was fed avocado to keep i t alive. When 5 grams avocado daily were added to its diet, an immediate better- ment in its condition was noted. I ts paralysis soon disappeared and it began to gain in weight rapidly. The growth of these animals, and the point at which avocado was added to the diet of the remaining rat, is indicated by Curve 3.

Curve 4 shows the growth of the rats fed on 1.5 grams of avo- cado daily in addition to the vitamin-B-free diet. No cases of polyneuritis developed in any of this group of rats. There was decided increase in weight over the vitamin-free rats, though their weight did not equal that of the rats with the yeast.

C u m 5 shows the growth of the rats fed on 3.5 grams of avocado daily. There is a large increase in weight of these rats over those being fed on 1.5 grams daily. They appeared normal rats in every way.

Curve 6 shows the growth of the rats fed on 5.0 grams of avo- cado daily. An increase in weight over those on 3.5 grams is shown, although their growth does not equal that of those on 0.5 gram of yeast. No greater amount than 5.0 grams of avocado was used because of the difficulty in getting the rats to eat more and because of this excess food changing too markedly the proportion of the other food materials in the diet.

From these growth curves it may be seen that the group on 0.25 gram of yeast reached a plateau a t the fourth week. The growth curve for those on 3.5 grams of avocado and on 5.0 grams of avocado crossed this curve and continued to show an increase in weight throughout the duration of the experiment. The group on 1.5 grams of avocado continued to show an increase throughout, but never quite reached the weight of those on 0.25 gram of yeast.

By inspection of this graph it is evident that the vitamin B content of approximately 3 grams of avocado would equal that of 0.25 gram of yeast as indicated by the growth curves. From this ratio 12 grams of avocado is the equivalent of 1 gram of yeast, or 12 ounces of avacodo is equivalent to 1 ounce of yeast.

The average weight of the edible portion of the Fuerte avocado is approximately 12 ounce^.^ This would make the vitamin B content of one Fuerte avocado of Calavo grade approximately the equivalent of one ounce of dry yeast.

Examination of Graph IIshows that the quantity of avocado necessary per day to give net maintenance during this period, which is the generally accepted unit of vitamin B, would be approximately 1 gram. It may he said, therefore, that the vitamin B content of avocados in terms of units is approxi- mately 450 units per pound.

Conclusion

From this investigation it is apparent that the avocado ranks extremely high as a food source of vitamin B. If what is known as vitamin B is in reality two vitamin fac- tors, one antineuritic and one growth-promoting, as recent investigations seem to i n d i ~ a t e , ~ it is evident that the avocado contains both factors since it prevents the paralytic symptoms of vitamin B deficiency and also promotes growth.

Note-The determinations of vitamins A, C, D, and E in avo- cados are now under investigation in this laboratory.

.

3 U. S. Dept. Agr., Bull. 1073, p. 15. 4 Sherman and Axtmayer, J . Bid Chem , 75, 207 (1927).

Determination of Unsaturateds in Lubricating Oils' A Modification of the Francis Method

Frederick S. Bacon

-4RTHUR D. LITTLS. INC., CAMBRIDGE, MASS.

HE method described by Francis2 for the determination of unsaturateds by titration with bromide-bromate T solution has proved very satisfactory and is used es-

dusively in this laboratory in all research work. This method has several advantages over that described by Mc- Ilhiney3 in that absolute neutrality of all reagents is not re- .quired and the presence of water during bromination has no deleterious effect. In the McIlhiney method any acid in the reagents would have the same effect as hydrobromic acid, and water in the carbon tetrachloride would cause the for- mation of bromohydrin as well as hydrobromic acid.

The principal difficulty in the operation of the Francis method has been encountered in testing dark-colored oils such as lubricating oils or untreated cracked gasolines. With such materials it has been impossible to establish the end point because of the inability to note when there was an ex- cess of bromine and when the last traces of the iodine have completely reacted with the thiosulfate.

A slight modification of the original method has been worked out which gives satisfactory check results on such dark-colored materials. It consists of adding to the sample of oil to be tested 10 cc. of c. P. benzene or gasoline of known

1 Presented under the title "Unsaturated and Molecular Weights of Commercial Lubricating Oils. Modification of Francis Method of De- termination of Unsaturateds in Oils" before the Division of Petroleum Chemistry at the 74th Meeting of the American Chemical Society, Detroit, Mich., September 5 to 10, 1927.

8 IND. END. CHEM., 18, 821 (1926). 8 J. Am. Chem. SOC., 91, 1084 (1899).

unsaturated content, then a slight excess of bromide-bromate solution as shown by the color in the water layer, using po- tassium iodide solution as the indicator, then running past the visible end point with thiosulfate and shaking vigorously to insure complete removal of the iodine from the oil. The two layers are allowed to separate, the lower carefully drawn off, starch added, and then the excess thiosulfate titrated with iodine to blue color. Very thorough and vigorous shak- ing is essential.

This more involved procedure appears necessary to se- cure accurate results in all cases. The 1- to 10-cc. sample to be analyzed is mixed with a t least 10 cc. of c. P. benzene or gasoline of known unsaturated content in order to facili- tate the separation of the oil and water layer before the iodine titration. The addition of the benzene as a diluent also prevents a peculiar viscosity effect of certain types of oil, such as neutral white oil, which prevents complete reaction be- tween the olefins and bromine and therefore great difficulty in checking results. Benzene as well as gasoline will brominate somewhat if exposed to very strong light or sunlight, even under the small concentration of bromine employed. This is shown by the following bromine numbers of c . P. benzene: in moderate light, 0.027; in sunlight, 18.00.

The first end point to an excess of bromine which shows yellow in the ordinary method is very indefinite in colored samples. It has been found that a large excess of bromine may be present without giving a definite color indication in the water layer. When titrating with thiosulfate the iodine