Detection of Gelatin in Dairy Products NIORRISB. JACOBSAND LEONJAFTE Department of Health of the City of New York, 139 Center St., New York, N. Y.
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ELATIN has for many c o n c e n t r a t e d s o l u t i o n s of A method f o r the detection of gelatin in dairy years been used as a gelatin* products, especially for sour cream and cultured t h i c k e n i n g a g e n t in Steinizer (Q), in a study of the mi'ks1 is presented* Basic lead nitrate is used dairy products. In ice cream it properties of g e l a t i n and glue as a protein precipitant and charcoaE in comparison with other prohas of course been accepted beis used to adsorb the pseudo-gelatins formed in cause it improves the texture as teins, noticed that lead acetate well as the standing qualities of solution did not precipitate gelathe souring process. The details of the method this product. However, in milk tin, whereas it did precipitate and its comparison with ofher methods as to chondrins and mucins. and cream and like products, sensitivity and applicability are given. It is Straub (11) s t a t e s t h a t if i t s use h a s g e n e r a l l y been found less sensitive but more definite lhan the gelatin solution is shaken for 20 f r o w n e d u p o n because of a oficial or Stokes method. to 30 minutes with a few cubic tendency to use it to conceal incentimeters of thionin solution, ferior products. The method mainly used €or its detection in dairy products a dark blue color results, whereas if agar-agar is treated the in the past has been that of Stokes (lo),which will be de- same way, a violet coloration is obtained. Liesegang (6) found that if 40 per cent tertiary potassium scribed in detail later. This method, however, in the case of slightly soured milk or slightly soured sweet cream, and phosphate and 10 per cent cupric chloride are allowed to difespecially in the case of sour cream, fails to give a decisive fuse into gelatin gel, the green tertiary cupric phosphate does result in the absence of gelatin. This necessitates either a not result, but a deep violet transparent color. A waterlaboratory rule-of-thumb method, such as the density of tur- gelatin solution may be used as well, but tertiary potassium bidity before considering a test positive, or a resort to a test phosphate causes a precipitate of gelatin. However, in 1914 distinguishing between a gelatin-picrate precipitate and a (4) he pointed out that tricalcium phosphate and cupric pseudo-gelatin-picrate precipitate such as the Seidenberg chloride gave a violet color without turbidity instead of a method (8). This test, besides being laborious, does not al- green precipitate in the presence of gelatin in solution. ways differentiate between the aforementioned types of preSTOKESMETHOD cipitate. In view of these facts, the authors have developed a method The official method given in the methods of the A. 0. A. C. from their experience with other thickeners (3) which will for the detection of gelatin in milk and milk products is the completely satisfy the condition of giving no precipitate in Stokes (10) method. This is performed in the following the case of a dairy product in which gelatin is absent and a manner: To 10 cc. of milk or cream or milk product, add an heavy precipitate or a marked turbidity for every dairy prod- equal volume of acid mercuric nitrate solution (mercury disuct containing gelatin, including sour cream. Furthermore, solved in twice its weight of nitric acid and this solution dithis method needs no laborious confirmatory tests. luted to 25 times its volume with water). Shake the mixture, The properties of gelatin as a colloid have been discussed add 20 cc. of water, shake again, allow to stand 5 minutes, and so frequently in the literature that there is no need for a fur- filter. If much gelatin is present, the filtrate will be opalesther discussion along these lines here. However, there are cent and cannot be obtained clear. To a portion of the filmany other properties of gelatin so little known and yet so trate contained in a test tube, add an equal volume of satupertinent to its detection that it would be wise to review them. rated aqueous picric acid solution. A yellow precipitate will Moreover, there are a few methods used for the estimation of be produced in the presence of any considerable amount of gelatin which are also used for its detection which will be gelatin, whereas smaller quantities will be indicated by a reviewed. cloudiness. I n the absence of gelatin, the filtrate will remain Trillat ( l a ) estimated gelatin in gums and alimentary sub- perfectly clear. Acid mercuric nitrate is added to the milk product in order stances by weighing the precipitate produced by formaldehyde. Henzold (2) boiled the material supposed to contain gelatin to precipitate all the proteins except gelatin. I n the souring in water and filtered. The filtrate is boiled with excess 10 process certain decomposition products are formed whose exper cent potassium dichromate. After cooling, if gelatin is act nature is unknown. Patrick (7) found that these subpresent, a few drops of concentrated sulfuric acid produce a stances had properties very similar to gelatin, and called them "pseudo-gelatins." They are not completely precipiwhite, flocculent precipitate. Greifenhagen (1) used zinc sulfate to precipitate both gela- tated by acid mercuric nitrate, and they give a precipitate tin and proteoses. The zinc sulfate precipitate is redissolved with picric acid and with tannic acid. Consequently, the and reprecipitated by use of mercuric chloride. Since mer- Stokes method is indeterminate for milk and sweet cream on curic chloride precipitates the proteoses only, the filtrate will the verge of souring, and fails completely for sour cream. contain only gelatin, which can be estimated by determining Furthermore, acid mercuric nitrate itself will, if present in large excess, cause a turbidity in the presence of picric acid, the nitrogen. A. and L. Lumiere and A. Seyewitz (6) made a series of and a turbidity will develop on standing even if the acid studies of the properties of 10 per cent gelatin solutions. mercuric nitrate is not in excess. They found that it is precipitated by phosphotungstic and SEIDENBERGMETHOD phosphomolybdic acids, chlorine and bromine water, and Seidenberg (8) developed a method to distinguish between ferric, manganic, vanadic, ceric, uranic, and mercuric salts. They made a rather exhaustive study of the properties of the precipitate produced by gelatin and that produced by 418
October 15, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
pseudo-gelatin. His method is as follows: After performing the Stokes test, shake the solution and precipitate in a large test tube very thoroughly, allow to stand, decant off the clear liquid, and collect the precipitate on a filter. Wash with water containing 2 to 3 drops of ammonia per 100 cc. until the washings are slightly alkaline to litmus, then with water alone until they are neutral. Transfer the precipitate or the precipitate and filter to a small beaker, add 10 to 20 cc. of water, heat to boiling, and filter hot into a test tube. The filtrate will contain the gelatin-picrate but not the protein. Cool and test for gelatin by adding an equal volume of the picric acid solution. This method was advocated for sour creams, but failed to solve the problem completely because sometimes on samples known to contain gelatin, a positive test for gelatin with picric acid was not obtained after subjecting the sample to the Seidenberg treatment. BASICLEADNITRATEMETHOD I n view of the limitations of both the Stokes and Seidenberg methods, the authors have devised and developed an entirely new method for the detection of gelatin in dairy products. This method is based on the separation of gelatifi from the other proteins in the milk product and then the subsequent identification of gelatin. This is done first by the use of a protein precipitant, and second by the use of an agent to adsorb the pseudo-gelatins and proteoses. The protein precipitant is basic lead nitrate and the adsorbing agent is calcined charcoal. The basic lead nitrate consists of two solutions added separately to the solution or mixture to be clarified. Solution 1: lead nitrate, 250 grams to 500 cc. Solution 2: sodium hydroxide, 25 grams to 500 cc. To BO cc. of milk or milk product, add 3 cc. of lead nitrate solution and stir, add 3 cc. of sodium hydroxide solution and stir, add 5 cc. of water and stir, add 0.1 gram of calcined charcoal and stir thoroughly, allow to stand for 5 minutes, and filter. To 3 CC. of the filtrate add 2 drops of concentrated nitric acid and then a few drops of freshly or recently prepared 5 per cent tannic acid solution. In the presence of gelatin there is a white or brownish voluminous precipitate. In the absence of gelatin the solution remains perfectly clear. As a confirmatory test, add to a portion of the filtrate (no addition of nitric acid is now necessary) an equal volume of freshly filtered saturated aqueous picric acid solution. I n the cage of considerable quantities of gelatin, there is a heavy precipitate of gelatin picrate. I n the case of smaller quantities, there is a turbidity which develops within 2 minutes. In the absence of gelatin, the filtrate will remain perfectly clear even on standing. For the tannic acid test the addition of nitric arid is essential, for otherwise tannic acid will always give a precipitate. For the picric acid test there is no need to use acid. The addition of acid in this case reduces the sensitivity of the test, because gelatin picrate is somewhat soluble in nitric acid. BASICLEADACETATE METHOD As an alternative method to the one given above, the authors suggest the following. It will not give as good results as the previous method, but nevertheless is better for sour creams than the Stokes method. It is based on the same principle as the preceding one, but uses dry basic lead acetate instead of basic lead nitrate solution as the protein precipitant. The adsorbing agent used is the same. The dry basic lead acetate may be made in the manner given in the methods of the A. 0. A. C. or in any sugar handbook. To LO cc. of milk or milk product, add 1 gram of basic lead
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acetate and stir. Add 15 cc. of water and stir, add 0.1 gram of calcined charcoal and stir thoroughly, allow to stand for 5 minutes, and filter. To 3 cc. of the filtrate add 3 cc. of water, 2 drops of concentrated nitric acid, and then a few drops of freshly or recently prepared tannic acid. ' A heavy voluminous white or brownish precipitate shows the presence of gelatin. To another portion of the filtrate add an equal volume of water and 2 to 3 drops of concentrated, nitric !mid, and then add an equal volume of freshly filtered saturated aqueous picric acid so1ut)ion. A heavy precipitate of gelatin picrate or a pronounced turbidity which develops in 2 minutes shows the presence of gelatin. In the absence of gelatin the filtrate will remain clear for at least 10 minutes. The main drawback of this method is that after adding picric acid, a crystalline precipitate settles out on standing if the filtrate from the basic lead acetate treatment is not diluted. However, this precipitate is markedly different from gelatin picrate or pseudo-gelatin picrate, and consequently can easily be distinguished.
EXPERIMENTS ON ADSORBING AGENT In order to determine the best adsorbing agent for the pseudo-gelatins a series of experiments were run with various adsorbents and clarifying agents. The results of thew are given in Table I. TO DETERMINE ADSORBING TABLE I. EXPERIMENTS
AGENT
(All results considered after 2 minutes) PICRIC ACID TANNIC ACID Sour cream Sour cream Sour cream with Sour cream with ADBORBENT without gelatin gelatin without gelatin gelatin Fuller's earth Clear soln. Ppt. Faint ppt. Heav,y ppt. Magnesium carbonate Turbid Turbid Ppt. Heavy ppt. Calcined charcoal Very clear Ppt. Very clear soh. Heavy ppt. soh. Kaolin Turbid Ppt. Heavy ppt. Heavy ppt. P t Ppt. Heavy ppt. Heavy ppt. Pumice Cfieir soh. Turbid Faint ppt. Blood charcoal Heavy ppt. Alumina cream Ppt. Ppt. Heavy ppt. Heavy ppt.
These tests were performed in the following manner: Sour cream known not to contain gelatin was subjected to the basic lead nitrate treatment substituting in each case 0.1 gram of the adsorbents given in Table I. Columns 1 and 3 show the results with picric and tannic acids. Then sour cream known to contain gelatin was again tested by the basic lead nitrate method, substituting in each case 0.1 gram of each adsorbent. These results are shown in columns 2 and 4. It can be seen a t a glance that calcined charcoal adsorbs the pseudo-gelatins the most, and gelatin itself the least, making it ideal for use in this test. The authors found that decolorizing carbons were better adsorbents to use in this test than carbons used for physiological or pigment purposes. Of these types of decolorizing carbons, Eimer and Amend's calcined charcoal, Darco, and Norit were found to be about equally efficaloious as the adsorbing agent. COMPARISON OF METHODS SENSITIVITY.The basic lead nitrate method will give a good test for gelatin for one part of gelatin in 2000 of the milk product. The actual sensitivity of this reaction is grleater than one part in 2000 but, owing to the addition of the adsorbing agent, it is lowered. This is compensated by obtaining an exceedingly clear filtrate and negative test. Moreover, this disadvantage is completely balanced by the fact that the use of gelatin in dairy products requires a gneater proportion than one part in 2000 if the effect the gelatin is supposed to produce is really to be obtained. However, the method is only sensitive to one part in 400 for such products as ice cream or ice cream mix, particularly if these products contain egg material.
ANALYTICAL EDITION
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T A ~ LIT. E COMPARISON OF METHODS FOR APPLICABILITY
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(All tests considered after standing 2 minutes)
SWEETCREAM Without selatin With gelatin Very faintly cloudy Heavy turbidity Heavy ppt. Heavy ppt.
7 SOURCREAM Without gelatin With gelatin Turbid Heavy turbidity Heavy ppt. Heavy ppt.
Picric acid Tannic acid
Very faintly cloudy Clear soh.
Marked turbidity Heavy ppt.
Very faintly cloudy Clear s o h .
Marked turbidity Heavy ppt.
Picric acid Tannic acid
Perfectly clear s o h Perfectly clear soh.
Turbid Heavy ppt.
Perfectly clear soh. Perfectly clear s o h
Turbid Heavy ppt.
METHOD Stokes
REAQENT Picric acid Tannic acid
Basic lead acetate Basic lead nitrate
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In order to make certain as to whether this method would detect any type of gelatin, twenty samples of gelatin of various grades and types, one sample of glue, and one sample of isinglass were respectively incorporated with sour cream, or milk or cultured milks which had previously been found not to contain gelatin, Subjecting these prepared samples to the basic lead nitrate test yielded a positive test for gelatin for every type of gelatin or glue used. These tests were performed on sour cream samples containing 0.25 per cent gelatin. A further series of experiments was run to determine whether time would alter the sensitivity of this test. This set of experiments a t the same time reproduced plant conditions, for a dairy product to be tested for gelatin seldom comes directly to the analytical laboratory from its original source, hence time elapses before the test for gelatin is made. To three samples of sour cream which had been found not to contain gelatin by the basic lead nitrate method three samples of gelatin were added. For each cream and each sample of gelatin, a 0.25 and 0.5 per cent mixture of gelatin in cream was made. These six prepared samples were tested for gelatin over a period of one month a t various intervals. They continued to give a decisive test for gelatin throughout the entire period. Practically every type of dairy product that might contain gelatin was tested by this method to determine whether it would apply in every case. The following products were tested: milk, sweet cream, sour cream, sweetened condensed milk, unsweetened condensed milk, and cultured milks. Ice cream samples were also tested and in every case gave a positive test for gelatin, as might be expected from the fact that nearly all ice creams contain gelatin. For unsoured ice cream and ice cream mix, the amount of adsorbent added and the length of standing time must be strictly adhered to in order to obtain results. APPLICABILITY. The basic lead nitrate method was rigidly tested for complete and practicable applicability in three ways, First, it was compared with the Stokes method. This was done in the following manner: The Stokes method we know works for sweet creams. Consequently, a sweet cream known not to contain gelatin was tested by the Stokes, the basic lead nitrate, and the basic lead acetate methods. To a portion of this cream which was found negative for gelatin by all three methods, some gelatin was added, the sample was again tested by all three methods, and all three gave a positive reaction. Then that portion of the sweet cream to which no gelatin was added was allowed to sour. This soured cream was now tested for gelatin by all three methods. The Stokes test was now indeterminate because of a turbidity, the basic lead acetate method gave a very faint turbidity, whereas the basic lead nitrate method yielded a
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perfectly clear negative result. Gelatin was now added to the remainder of the soured cream and was again tested by all three methods. The results of this experiment are given in Table 11. It may be seen in the table that the Stokes method gives a positive gelatin test with a soured sweet cream containing no gelatin. It may also be seen that the tannic acid test for gelatin cannot be used in conjunction with the Stokes method. The second test of the basic lead nitrate method was performed in the following manner: Samples of cream and cultured milks containing and not containing gelatin were submitted to three chemists. These chemists did not know which samples contained the gelatin and which did not contain it. By the use of this method every sample of cream or of cultured milk with gelatin and those without it were reported correctly by each of the three. The third test of this method was performed by subjecting various grades of gelatin and glue and isinglass to the test. These results have been discussed in the above text.
DISCUSSION The basic lead nitrate method is one which, in so far as the picric acid reaction for gelatin is concerned, is less sensitive than the Stokes method, but which far surpasses the Stokes test in the following particulars: 1. It gives a perfectly clear blank or negative result in the absence of gelatin for sour cream and cultured milks, as well as for any other dairy product, for the reactions with tannic acid and picric acid. 2. The filtrate can be tested by one reagent-namely, tannic acid-and confirmed by another reagent-namely, picric acid. 3. It is easy t o perform and dispenses with the Seidenberg addition to the Stokes test.
ACKNOWLEDGMENT The authors wish to express their thanks to Reginald Miller, chief chemist, for his aid and cooperation in this work.
LITERATURE CITED (1) Greifenhagen, Konig, a n d Scholl, Biochem. Z., 35, 217 (1911). (2) Hensold, J. SOC.Chem. Ind., 19, 1042 (1900). (3) Jacobs a n d Jaffe, IND.ENQ.CHEM.,Anal. Ed., 3, 210 (1931). (4) Liesegang, Boll. chim.-farm., 53, 736 (1914). ( 5 ) Liesegang, 2. Chem. Ind. Kolloide, 5, 248 (1909). (6) Lumiere, A., a n d L., and Seyewits, A., Bull. SOC. chim., 3-4, 743 (1908). (7) Patrick, U. S. Dept. Agr., Bur. Chem. Bull. 116, 26 (1914). (8) Seidenberg, J. IND.ENQ.CHEM.,5, 927 (1913). (9) Steiniser, Kunststoffe, 5 , 73 (1915). (10) Stokes, Analyst, 22, 320 (1897). (11) Straub, 2. Nahr.-Genzissm., 27, 801 (1914). (12) Trillat, Compt. rend. 127, 724 (1898). RECEIVEDJune 7, 1932.
