April, 1950; BLACK AND SCHWARTZ 185 The Estimation of Chitin and Chitin Nitrogen in Crawfish Waste and Derived Products BY Miss M. M. BLACK AND H. M. SCHWARTZ SvNorsrs-The determination of chitin and chitin nitrogen in crawfish waste and derived products has been subjected to a critical study. In the proposed method, chitin is isolated and either weighed as such or, in the case of the determination of chitin nitrogen, its nitrogen content is determined by one of the standard methods. When applied to crude chitin samples, the precision of the proposed method is of the order of 0.5 per cent. of the mean. I t is somewhat lower ( &3 per cent. of the mean) in the case of crawfish meals. The method is similar to the A.O.A.C. method for the determination of crude fibre. A comparison of the results obtained on a number of crawfish meals by the two methods shows close agreement.The chitin, chitin nitrogen and total nitrogen content of a number of crawfish meals are reported, and the importance of making a correction for chitin'nitrogen when calculating the protein content of such meals is stressed. APPROXIMATELY 90oO tons of waste are available annually from the production of canned and frozen crawfish tails on the west coast of the Union of South Africa and South-West Africa. At present only a limited proportion of the waste is converted into crawfish meal, which is used for poultry feed. An investigation into the possibility of producing chitin on a commercial scale from crawfish waste was undertaken by the authors. The results of this work will be published elsewhere.As part of this investigation the determination of chitin in crawfish waste and products derived from it was studied. The estimation of chitin nitrogen in crawfish meals used for feeding purposes was also studied. This is of interest because it is customary to sell the meal on the basis of its protein content, which is calculated from the total nitrogen content. Since chitin contains 6.90 per cent. of nitrogen, and since it is very doubtful whether this is available to animals, with the possible exception of ruminants, it is desirable to subtract the chitin nitrogen from the total nitrogen when calculating the protein content of crawfish meal. A survey of the literature revealed that the methods most commonly used for the estima- tion of chitin in plant and animal tissues are based on the isolation of chitin.lp2g3 Crustacean shells are usually tre9ted first with excess of hydrochloric acid to dissolve out the mineral matter.The sample is then subjected to repeated treatments with 4 to 10 per cent. aqueous sodium or potassium hydroxide. The residue is finally decolorised with dilute potassium permanganate and extracted with alcohol and ether. No attempt appears to have been made to determine the number of alkali treatments necessary to remove completely all non-chitinous material, or the extent to which repeated alkali digestions cause de-acetylation and subsequent loss of chitin. Chitin has also been determined by conversion to de-acetylated chitin (chitosan) by the action of very concentrated potassium hydroxide at 120" to 150" C., and then weighed as such.4 This method is unsatisfactory, however, since the amount of chitosan obtained varies inversely with the duration of the alkali treatment.3 EXPERIMENTAL In the present method, chitin is extracted and weighed as such. The following procedure was finally adopted. Estimation of chitin-Weigh 0.2 to 0.4g. of crude chitin or 2 g. of crawfish waste or meal into a 250-ml. beaker, add 50 ml. of N hydrochloric acid and heat on a boiling water- bath for 1 hour. At the end of this period, filter the contents of the beaker through a sintered glass crucible (porosity G1) or a piece of linen. Wash the beaker and the filter with boiling water until the washings are no longer acid. Then wash the residue on the filter back into the beaker with 100 ml.of 5 per cent. w/v sodium hydroxide solution, and digest on a steam- bath for 1 hour. The residue at this stage should consist of chitin together with any silica186 present in the sample. Filter it through an alunldum crucible or through a Gooch crucible prepared with ignited asbestosJ5 wash thoroughly with boiling water and finally wash twice with about 15 ml. of acetone. Dry the crucible and contents at 110" C. to constant weight. Incinerate the contents of the crucible in an electric muffle-furnace or over a Meker burner at a dull red heat until all carbonaceous matter is consumed. Cool the crucible and re-weigh. Report the loss in weight as chitin. Estimation of chitin nitrogen-Carry out the acid and alkali digestions as described above. Filter the residue left after the alkali digestion through a sintered-glass crucible or through a piece of linen, and wash thoroughly with hot water.Transfer the chitin to a 250-ml. Kjeldahl flask, using the least possible amount of water. Evaporate the water to less than 5 ml., taking care to avoid bumping. Add 25 ml. of concentrated sulphuric acid and 10 g. of potassium sulphate (0.25g. of copper selenite or any other catalyst may also be added if desired) and complete the nitrogen determination in the usual way.6 RESULT s BLACK .4XD SCHWARTZ: THE ESTIMATIOS OF CHITIN AND CHITIN [Vol. 75 NUMBER OF ALKALI TREATMENTS REQUIRED- Previous workers have used at least three alkali treatments to purify their chitin in the estimation of chitin in both animal and plant r n a t e r i a l ~ .~ ~ ~ ~ ~ The number of alkali treatments necessary to ensure the removal of all non-chitinous organic matter was investigated. The isolation of chitin from crawfish meal samples was carried out by the recommended procedure. The residue after the first alkali digestion was dried and weighed and then subjected to a TABLE I EFFECT OF SUCCESSIVE TREATMENTS WITH 100ML. OF 5 PER CENT. NaOH ON THE WEIGHT OF THE RESIDUE: BEFORE IGNITION Weight of residue after 1st treatment, 2nd treatment, 3rd treatment, €5 g. g. 0.2749 0.2701 0.2660 0.2410 0.2369 0.2 3 2'7 0.2554 0-2508 0.24513 0.2074 0.2054 0.2030 0.2543 0.2507 0.250 1 0.2992 0.2946 0.2916 0.2542 0.2499 0.2488 A I 7 Loss in weight* ind treatment, 3rd treatmen;, 1.8 3.2 1-7 3.5 1-8 3.7 1.0 2.1 1.4 1.7 1.4 2.4 1.7 2.1 A % % * As percentage of weight of residue after 1st alkali treatmen!. TABLE I1 DE-ACETYLATION OF CHITIN UXDER CONDITIONS OF ESTIMATION No.of alkali treatments Weight of chitin isolated, g. 0.1821 0.1843 0.2532 0.2660 0.2660 0.2327 0.2459 Weight after acetic acid treatment, g. 0.1817 0.1834 0-2522 0.2639 0-2651 0.2313 0.2451 De-acetylated chitin, 0.2 0-5 0.4 0.8 0-3 0.6 0.3 % second and finally a third treatment with 100 ml. of 5 per cent. sodium hydroxide solution, The results are summarised in Table I. It will be seen that the second and third alkali treatments cause small losses in the weight of the residue obtained after the first treatment, These losses, however, are only of the order of 2 to 4 per cent.of the weight of the chitin in the sample. This is of the order of experimental error of the method, and consequently one treatment with alkali is deemed sufficient in the estimation of chitin in crawfish meals andApril, 19501 NITROGEN IN CRAWFISH WASTE AND DERIVED PRODUCTS 187 derived products. That one alkali treatment suffices to remove all but a trace of non- chitinous organic matter was further demonstrated by the fact that fish meals, which were known not to have been admixed with crawfish meal, gave less than 0-2 per cent. of residue when treated according to the proposed method (Table I11 ( a ) ) . The de-acetylation of chitin even after three treatments with 5 per cent. sodium hydroxide solution under the conditions employed in the determination is negligible.This was demon- strated by heating samples of chitin isolated by the proposed method with 3 per cent. v/v acetic acid on a steam-bath for 1 hour. Under these conditions de-acetylated chitin dissolves readily. The loss in weight of the chitin preparations, however, was less than 1 per cent. (Table 11) , The chitin isolated from crawfish wastes by the proposed method was pure white in colour. Most of the colour was removed by the alkali treatment and what remained was taken out by washing with acetone. Further decolorisation with dilute potassium perman- ganate as employed by other workers is thus unnecessary for products derived from crawfish waste. PRECISION OF METHOD- Pure chitin was prepared from crawfish shells by treatment with hydrochloric acid, followed by repeated treatment with 5 per cent.sodium hydroxide. Any de-acetylated chitin produced in the process was removed by treatment with 3 per cent. acetic acid. The purified chitin was added to samples of pilchard meal which had been shown to contain less than 0-2 per cent. of chitin. Determination of the chitin content of the mixtures by the proposed method gave recoveries of 98.2 to 100.1 per cent. (Table 111). The recovery of chitin added to fish ineals was studied. rrA4BLE 111 RECOVERY OF PURIFIED CHlTIN ADDED TO FISH MEALS (a) BLANK DETERMINATIONS ON PILCHARD MEALS Weight of meal Sample taken, isolated, Weight of chitin g. g . 1 2.0402 o*oooo 2.0690 0~0001 2- 1369 1,9300 (L) RECOVERY OF ADDED CHITIN Weight of Pilchard pilchard meal meal used taken, g - 1 2.2344 1-9917 2 2.7097 2.0703 8.7022 2.2167 Chitin in pilchard meal, g.nil nil 0.0032 0.0024 0.0032 0.0027 0.0023 0.0026 Purified chitin added, g. 0.2015 0.2081 0.3354 0.43 16 0.1969 0.2634 % nil nil 0.1 1 0.13 Chitin found, Recovery, g- % 0.2018 1 oo. 1 0.2070 99.5 0.3330 98.4 0-4270 98.3 0.1964 98.2 0.2639 98-7 Determination of the chitin content of thirteen samples of crude chitin, containing 60 per cent. or more of chitin, was carried out in duplicate by the method described. The difference between duplicate deterininations ranged from 0-2 to 1.6 per cent. of the mean. The average deviation from the mean was 0.4 per cent. When the method was applied to crawfish meals, the agreement between replicate determinations was not so close. Duplicate, and in some cases quadruplicate, determinations were carried out on thirteen samples of crawfish meal from various sources.The deviation of a single determination from the mean for the sample ranged from 0.2 to 10.7 per cent. The average deviation from the mean was 3.0 per cent. One reason for the lower precision of the method when applied to crawfish meals appears to be the difficulty of sampling the meal properly, even after it has been finely ground. This was particularly noticeable when the meal contained a high proportion of sand.188 BLACK AND SCHFVARTZ: THE ESTIMATION OF CHITIN AND CHITIX [VOl. 75 The precision of the method for the determination of chitin nitrogen in meals is of the same order as that for the determination of chitin, The determination of the chitin nitrogen content of nine crawfish meals was carried out in duplicate.The difference between duplicates ranged from 1.4 to 9-1 per cent. of the mean. The average deviation from the mean was 1-8 per cent. COMPARISON OF THE METHOD WITH THE A.O.A.C. METHOD FOR THE DETERMINATION OF CRUDE The method described here for the determination of chitin is very similar in principle to the A.O.A.C. method for the determination of crude fibre.5 A comparison of the values obtained by the two methods for a number of crawfish meals is given in Table IV. The results in each case refer to the dry, fat-free meal. The values obtained by the two methods FIBRE- TABLE I V COMPARISON of; VALUES Meal sample FOR CHITIN AN11 CRUDE IY CRAWFISH MEALS Chitin, 11.4 9.4 11.2 10.5 13.1 11.5 13.8 % FIBRE (A.o.A.c.METHOD) Crude fibre, 11.7 10.5 11.6 10.2 13.1 11-8 15.4 % are in very close agreement, except in two cases (samples 4 and 9), and here the differences are within the limits of the experimental errors clf the methods. The A.O.A.C. method for crude fibre determination may therefore be used to determine chitin in crawfish products. The method described here is preferred, however, since it requires less rigid adherence to specified conditions. THE CHITIN AND CHITIN NITROGES COXTENT OF CRAWFISH MEALS- The chitin, chitin nitrogen and total nitrogen content of nine crawfish meals are recorded in Table V. The chitin nitrogen content as determined by the proposed method is generally TABLE V CHITIN, CHITIN NITROGEX AND TOTAL NITROGES COXTEXT OF CRAWFISH MEALS Chitin nitrogen, % r - y Chitin nitrogen/ Chitin, (Calc.) (Found) Total nitrogen, total nitrogen, Ye O/ % / O 13.1 12.1 11.4 11.2 10.5 13.1 11-5 13.8 13-6 0.91 0.84 0.79 0.77 0-73 0.90 0-79 0-96 0.94 0.75 0.86 0.73 0.68 0-82 0-81 0.76 0.94 0.94 7.06 6.29 7.70 9.79 9-20 7-37 9-12 8.16 7.91 10-7 13.6 9.5 7.0 8.9 11.1 8.3 11.6 11.9 slightly lower than that calculated from the chitin content of the meal, using the theoretical value of 6.90 per cent.for the nitrogen content of chitin. This is to be expected, since even carefully purified chitin samples generally contain less than the theoretical amount of nitr0gen.l In the meals examined, the chitin nitrogen ranged from 7.0 to 13.6 per cent. of the total nitrogen in the meal. The importance of making a. correction for chitin nitrogen in calculating the protein content of crawfish meals is therefore apparent. We are indebted to the South African Council for Scientific and Industrial Research UJe are also indebted to the Fishing Industries Research for permission to publish this work. Institute, Cape Town, for supplying a number of crawfish meals used in this work.April, 19501 NITROGEN IN CRAWFISH WASTE AND DERIVED PRODUCTS 189 REFERENCES 1. 2. 3. 4. 5. 6. Ibid., p. 26. Pringsheim, H., and Kriiger, D., “Handbuch der Pflanzenanalyse,” Vol. 3/1, p. 77, Vienna, Julius Bergmann, W., Ann. Entomol. SOC. Am., 1938, 31, 315. Lafon, M., Compt. Rend., 1941, 212, 456. Tauber, 0. E., J . Morphol., 1934, 56, 61. “Official and Tentative Methods of Analysis of the Association of Official Agricultural Chemists,” Springer, 1932. 6th Edition, 1945, p. 408. 17hTS AND PROTEINS UNIT OF THE NATIONAL CHEMICAL RESEARCH LABORATORY UNIVERSITY OF CAPE TOWN RONDEBOSCH, SOUTH AFRICA Sepember, 1949