An Electrometric Method for Detection of Relative Freshness of Haddock MAURICEE. STANSBYAND JAMESM. LEMON,U. S. Bureau of
D
URING the course of an investigation conducted by the Bureau of Fisheries concerning the handling of fresh fish, it became highly desirable that an index as to the state of freshness of the samples to be examined be available. Such a test must not only be reliable, but also must not require too much time to execute. It is not necessary that such a test should tell how long it has been since the fish was caught, but it is important to obtain a knowledge of how much longer the fish may be expected to keep, if handled properly. Since nearly all the work in the Gloucester, Mass., laboratory dealing with the handling of fresh fish has been with haddock, the test was designed to give a measure of the freshness of haddock only. While it seems probable that the results to be discussed might, in a modified form, be applicable to other varieties than the haddock, no comprehensive tests have been made with any other varieties. CHANGES OCCURRING WHENFISHDECOMPOSE Analyses (3,7 ) have shown that haddock consists of about 80 per cent water, 16.5 per cent protein, 1.28 per cent ash, and 0.3 per cent fat. The only substance whose decomposition is of any importance, during the spoilage of the flesh of these fish, is the protein. A measure of the protein hydrolysis or decomposition will give an indication of the freshness of the flesh. One of the first changes occurring after the death of a fish is the onset of rigor mortis. Among a number of changes associated with Tiger mortis may be mentioned an increase in lactic acid content and a very slight increase in hydrogenion concentration. Of course, the most noticeable change observed is that the fish becomes stiff. Nothing definite can be said as to the duration of rigor mortis, or as to the time elapsing between the death of the fish and the onset of rigor. These seem to depend to a great extent upon a variety of factors, such as the method of capture and the temperature at which the fish is kept. However, it may quite generally be said that, if rigor mortis is present, the fish is of the highest quality. While the fish is still in rigor mortis or immediately after i t has passed off, a second process begins. All fish secrete certain enzymes, which during the life of the fish perform various normal functions. However, after death a process known as autolysis begins, and these enzymes start to hydrolyze the highly complex protein of the fish muscle into the simpler polypeptides, peptones, and amino acids. A second process, usually accompanying autolysis, is bacterial decomposition. The first result of the action of bacteria is the formation of the same type of compounds as are produced during autolysis, such as polypeptides and amino acids. These, however, are only intermediate products which are later decomposed into a wide variety of substances. Many of these have disagreeable odors and some of them may be toxic. Most of the end products of bacterial decomposition of fish are basic, so that a rise in pH is observed. When bacterial decomposition occurs at low temperatures, as when the fish is packed in ice, the principal products seem to be the intermediate ones. Although fish decomposition is usually classified as bac-
Fisheries, Gloucester, Mass.
terial or autolytic, a more satisfactory basis, as far as a teat for freshness is concerned, is based upon the type of products formed. Two types of changes occurring may be defined as follows: Primary changes are those changes in fish flesh which lead to the formation of amino acids from protein or to any intermediate products such as polypeptides and peptones. Secondary changes include those changes of the fish flesh which lead to the formation of products which detract from the value of the fish, usually contributing disagreeable flavors or odors. Some of these products are ammonia, amines, indole, hydrogen sulfide, and skatol. There are two objections to the occurrence of primary change in the flesh of fish. First, as the protein molecule hydrolyzes, the fish becomes softer. I n extreme cases juices run from the fish. These juices or LLdrip”contain dissolved protein, amino acids, and minerals. Such a condition, while detracting from the value of the fish, in no way has any actual harmful effect. The second objection is that a fish in which extensive primary changes have occurred is much more readily decomposed into secondary decomposition products when exposed to high temperatures and the action of bacteria. Gibbons and Reed (11) have shown that where extensive autolysis (largely primary changes) has taken place, the fish is much more rapidly decomposed by bacteria than is a fresh fish. Secondary decomposition is generally considered to be the result of bacterial action. The end products of these reactions are chiefly basic. It is this type of decomposition which is usually considered as fish spoilage, and which renders it inedible. When haddock are packed in ice, primary changes are the chief types of degradation occurring, at least until after the fish have been in ice for several weeks. After the haddock have been removed from the ice, secondary decomposition sets in very rapidly and true spoilage occurs. Since haddock are often packed in ice for about two weeks before they reach the consumer, the detection of the primary changes is of the highest importance. Numerous tests for the degree of freshness of fish flesh have been proposed. Most of these depend upon the formation of some one end product of bacterial or enzyme action. Among these may be mentioned the test for hydrogen sulfide of Almy (I), and the test for ammonia of Tillmans and Otto (16). Other tests have been described by Fellers, Shostrom, and Clark (9), Clough (8),Tressler ( l 7 ) , Smith (IS), Tauti, Hirose, and Wadi ( 1 4 , Tillmans, Hirsch, and Kuhn ( I F ) , Benson and Wells ( b ) , and Carey (6). Since these tests consider only secondary changes, and disregard the primary ones, their value is limited. An excellent discussion of the organoleptic list is given by Anderson (8).
THEORY OF
THE
METHOD
I n developing the present method, it was thought more advisable to follow the disappearance of the protein molecule than to observe the formation of any one end product. It was found that the buffer capacity (18)of a haddock solution decreased as primary changes occurred in the flesh. By choosing a pH range over which the buffer capacity of the inorganic constituents of the fish (mostly phosphate) was a t 208
May 15, 1933
IXDUSTRIAL AND ENGINEERING CHEMISTRY
a minimum, a sensitive index of the primary chan,ves was obtained. Such a range is between pH 6.0 and 4.2. One of the principal products formed as a result of bacterial action is ammonia. By titrating the fish solution to a pH of 6.0 an indication of the amount of ammonia formed and hence the amount of secondary decomposition present is obtained. The test then consists of titrating a fish solution first to about p H 6.0, then to about pH 4.3. The amount of acid used in the first step should be proportional to the amount of end products of bacterial decomposition, or secondary decomposition, and the amount of acid to go from pH 6.0 t o pH 4.3 should be inversely proportional to the amount of primary changes or protein hydrolysis having taken place.
hydrochloric acid required to bring the electromotive force to E = 0.100 volt, using the technic described. This is assumed to be proportional to the amount of secondary decomposition present. In a similar way the value of A is defined as the number of milliliters of 0.0165 N hydrochloric acid required to change the E value from 0.100 to 0.200 volt. This corresponds to a p H change of from about 6 to 4.3, and is inversely proportional to the amount of primary changes present in the fish. TABLEI. E. M. F. OBTAINED AFTER ADDITIONOF HYDROCHLORIC ACID TO FIBHSOLUTION ACIDADDED Ml ,
METHODOF MAKINGTHE TEST At least 20 grams of the fish flesh, freed from all bones, should be ground as fine as possible in a meat grinder. Five grams of this are then weighed out and transferred to a 150-ml. bottle, 70 ml. of water are added from a graduated cylinder, and the bottle is stoppered and shaken for at least 10 minutes in a shaking apparatus. The contents of the bottle are then transferred to a 200-ml. Erlenmeyer flask, and an excess (about 0.3 gram) of quinhydrone added. The bottle is rinsed with 30 ml. of water, and the contents added to the flask. The flask and contents are shaken for an additional 2 minutes, and the contents then transferred to a 250-ml. beaker. A platinum electrode and saturated calomel half cell are then dipped in and connections made to a potentiometer. Readings are taken until a “constant” value is obtained. Successive portions of 0.0165 N hydrochloric acid are then added from the buret, stirring the solution and waiting for equilibrium to be attained between each addition. Several potentiometer readings are obtained between E = 0.200 and E = 0.100. The entire titration requires between 15 and 30 minutes. The acid should not be added too ra idly especially between E = 0.140 and E = 0.170, since otgerw$e A drifting potential is obtained near E = 0.200. The amount of acid used t o reach E = 0.100 (pH = 5.97) is calculated as well as the amount used to go from E = 0.100 to
8.0 8.0 8.0 8.0 8.0 8.0 9.0 9.0 9.0 9.0
INTERPRETATION OF RESULTS In all the following discussions, the value of B will be taken as the number of milliliters of 0.0165 N hydroohloric acid required to bring the pH of 5 grams of fish in 100 ml. of water to 6.0. More exactly, since these pH values neglect protein error, the value of B is the number of milliliters of 0.0165 N
TIXESOLUTION STOODAFTER LAST
E. M. F.
ACIDA D D I T I O N Minutes 0 0.25 1 2 3 5
Volts 0.075 0.070 0.055 0.054 0.054a 0.054
n
0.1025 0,099 0.0975 0.0975a 0.0970 0.0965 0.1060 0.1055 0.1050 0 . l05Oa
1 2 3 4 7 0 0.5 1 2
20 20 20 20 20 20 20 20 20 31 31 31 31 31 31 31 35 35 35 35 35 35
E = 0.200 (pH = 4.28).
The pH values given in the last sentence are calculated without regard to protein error. Care should be taken that equilibrium is always attained before taking a reading, since otherwise erratic results will be obtained. KO absolutely constant e. m. f. is ever obtained. Usually there is a rapid fall to the point taken as the reading. The e. m. f. then slowly drifts, usually downward. The amount of drift of potential varies with three factors. First, fresh fish produce less drift than stale ones. Second, after a large addition of acid, there is more drift than after a smaller addition. Third, a t the end of the titration, near E = 0.200 volt, there is a considerably greater drift than a t the beginning. With a little practice, it is not difficult to obtain the correct reading. The drift of potential seems to be caused by the presence of some of the fish protein suspended in the solution, since with filtered solutions very little drift occurs. However, the solution cannot be filtered, since the sensitivity of the test depends upon the presence of these suspended particles which contribute to the buffer capacity. This test was run on a large number of samples of fish preserved in various ways. Some fish were packed in ice whole, and a separate fish withdrawn for each test. In other cases the fish was cut into small pieces, wrapped in moistureproof cellophane, and packed in ice. I n still other cases the fish was ground and stored a t various temperatures.
209
0
0.176 0.172 0.169 0.167 0.166 0.165 0.164 0.163 0.1625& 0.199 0.196 0.195 0.194 0 . 194a 0.1935 0.1930 0.212 0.210 0.2105 0.2095 0.2095 0.2090
0
1 2 3 4 5 6 0
1 2 3 4 5 E. M. F. taken as the reading,
The following data show the average values of A for haddock kept packed in ice from the time they were caught, samples being removed for analyses a t the intervals indicated. The results are the average of about 200 determinations. The last column shows the maximum deviations which can be obtained for different samples of haddock in identical stages of decomposition.
TABLE11. AVERAGEA VALUESFOR HADDOCK PACKED IN ICE
DAYS IN
ICE
CONDITION
0 . 2 5 (6 hours) Fresh, stiff 1 Fresh, stiff 2 Fresh, slightly stiff 3 Fresh 4 Fresh 6 Fresh, slight sweet odor 8 Somewhat sweet odor, slightly soft 12 Sweet odor, a little soft 18 Verv sweet odor. soft 21 Intense sweet odor, very soft
A ‘ 26 31 27.5 26 24 22.5
MAXIMUH ERROR 0.3 0.3 0.3 0.3 0.4 0.4
20 19 17
0.5 0.6 0.8
15
1.0
From these data it will be seen that a fresh fish about 6 hours out of the water has an original A value of about 26, and that this value rises, as rigor mortis appears. As rigor mortis passes off, the value of A diminishes from a maximum of about 31 to a minimum of about 15. The decrease in accuracy with increase in decomposition is due to the greater difficulty of obtaining a constant e. m. f. for stale fish.
210
ANALYTICAL EDITION
Vol. 5 , No. 3
One cause of the initial rise in the A value is the formation of lactic acid. The presence of lactic acid tends to increase the A values in two ways. First it acts as a buffer to some extent around a pH of 4.3, thus requiring more acid to reach this pH value and increasing A. Moreover, Beatty (4) has shown that when lactic acid forms, the protein becomes more soluble. Since the protein, itself, acts as a buffer, an increase in protein solubility increases the buffer capacity, and hence the value of A. Sharpe (12) and Fiske and Subbarow (IO) have shown that certain phosphorus-containing organic substances are converted into orthophosphates after the death of the fish or animal of which they are a part. Since orthophosphates act as buffers to a small extent over the pH range of the A value, the formation of phosphates may be a contributing factor to the initial rise in the value of A. The exact cause of the fall of the A value as primary decomposition proceeds is uncertain, but may be due in part to a change in solubility of the protein. It has been shown that when a frozen fish is stored, a transformation of a part of the fish protein to a less soluble form occurs. It is possible that such a reaction occurs in the case of fish packed in ice. Another possibility is that the buffer capacity of the primary decomposition products of the fish over the pH range measured is less than that of the protein in the fresh fish. However, whatever the exact cause may be, it is certain that during primary decomposition the protein molecule does break down into simpler compounds, and that accompanying this change a loss in buffer capacity of the fish SUSpension over the pH range 4.3 to 6.0 simultaneously occurs. The initial rise in the value of A from 25 to 31 followed by its decrease makes all the values of A between 25 and 31 correspond to two conditions of the fish. However, this is of no practical importance. The initial rise in the value of A is very rapid, a value of 31 usually being reached in less than 24 hours. Moreover, fish in which the value of A is rising are in the state of rigor mortis which can readily be recognized. Therefore, since haddock are almost invariably one day old before they are landed, the initial rise in the values of A can be neglected. Usually haddock obtained from gill netters, the freshest of any, have an A value no higher than 28 and the value is falling. For trawl fish the value is ordinarily mound 23. When a haddock is first caught, it probably has a b value between 7 and 8. If no pronounced bacterial decomposition sets in, the value of B falls to a minimum of 5 when lactic acid forms, and the protein begins to hydrolyze. This initial fall in the value of B is due to the combined effect of the formation of lactic acid, and the consequent lowering of the pH, and to the loss of buffer capacity of the flesh. In the initial stages of hydrolysis, any value of B less than 8 may be said to indicate a fish in which no marked bacterial decomposition has occurred. However, a haddock which has been caught quite recently, and has an A value of 24 or more, should not have a B value of more than 8. If the value of B in such a: case does exceed 8, suspicion is raised that the fish has a t some previous time been allowed to stand at too high a temperature. If a haddock is allowed to spoil while packed in ice, the actual spoilage, as indicated by the formation of a putrid odor, does not occur until after three weeks or more. Usually after this time offensive odors develop rapidly. Simultaneously the value of B, which up till then has not exceeded 8 or 9, sharply rises to 12 or more. In case the spoilage occurs at a higher temperature, the value of B rises more rapidly, a value of 20 being reached in extreme putrefaction. I n such a case a value of about 15 indicates the fish to be barely edible.
,
May 15, 1933
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INDUSTRIAL ARD ENGINEERING CHEMISTRY
I n interpreting the results of this test, it is necessary to consider the values of both A and B. The value of either alone is meaningless. The chart (Table 111) assumes that maximum rigor has been reached or passed away. For haddock, this condition will virtually always be realized, The estimate of the time since the fish had been caught assumes that the fish was well packed in ice since catching. This value is necessarily only approximate. The probable odors which definitely accompany the varying degrees of secondary change are given, and the italics indicate a second type of odor which can be detected when sufficient primary change has taken place and the odor due to secondary change is not too intense. No attempt has been made to determine the amount of decomposition or changes which must be present to make a fish inedible. This is largely a matter of personal opinion, and the establishment of any A and B values as a limit for a fresh fish would be purely arbitrary. Such a procedure is beyond the scope of this paper.
TESTBASEDON CONDUCTOMETRIC TITRATION An attempt was made to develop a conductometric titration, to replace the potentiometric determination of the amount of primary decomposition present in the fish. Enough acid was added to a 5-gram sample of the fish suspended in water to bring the pH to 6.0. The conductivity was then determined, a definite quantity of acid added, and the increase in conductivity ascertained. This increase in conductivity, designated by R, should be proportional to the value A. This was found to be reasonably true, as can be seen from the following data: TABLEIV. RESULTSOF CONDUCTOMETRIC TITRATION R
A 29.0 26.1 24.8 23.9 22.5
23.0 21.3 31 29 20.2
20.7 17 15.8 15.6
41.7 65 30.3 40.5
CONDITION Very fresh, still slightly stiff, fresh odor Fresh Fresh, aweet odor, slightly soft Fres'h, slightly fishy odor Fairly good condition, sweet odor, slightly soft Fairly good condition, fresh odor, quite soft Stale, very soft Very sweet odor, aoft Extromely sweet odor, stale, aoft
The method is extremely simple, requiring only about 3 minutes to execute. However, i t cannot be recommended
211
for a test, since differences in initial conductivity caused by brining the fish before the test was made (a common commercial practice) cause large deviations in the results. While this test has been applied principally to fresh haddock, the authors are of the opinion that a great variety of applications are possible. The tests have also been applied to cod and pollock with results indicating that it is applicable. It is possible that the method may be applied in the case of various meats and packing house products. SUMMARY A reliable test for the freshness of fish has been described, based on buffer capacity measurements. The test requires less than one hour to perform, It gives information, not only as to the accumulation of bacterial end products, but, what is even more important, as to the amount of protein breakdown taken place. (1) Almy, L. H.,
LITERATURE CITED J. Am. Chem. SOC.,49, 2540 (1927).
(2) Anderson, A. G., Fishery Board of Scotland, 26th AnnuaJ Rept., Part 111, p. 37 (1907). (3) Atwater, W. O., Rept. of U. S. Commissioner of Fish and Fisheries, 1888, p. 679, Appendix X (1891). (4) Beatty, 9. A., Biological Board of Canada, Annual Rept., p. 47 (1930). (5) Benson, R . L., and Wells, H. G., J . Biol. Chem., 8 , 61 (1910). (6) Carey, W. E., Am. J . Pub. Health, 6,124 (1916). (7) Clark, E. D., and Almy, L. H., J . Biol.Chem., 23,483 (1918). (8) dlough, R. W., Pub. Puget Sound Biol. Sta., Univ. Wash., 3, 195 (1922). (9) Fellers, C. R., Shostrom, 0. E., and Clark, E. D., J . Bact., 9, 235 (1924). (10) Fiske, C. H., and Subbarow, Y., Science, 67, 169 (1928). (11) Gibbons, N. E., and Reed, G. B., J. Bact., 19, 73 (1930). 112) Shame. J. G.. DeDt. Sci. Ind. Research fBrit.). Food Investieation Board R e s . , p. 202 (1931). (13) Smith, C. S., Biochem. Bull. 3 (1913). (14) Tauti, M., Hirose. I., and Wadi, H., J. I m p . Fisheries Inst. (Japan), 26, 79 (1931). (15) Tillmans, J., Hirsch, P., and Kuhn, A., 2. Untersuch. Lebensm., 53, 44 (1927). (16) Tillmans, J., and Otto, R., Ibid., 47, 28 (1924). (17) Tressler, D. K., U. S. Bureau of Fisheries, Document No. 884, pp. 29-31. (18) Van Slyke, D. D., J. Biol. Chem., 42, 525 (1922). ~
I .
I
RECEIVED February 3, 1933.
Determination of Zirconium in Steels Selenious Acid Phosphate Method STEPHEN G. SIMPSON WITH WALTER C. SCHUMB, Massachusetts Institute of Technology, Cambridge, Mass.
I
N A previous paper (1) the selenious acid method for zirconium (8,s) was shown to be applicable to the determination of airconium in alloys. It was shown, however, that tungsten may be brought down with the final precipitate and an additional step in the procedure was found necessary. I n view of the fact that those elements which tend to cause interference in the selenious acid method do not do so in the phosphate method, and vice versa, it seemed advisable to attempt to combine the two methods in the hope of obtaining a rapid, reliable method for zirconium in alloys in the presence of any element which might ordinarily be present. By thus combining these two procedures, a double precipitation of zirconium is made (advantage over the usual phosphate method) and tungsten and other elements should not interfere (advantage over the plain selenite method). As to the order of precipitation, the difficulty of dissolving a zirconium phosphate precipitate would indi-
cate the desirability of precipitating the zirconium first as selenite and then as phosphate rather than in the reverse order. By using this order the zirconium selenite is dimolved in fairly concentrated sulfuric acid in whioh it is readily soluble, rather than in dilute hydrochloric acid, in which (as in the case of the plain selenite method) it sometimes dissolves with difficulty.
PROPOSED METHOD Dissolve 3 grams of steel in 40 cc. of concentrated hydrochloric acid. When solution is complete or nearly so, add sufficientconcentrated nitric acid to oxidize the iron. Eva oraBe t o dryness on the steam plate, moisten the residue wit{ 6 N hydrochloric acid, evaporate t o dryness, heat at 105' C. for 30 minutes, and treat with 30 cc. of 6 N hydrochloric acid and 50 cc. of hot water. After all soluble salts have dissolved, filter and wash the residue with dilute hydrochloric acid and finally with hot water. Treat the filtrate and residue (each of which may contain zirconium) separately as follow :
