NOTES AND COMMENTS BY THE COLLABORATORS. C. S. Brinton used the tables of Rota and others given by Allen, a Schultz and Julius, "Organic coloring matters," and Circulars 25 and 35, Bureau of Chemistry. Considerable difficulty was encountered in some cases in isolating color from fruit pulp and sirup. Double-dyeing method was used for extracting color from material, and color was obtained in aqueous solution by extracting wool with ammonia. Sample VI gave considerable trouble, and definite report was not made. F. O. Woodruff used chiefly tables of Green, Yoeman, and Jones, also tables in Allen and in Schultz and Julius, and Circular 35, Bureau of Chemistry. He says: "Three difficulties attending identification are: (1) A commercial dye from different manufacturers varies in purity and therefore in properties, though bearing the same or a synonymous trade name; (2) amount of color on dyed fiber or in color solutions affects the nature of the reactions therewith; (3) ordinary description of color reactions varies with the observer and does not allow of fine distinctions." Hare, Mitchell, and Pringle used Rota's table and those in Circular 35, Bureau of Chemistry. They comment as follows: "We find Rota's scheme quite valuable in assisting us in the general classification of the dye. An accurate and complete color chart would be a great aid, especially to those not used to making sharp color distinctions." E. J. Shanley used the tables in Allen and Circular 35, Bureau of Chemistry. It is recommended RECOMMENDATIONS. (1) That an effort be made to obtain authentic samples of vegetable or natural coloring matters, such as are used in food products. This work should be assigned to such men as are in a position to obtain authentic samples, for it is well-nigh impossible for one person to obtain any considerable number of such samples and to ascertain their source and method of preparation; (2) That characteristics of vegetable coloring matters and methods for identification be studied; (3) That synthetic preparations of pure colors for standards be made; (4) That the separation and identification of mixed colors be studied. The president announced the following appointments as members of Committee A on recommendations of referees: R. J. Davidson, J. P. Street, J. G. Lipman, B. L. Hartwell, and W. A. Withers. The association adjourned until 2 o'clock. THURSDAY AFTERNOON SESSION. REPORT ON MEAT AND FISH. By F. C. WEBER, Associate Referee. In view of the fact that no work has ever been reported to the association on this subject, it seemed to the referee that some results showing the degree of accuracy of some of the chemical methods ordinarily employed in separating protein nitrogen, and at what point they show deterioration of meats, might be of interest. Owing to the nature of the work and the difficulty of keeping samples uniform, no attempt was made to secure collaborative work. SAMPLES. The determinations here reported were made on three samples of chicken meat. Six young market chickens were obtained, killed, dressed, and allowed to stand in the ice box over night. The next morning the flesh was separated from the bones a Commercial Organic Analysis, vol. 3, part 1. b Soc. Dyers and Colorists, 1905, 21: 236. and skin and thoroughly ground and mixed by passing six times through a meat chopper. It was then divided into two equal portions, one marked "fresh" and the other, after the addition of 0.1 per cent of boric acid, was marked "preserved." The third sample represents the meat from three cold-storage drawn chickens, in storage twenty-six months, treated in the same manner as above, but without the addition of boric acid, and marked "stored." Each sample was placed in a screw-cap Mason jar and allowed to stand for one week, at laboratory temperature during the day, and in an ice box at night. During this time samples were taken for analysis on the first, second, third, sixth, and seventh days of standing. Every precaution was observed to guard against loss of moisture during the removal of the sample, as a result of which the moisture content remained very constant. METHODS. The following determinations were made at each of the periods cited: Moisture, total nitrogen, ammonia nitrogen, and, in the aqueous extract at room temperature and with ice water, nitrogen was determined as total, coagulable, amido, and ammonia. The difference between the sum of the coagulable and amido nitrogen and the total soluble nitrogen is considered as proteoses and peptones. The fat was determined once at the beginning of the experiment. Moisture was determined on a 2-gram sample, dried in a water oven for ten hours. The loss of weight was calculated as moisture. Fat. The dried sample from the moisture determination was ground with dry sand and extracted with anhydrous ether in a Knorr extractor for twenty-four hours for the determination of fat. Nitrogen determinations were made in the Nitrogen Section of the Bureau of Chemistry by Mr. H. W. Houghton, using the Gunning modification of the Kjeldahl method. The ammonia nitrogen was determined on from 5 to 10 grams of sample distilled from a 750 cc flask, after the addition of 250-300 cc water and 10 grams magnesium oxid. The distillate was collected in standard acid and the ammonia nitrogen determined after a one-half hour distilling, 150 cc being distilled off. The distillation was continued for three half-hour periods, 150 cc of water being returned to the flesh between each distillation. The results reported represent the sum of the three halfhour periods. Water-soluble nitrogen [at room temperature (23°-25° C.) and with ice water (8° C.)]: Twenty grams of the well-mixed sample of meat were weighed into a 450 cc Erlenmeyer flask, 250 cc of water added, and shaken for three hours in a shaking machine. In the case of ice-water extract chopped ice was added from time to time, the volume in the flask being kept constant by decanting the excess of water into a second flask. After being shaken the required length of time, the flasks were placed in the refrigerator over night, a small quantity of thymol and phenol having been added as a preservative. The next day they were poured through linen bags and extracted with room temperature and ice water, respectively, by vigorous manipulation with the hands and successive portions of water, till the final extract gave a negative biuret reaction. The extraction was very tedious and required, at first, an entire day for completion, using from 2,200 to 2,500 cc of room-temperature water, and from 1,800 to 2,000 cc of ice water. The room-temperature extract was made up to a volume of 2,500 cc, while the ice-water extract was made up to 2,000 cc throughout the experiment, though the latter extractions, particularly on the last two days, were completed with from 1,400 to 1,800 cc water. After making to volume and thoroughly mixing, the solutions were filtered through 6-inch funnels containing a 38.5 cm S. & S. 588 folded filter paper. The first 750 cc which ran through was discarded (in the case of the room-temperature extract this was used for the ammonia determination); the second quantity, 600 cc to 800 cc, was used for the water-soluble nitrogen determinations. The filtration of the solutions of the first three extractions was very simple, the solutions running through the paper readily, though the second portion was still somewhat cloudy. As the samples spoiled, the extraction became more easy and the filtration more difficult, until on the last two days it was quite difficult to obtain sufficient solution to make the determinations. This filtered extract was entirely clear. The total nitrogen in the aqueous extract was made on 100 cc of the solution. Ammonia nitrogen was determined on 500 cc of the room temperature extract, by distillation with magnesium oxid. The coagulable protein nitrogen was determined in a sample of 200 cc of the water extract. This was placed in a 300 cc evaporating dish and evaporated on the steam bath to a volume of 40 cc. The solution was neutralized with tenth-normal sodium hydroxid, using phenolphthalein as indicator, then replaced on the steam bath and allowed to evaporate for ten minutes, filtered on a plain filter, and washed with hot water. The filter and precipitate were transferred to a Kjeldahl flask and the nitrogen determined. Amido nitrogen: The coagulable protein filtrate was made up to 100 cc volume and 50 ec employed for the amido nitrogen determination. The 50 cc were placed in a 100 ec graduated flask, 15 grams of sodium chlorid added, and the flask well shaken and placed in an ice box. A 24 per cent solution of tannin was prepared, filtered, and placed in the ice box. After one hour 30 cc of the 24 per cent tannin solution were added to each flask and the two flasks filled to the mark with ice cold water. The flasks were thoroughly shaken and stood in the ice box over night. A blank must be carried out simultaneously, as the best tannin contains some nitrogen. The solutions are filtered into 50 ce flasks and the nitrogen determined in the 50 cc. The nitrogen figure thus obtained multiplied by two, minus the nitrogen of the blank, gives the amido nitrogen in 50 cc of the coagulable filtrate. The sum of the amido and coagulable nitrogen subtracted from the total soluble nitrogen is considered as proteoses and peptones. No effort was made to separate the albumoses, proteoses, and peptones. All the results are calculated to a moisture and fat-free basis and are also expressed in per cent of the total nitrogen of each day's analysis. The ice water extractions were made by Mr. H. L. Amoss and the coagulable and amido nitrogen separations by Mr. F. C. Cook, both of the Bureau of Chemistry. The methods as selected, while not representing all that might have been employed, were those that have been generally used in the Bureau of Chemistry, and it is hoped that the work may be used as a starting point in this subject and serve to show the accuracy of the methods when applied to meats in a progressive state of deterioration. DISCUSSION OF RESULTS. The moisture results show very little change throughout the period, the average in the case of the fresh and preserved samples being 73.00 and 71.70 per cent for the storage sample. There was 4.12 per cent of fat in the fresh chicken and 4.09 per cent in the storage. The results on total nitrogen (see table, page 48) are as uniform throughout as the nature of the material and the accuracy of sampling would permit, and serve to show that there is no gaseous loss of nitrogen, while the ammonia nitrogen (that determined directly on the sample, as well as that determined in the extract) is markedly increased throughout and very uniform, particularly in the case of the stored and preserved samples. The amount is quite small at the time of the first analysis and remains so till the third analysis (made after standing two days), when the storage sample contains a little more than the other samples. From this point the increase is rapid. The variations in percentage amounts are from practically 1 per cent in all cases on the first analysis, to 11, 15, and 13 per cent for the fresh stored and preserved samples, respectively, on the last analysis, after seven days standing. The ammonia results on the water extract were unfortunately not made on the first day. They show practically the same results as those determined directly, but are not quite so uniform and not so high in amount. In the case of the formation of ammonia, the increased amount seems to begin to be formed after two days standing. In connection with these changes it may be well to state here the changes in the samples which could be observed macroscopically. At the time of first analysis the samples were fresh, the storage sample showing a characteristic dried appearance. After standing one day they were practically the same, though what may be termed a slight fermenting action seemed to be taking place. On standing two days the samples had begun to deteriorate, especially the fresh and stored sample, while the preserved sample appeared fairly fresh. After three days standing, the deterioration was more marked. A slight odor of spoiled meat was noticeable, more markedly in the fresh and stored meat than in the preserved. After standing six days the odor was quite bad; the samples had lost their texture and there was no doubt that they had spoiled. No difference in their physical condition could be detected after standing seven days that was not noticeable after six days standing. The nitrogen determined in the water-soluble material at room temperature shows the total nitrogen extracted to be largely increased during the experiment, the first decided increase showing in the samples after standing two days. The coagulable nitrogen shows but a slight tendency to increase, the most marked and uniform change being in the stored sample. The amido nitrogen is not very uniform and shows a tendency to decrease especially where the samples are in an advanced stage of putrefaction. The nitrogen here termed proteoses and peptones is markedly increased during the final days of the experiment, the storage sample again showing a more uniform change. The increase of ammonia nitrogen in the water extract conforms to that determined directly, but is not quite so large in amount. The nitrogen in the ice water extract in the various forms separated shows the same general trend as does that of the room temperature extract, though the amounts extracted are usually not so large. The graphic charts, figs. 3 and 4, show these changes more plainly. It is quite noticeable throughout that the results on the storage sample are very uniform and progressive and, moreover, in all but two instances, in all the determinations, the results on the first analysis show the storage sample to be lower in the various constituents than the fresh samples. The same general tendency seems to run throughout the experiment though one would expect the storage meat to deteriorate more rapidly. Taking into consideration the variations in the determinations and the limitations of the methods themselves, there does not appear to be a very clearly defined point at which deterioration can be said to begin, unless it is shown by the ammonia and water-soluble total nitrogen determinations. The increase in these constituents coincides with the macroscopical observation and physical appearance of the sample. The use of ice water in the extraction is unnecessary, as the methods employed are not of sufficient accuracy to detect the greater changes from day to day in the early stages, much less any change which may be due to enzymic action during the process of extraction. It seems probable from the results that the determination of ammonia may be a valuable asset in showing the first indications of changes, as these results are the most uniform and progressive. A large amount of work has recently been done on the methods for the determination of ammonia in animal and vegetable materials. Richardson a after considerable experimenting on the ammonia nitrogen determination, and in which he extracted the meat with 60 per cent alcohol and distilled with magnesium oxids, aspirating air through the flask, and distilling under reduced pressure, finally adopted the method as outlined above as best suited to the purpose. His results on pure ammonium chlorid distilled in a vacuum with magnesium oxid and 60 per cent alcohol are nearly theoretical. This is in substance the method as now employed in the determination of ammonia in urine and might be adapted to this work. a J. Amer. Chem. Soc., 1908, 30: 1515. |