June, 19511 ANALYSIS OF MEAT EXTRACT 320 Analytical Methods Committee RECOMMENDATIONS OF THE MEAT EXTRACT SUB-COMMITTEE Analysis of Meat Extract THE Analytical Methods Committee has received the following Report from the Meat Extract Sub-committee, and its publication has been duly authorised. CONSTITUTION OF THE SUB-COMMITTEE The Sub-committee consists of: G. Taylor, O.B.E., F.R.I.C. (Chairman) ; R. Gordon Booth, Ph.D. (from December, 1949) ; Osman Jones, F.R.I.C. ; the late E. C. Keeley, B.Sc., A.R.I.C. (until December, 1949) ; J. King, O.B.E., F.R.I.C. ; G. Spall; R. G. Westall (nominee of Dr. E. C. Bate-Smith); H. G. Rees, B.Sc., Ph.D., A.R.C.S., D.I.C., F.R.I.C. (Honorary Secretary). The Sub-committee wish to place on record the loss sustained by the untimely death of Mr.E. C. Keeley on December ZOth, 1949. INTRODUCTION The terms of reference of the Sub-committee are: “to consider whether standard analytical methods are necessary for meat extracts and similar products and to carry out such investigations as may be deemed necessary. ” The Sub-committee agree that Standard Methods of Analysis are required and that they should be so formulated to give the maximum information as to the detection of possible adulterants . They further consider, however, that it is desirable that a standard method for this purpose should be one that is both sufficiently accurate and precise for all commercial purposes and also capable of adoption in any analytical laboratory; on this basis, in fact, the experi- mental work has been designed and the suggested methods have been formulated.The Sub-committee do not regard the prescription of standards of composition for meat extracts and similar products as coming within their terms of reference. Nevertheless they have thought it desirable to define products that they regard as being within the general330 ANALYSIS OF MEAT EXTRACT [Vol. 76 description of meat extracts and similar products, in order to indicate suitable methods of analysis. These definitions are given immediately below. DEFINITIONS- Meat extract-The product obtained by extracting fresh lean meat with boiling water and concentrating the liquid portion after removal of fat. Meat stock-The product obtained by the extraction of meat trimmings, bones, rind and edible offal with boiling water and concentrating the liquid portion after removal of fat, with or without the addition of salt.Bone stock-The product obtained by the extraction of fresh trimmed bones with boiling water, with or without pressure, and concentrating the liquid portion by evaporation after removal of fat, with or without the addition of salt. Essence of beef-The product obtained by the extraction of minced beef with boiling water, such extraction being sufficiently prolonged to produce a jelly on cooling. Meat juice-The fluid portion of fresh lean meat obtained by pressure and concentrated by evaporation at a temperature below the coagulating point of the soluble proteins. Similar firoducts-Products or composite products more or less simulating the charac- teristics of the various extracts obtainable from meat or bones but containing products other than meat, for example, yeast extract, hydrolysed protein, vegetable soup stock, meat extract cubes or gravy cubes and soup powders.SCOPE OF THIS REPORT- This Report of the Sub-committee deals only with meat extracts as defined above, but is generally applicable to materials other than “similar products.” Additional methods of analysis are necessary to cover these and, to a less extent, meat stock and bone stock, and it is the intention of the Sub-committee to undertake further work for this purpose. DISCUSSION AND EXPERIMENTAL WORK To assess the quality and genuineness of meat extracts it was considered that the deter- minations listed below should satisfactorily serve the purpose for products of the nature of meat extracts, and accordingly only such determinations are dealt with in this Report- Water.Ash. Chloride. Total nitrogen. Total creatine and creatinine (determined as creatinine). Some consideration was also given to organoleptic tests, but although these are of con- siderable value to an observer with a trained pala.te, it was decided that they cannot be recom- mended for an analyst without this experience. For the purpose of a more detailed analysis, particularly to assess and estimate adulterants or the addition of non-meat ingredients, it was thought that some or all of the further deter- minations specified below would be necessary-- Phosphate. Fat. Soluble and insoluble nitrogen. Amino nitrogen. Gelatin. Tannic acid precipitate. Nicotinic acid.Starch. Qualitative tests for extraneous ingredients. PREPARATION OF THE SAMPLE FOR ANALYSIS- Meat extracts contain significant proportions of constituents that tend to separate after the extract is filled into the containers; for this reason, and owing to their viscous condition when cold, the extracts are usually transferred slightly warm to the containers. On storage there is a tendency for creatine, together with some mineral matter, principally phosphate, to separate out. I t is therefore necessary to mix the sample thoroughly beforeJune, 19511 ANALYSIS OF MEAT EXTRACT 331 taking portions for analysis. A cautious warming will expedite the blending of pasty samples and any sediment must be thoroughly incorporated. However carefully such mixing is carried out there is still a danger that very small portions of the mixture do not accurately represent the bulk. Accordingly it was decided that a satisfactory method of overcoming this difficulty would be to take a portion of 10 & 1 g dissolved in water to a bulk of 100 ml and to use aliquots of this solution for the various deteminations. It is desirable that a specially calibrated pipette be used as it has been realised that use of a pipette with such a solution may lead to a slight error due to differences in viscosity and surface tension from those of water.DETERMINATION OF WATER- Considerable discussion ranged around the question of determining the water content of meat extract, since it was accepted from the beginning that such a determination must be an arbitrary one, there being no assurance that any method would yield a true figure for water, that is to say for free water or free and bound water as distinct from water produced by protein breakdown. The Sub-committee were fortunate in having at their disposal copies of a recent monograph reprinted from the Journal of the Co.unciZ for ScientiJic and Industrial Research, AuskaZia,l in which the theoretical considerations are dealt with in considerable detail.They accepted the conclusions given in the monograph, viz., that it is not possible to obtain a true value for the water content by any known method and that therefore some arbitrary method carefully defined and rigorously followed should be adopted. As a basis for experimental work by members of the Sub-committee, it was agreed that an empirical standard method could be based on the Society's method for sweetened condensed milk.2 Comparative determination of loss of weight at 100" C were then made under various conditions of time, size and type of dish, weight of sample, and with and without sand.The results of this experimental work led the Sub-committee to the conclusion that the method based on drying for a fixed time 1 g of meat extract after solution in water yielded results satisfactorily comparable with the method using sand and was therefore to be preferred on account of its simplicity. A series of collaborative determinations was carried out by all the members of the Sub-committee on two samples of meat extract having moisture contents of approximately 20 and 27 per cent.Statistical examination of these results by Dr. E. C. Wood showed that the variance between laboratories was very significantly more than the variance within laboratories, The precision of the method from both points of view is summarised in the following statistics- This means that 19 out of 20 determinations of moisture on the sample, in the same laboratory, should differ from their mean by not more than 0.2 per cent.; but if one determination were made in each of 20 different laboratories, 19 out of 20 should differ from their mean by not more than 1-1 per cent. DETERMINATION OF ASH AND CHLORIDE- Ash-The direct ashing method was compared experimentally with a method involving charring, leaching out and complete ashing. As the results by the direct ashing method were found not to be significantly different from those by the alternative method, it was decided that the former should be recommended.Due care must be exercised to ensure that there are no losses due to decrepitation or volatilisation. The latter may be avoided by ensuring that a temperature of 550" C is not exceeded. As an alternative method when no temperature control is available for this purpose, details of a leaching-out process are included, Chloride-Methods examined included determination of chloride obtained (a) by direct ashing, ( b ) by ashing and leaching and (c) directly in the extract solution. An ashing process is recommended, but it is essential to avoid fusion of the ash and possible loss by volatilisation. Standard error, calculated from the within-laboratories variance = 0.096% Standard error, calculated from the between-laboratories variance = 0-501 yo DETERMINATION OF TOTAL NITROGEN- The determination of nitrogen has been so thoroughly examined by various workers that the Sub-committee considered it necessary only to survey the literature, particularly as regards the time of digestion and the type of catalyst, and to carry out collaborative work on given samples of meat extract by the accepted method.332 ANALYSIS OF MEAT EXTRACT [Vol. 76 The method for the determination of total creatine and creatinine (recommended to be expressed as total creatinine) has been very thoroughly investigated in the laboratories with which members of the Sub-committee are associated, since it is realised that this value is probably the most important index of quality of a meat extract and that on which analysts will largely base their conclusions. All stages of the process, including the method of hydrolysis, influence of volume, and concentration of caustic soda and picric acid on the development and stability of colour, have therefore been examined in great detail. The recommended method is based on the results of this investigation; it also incorporates as far as possible the techniques readily available for the evaluation of colour.As creatinine zinc chloride is now obtainable in a crystalline form of guaranteed purity, it is recommended as a standard. "Creatinine Zinc Chloride, 99 to 100% (standard for creatine and creatinine determinations)" may now be obtained from The British Drug Houses, Ltd.TOTAL CREATINE AND CREATININE- RECOMMENDED METHODS OF ANALYSIS All reagents should conform to recognised analytical standards. Stock soZution-A cautious warming will expedite the blending of pasty samples and any sediment must be thoroughly incorporated. Take 10 & 1 g of meat extract, accurately weighed, and dissolve with successive small quantities of hot distilled water to ensure so1ut:ion of all soluble material; cool and make up to 100 ml. Shake the solution before taking aliquots for the various determinations. DETERMINATION OF WATER- Procedure-Pipette 10 ml of stock solution into a nickel dish approximately 3 inches in diameter, preferably fitted with a close-fitting lid (see Report of the Milk Products Sub- Committee2) and evaporate to apparent dryness on an open steam-bath, adjusting the level of the dish to ensure an even film.Transfer to a drying oven at 100" C, insulate the dish from the shelf and dry for 8 hours. Replace the lid, which has also been in the oven, before removal to the desiccator. Cool for 30 minutes before weighing. DETERMINATION OF ASH- Procedure-Evaporate 10 ml of the stock sol.ution in a platinum dish on the steam-bath and char thoroughly over a low flame. Complete the ashing (preferably in an electric muffle) at a temperature not exceeding 550" C to avoid loss of volatile ash. AEternatzve procedzcre-Evaporate 10 ml of the stock solution in a platinum dish on the steam-bath and char thoroughly over a low flame. Cool and extract with three portions of hot water (10, 5 and 5 ml).Decant through a filter-paper and wash the paper with a few ml of hot water. Return the filter-paper to the dish, dry on the steam-bath and incinerate the contents completely. After cooling, return the filtrate to the dish, evaporate it to dryness and heat at a temperature not exceeding 550" C, preferably in a muffle, until the weight is const ant. DETERMINATION OF CHLORIDE- Procedure-Take 20 ml of the stock solution in a platinum dish and evaporate to dryness with 10 ml of 5 per cent. sodium carbonate. Ignite as thoroughly as possible at a temperature not exceeding dull redness. Extract with hot water, filter and wash. Return the filter-paper and residue to the dish and moisten with a few drops of carbonate solution, evaporate and ignite to a white ash.Filter from any insoluble matter, wash thoroughly and add to the previous filtrate. Determine chloride by the Volhard method. Dissolve in dilute nitric acid. DETERMINATION OF TOTAL NITROGEN- Digestiout-Digest 5 ml of the stock solution with 25 ml of sulphuric acid, 10 g of anhydrous sodium or potassium sulphate and a catalyst (0.2 g of copper sulphate, 0.7 g of mercuric oxide or 50 mg of selenium). The time of digestion should be 3 hours after clearing, irrespective of the catalyst used. Distillation of ammonia-Dilute the digest with 100 to 200 ml of water, according to whether a steam-distillation or boiling method is used, and make it alkaline with 100 ml of 40 per cent. sodium hydroxide, free from carbon dioxide, with addition of sodium sulphideJune, 19511 ANALYSIS OF MEAT EXTRACT 333 where mercury has been used as a catalyst. The method of distillation and absorption of ammonia shall be left to the discretion of the analyst.Note-(;) The clear layer of a 40 per cent. solution of caustic soda is satisfactory for rendering Note-(ii) Methyl red is a satisfactory indicator if the ammonia is absorbed in standard sulphuric Note-((iii) An appropriate blank determination should be carried out. the solution alkaline. acid. DETERMINATION OF TOTAL CREATINE AND CREATININE AS CREATININE- Solutions required- Hydrochloric acid-A 2 N solution. Sodium hydroxide-A 2 N solution. Picric acid-A 1 per cent. solution (see Note ii, below). Stock creatinine zinc chloride solfition-1.603 g of pure crystalline creatinine zinc chloride made up to 1000 ml with 0.1 N hydrochloric acid.This solution is stable for a t least six months, and an aliquot should be diluted ten times, immediately prior to use, so that 1 ml = 0-1 mg of creatinine. HYDROLYSIS OF MEAT EXTRACT soLuTIoN-Heat under reflux 10 ml of the stock solution of the extract with 10 ml of 2 N hydrochloric acid in a boiling water-bath for a t least 2 hours, or autoclave for 20 minutes at 117" to 120" C. Cool the hydrolysed solution and add 10 rnl of 2 N sodium hydroxide solution. Dilute to a volume of (a) 250 ml for Duboscq method, or (b) 500 ml for the absorptiometer method. (a) Duboscq and other visual methods such as Nesslerising-Measure from a burette two aliquots of 7ml and 10ml from the 250-ml dilution (see Note i) into clean, dry 100-ml graduated flasks.Make each quantity up to 20 ml with distilled water, add 20 ml of 1 per cent. picric acid solution, then 2.5 ml of 2 N sodium hydroxide, and maintain at 20" * 1" C for 15 minutes. Filter, rejecting the filtrate until the solution is clear and bright. Compare with a standard made up at the same time and under the same conditions from 20 ml of standard creatinine zinc chloride solution (equivalent to 2 mg of creatinine). The colour can be read immediately and is stable for 30 minutes. h'ote-(i) These two aliquots will indicate the approximate percentage of creatinine ; an aliquot can then be calculated which, after development of colour and dilution, will compare closely with the standard . (b) AbsorPtiometer method-Measure an aliquot of 5 ml from the 500-ml dilution into a clean, dry 100-ml volumetric flask and make up to 20 ml with distilled water.Add 20 ml of 1 per cent. picric acid solution and 2-5 ml of 2 N sodium hydroxide; maintain at 20" 1" C for 15 minutes. Filter; reject the first few millilitres until the solution is clear and bright. Readings are made in the absorptiometer with an Ilford filter No. 604 and a l-cm cell; a reagent blank composed of all the reagents minus the creatinine is used. The colour can be read immediately and is stable for 30 minutes. Prepare a standard curve covering a range of 0 to 1.0 mg of creatinine. Into clean, dry volumetric flasks measure quantities of 2, 4, 6, 8 and 10 ml of the standard creatinine zinc chloride solution. Add distilled water to bring the volume of solution in each flask to 20 ml. To each flask add 20 ml of 1 per cent. picric acid solution and 2.5 ml of 2 A' sodium hydroxide. Maintain at 20" & 1" C for 15 minutes and dilute each volvme to 100ml. Note-(ii): 1 per cent. picric acid solution-This strength is chosen owing to the difficulty of maintaining complete solution a t a concentration of 1.2 per cent. during winter conditions. I t should be standardised against 0.1 N sodium hydroxide, phenol red being used as indicator. Note-((iii) In a determination where Nesslerising is involved i t is essential that colour comparisons be carried out under optimum conditions in order to obtain good contrast and reliable results. These include employment of a good north light with no artificial light illuminating the Nessler tubes. Due consideration should be paid to the colour vision of the operator: i t is desirable that this should be normal in the region of the colour to be observed. Dilute to 100ml with distilled water. Dilute to 100 ml with distilled water. These flasks contain creatinine in amounts of 0.2 to 1.0 mg. REFERENCES 1. 2. Riddle, A. R., J . Council Sci. & I n d . Res., Australia, 1944, 17, 291. Analytical Methods Committee, "Report of the Milk Products Sub-committee," AnaZyst, 1927, 52, 402.