ON C H E M I C A L METHODS U S E D IN M E A T RESEARCH

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1 99 ON C H E M I C A L METHODS U S E D IN M E A T RESEARCH 0. E. BRADY UN I V E R S 1 T Y O F M I S S O U R I p - I wish t o make i t c l e a r a t the onset t h a t t h e topic which has been assigned t o me does not presume any g r e a t knowledge of a n a l y t i c a l chemistry on my part. Indeed, t h i e branch of chemistry is a highly specialized f i e l d i n i t s e l f, The observations which I wiah t o make a r e therefore of r a t h e r limited perspective but, I t r u s t, of s u f f i c i e n t i n t e r e a t t o stimulate discussion on t h i e important phase of meat research. You are q u i t e aware, I am sure, that our knowledge of meat chemistry i s limited. That it may a t times appear otherwioe i a probably t r u e only i n r e t r o s p e c t. Certainly t h e r e is a close relationship between our knowledge of meat chemistry and the methods which a r e p r a c t i c a l l y available t o the meat research laboratory, There is, however, no i n t e n t on my part t o underestimate t h e import a n t technological advances which are bei- made. They o f f e r i n c r e a s i q l y g r e a t e r opportunities through chemistry and related sciences t o discover the t r u t h s f o r which we search. Let us n o t minimize t h e g r e a t distance that we must t r a v e l. I believe the polnt t h a t we are most a p t t o l o s e s i g h t of i s t h a t a c u t of meat which appears s o commonplace and which is often taken f o r granted is t h e handiwork of the master craftsman. Here i s a product of the c l a s s of M a m m a l i s ; the end r e s u l t of a series of complicated biological reactions i n volving not only t h e e f f e c t s inherent i n t h e germplasm but a l s o the cumulative e f f e c t of many environmental changes during the animal's l i f e and subsequent t o slawhter. Is it not surprising a l s o how f e w of us r e a l l y appreciate t h e g r e a t value of a trained sense of perception? These mnses when used t o evaluate complex charges i n meat c h a r a c t e r i s t i c s are usually much more e f f i c i e n t, comprehensive and adaptable than any other method of evaluation. Certainly, our chemical methods have been developed as a t o o l t o supplement and c o r r e l a t e with our senaory perceptions givin& us increased o b j e c t i v i t y. It must be admitted t h a t many of our chemical and physical chemical techniques, w h i l e not as precise as w e might l i k e, do give us a l e 0 an increased knowledge of the "whys" and "wherefores" and t h a t our r e s u l t s g a i n i n o b j e c t i v i t y, a characteri s t i c much t o be desired. I f e e l sure t h a t your program committee d i d not expect ne t o g i v e a resume of a r a t h e r l a n p r number of chemical methods and an evaluation of each, This would not be poesible even d i s r e g a r d i w the time l i m i t a t i o n. I do wish t o mention, however, a f e w a n a l y t i c a l procedure6 which have been ehown t o be applicable t o meats work. Methods f o r Moisture Analyses There are quantitatively. As most common form is water serve8 aa the a number of methods available for determiniw moisture you know, meat contain8 water i n d i f f e r e n t forms. The free water as d i f f e r e n t i a t e d from bound water. Bound diapereiona medium f o r the c o l l o i d a l mterial. The

2 100. lyophilic colloids which characterize meat possess a g r e a t a f f i n i t y f o r water. The determination of the moisture content of meat is purely a n empirical procedure determined by the three variables, time, temperature, and pressure. Thus, i t is impossible t o define the moisture content without defining the conditions under which the drying took place since the various moisture methode o n l y determine more o r less of the "moisture" i n the meat. Therefore, standard or o f f i c i a l methods should be used unless only comparative r e s u l t s are desired i n which case any standard method may be used. There a r e two o f f i c i a l methods of the A. 0. A. C. (1)Drying t o constant weight a t 95-l0O0C. under pressure not t o exceed 100 mm, of Hg. and (2) drying over H2SO4 i n a vacuum desiccator a t a pressure of not more than of Hg. t o a constant weight. There are a l s o a number of methods f o r determining bound water. Here again, the relationehip of f r e e, bound and t o t a l water must be expressed i n relationship t o the method used i n the determination. Methods f o r Fat Analyses I n order t o remove f a t from t i s s u e it i s necessary t h a t the t i s s u e s be dried and t h i s drying must take place i n such a manner t h a t there i s no oxidation. Drying i n a i r i s not t o be recommended. The A. 0. A. C. method, huwever, c a l l s f o r drying a t 1Olo t o 102OC. f o r 16 t o 18 hours o r a t a t e m perature of 125%. f o r 2 t o 3 hours followed by extraction with anhy(kou6 e t h e r f o r 16 hours. This is one reason why it would seem desirable t o give s p e c i a l consideration t o the method f o r f a t analysis now being used by t h e Xhnsas Agricultural Experiment Station. Dr. H a l l informs llls t h a t t h i s method was developed a t the University of Kentuclqy. It is e s s e n t i a l l y a modification of the Babcock b u t t e r f a t t e s t. A somewhat similar method w a s described by worker8 a t Oscar Mayer's i n The t i m e required f o r a determination when one blendor is used i s reported t o be about 30 minutes. Very close agreement i s reported between t h i s method and the A. 0. A. C. Soxhlet method of ext r a c t i o n. With both the Icanaaa and Oscar Mayer methods the samples a r e put through the Waring Blendor. Free Fatty Acid Determination The determination of f r e e f a t t y acid8 i s of value i n estimating the amount of deterioration due t o hydrolysis. This hydrolysis may be caused by the n a t u r a l enzymes o r by f a t s p l i t t i n g microorganisms. It i s a measure of hydrolytic rancidity. The f r e e f a t t y acids a r e t i t r a t e d against sodium hydroxide. This determination is a good indication of how fats and especially pork fats have been handled. Pork f a t s with even a small amount of hydrolysis w i l l render out l a r d with a low smoke point. An i n c r e a ~ e, f o r example, i n f r e e f a t t y a c i d from 0.14 percent t o 2.20 percent w i l l lower the smoke Free f a t t y a c i d values a r e generally expressed as point from 370 to 271%. percent o l e i c acid. The Iodine Absorption Value is a good indication of the degree of unsaturation. It ha6 been used widely t o indicate firmness of pork f a t, This method masure8 d i r e c t l y the t o t a l concentration of double bonds and i s therefore but an estimation of the degree of hardness. It would appear

3 101. desirable that a s a t i s f a c t o r y method 02 physically determining the hardness be developed. Workers a t the U.S.D.A. and others have shown that there is excell e n t agreement between iodine values and r e f r a c t i v e index and that they both serve as a s a t i s f a c t o r y measure of firmness of the adipoee t i s s u e. Recently there has been a g r e a t deal of i n t e r e s t i n oxidative ranc i d i t y i n procsesed and stored meat products. Dr. Beadle of the American Meat I n s t i t u t e has pointed out and I quote "No one chemical test has been devised which can accurately measure and c o r r e l a t e all the f a c t o r s which a c t simultaneously t o produce the odors and flavors we c a l l rancidity, O f the various t e s t s which have been submitted f o r the detection of oxidative rancidity, the determination of 'active oxygen' or peroxide content seems t o give r a t h e r good correlation of the data. During the oxidation of the f a t c e r t a i n oxygen compounds a r e formed which a r e 'active' in the they a r e capable of l i b e r a t i n g iodine from potassium iodide. The iodine can be quantitatively determined and thus be a measure of rancidity." The peroxide compounds a r e themelves intermediate compounds and are not responsible f o r rancid f l a v o r s and odors. I would l i k e t o emphasize t h a t peroxide values can only be proportional t o rancidity a8 long as peroxides are being formed a t a r a t e which i s f a s t e r than they a r e being decomposed. It is a l s o t r u e t h a t the temperature coefficient of peroxide f o r m t i o n i s g r e a t e r than that of peroxide decomposition s o t h a t with i n c r e a a i w temperatures the values become increasingly less r e l i a b l e as indicators of rancidity development. When using peroxide t e s t s under a new s e t of conditions it i s always first necessary t o be sure a correlation i s obtained with organoleptic rancidity. Erroneous r e s u l t s o r conclusion6 are a l s o quite often the r e s u l t of comparing peroxide values obtained under one s e t of conditions with those secured under another s e t of conditions. As stated previously, rancid odors a r e not due to the peroxides themselves but t o tho breakdown products resulting i n paxt from t h e i r action. The r a t e of breakdown is dependant i n pa.rt upon the temperature of the teflte. Therefore, the peroxide numbers w i l l be much higher f o r the same degree of ranc i d i t y i n the case of shelf storage t e s t s than i n the case of accelerated tests, It i s a l s o t r u e t h a t rancidity develops a t very low peroxide values where c e r t a i n antioxidants are used. As C. H. Lea s t a t e s "Basically a l l of the methods which have been employed i n t h e determination of s u s c e p t i b i l i t y ( t o oxidation) are s i m i l a r i n principle. Gxidati on i s accelerated under carefully controlled condi tiona while s t i l l preserving as far as possible the o r i g i n a l r e l a t i o n s between the various samples u n t i l the time necessary f o r the d e v e l o p e n t of spoilage has The progress of acbeen reduced from months or weelus t o days o r hours. celerated oxidation i a then followed by a m e l l and t a s t e, by d i r e c t measurement of the weight o r value of oxygen absorbed, by changes i n some physical charact e r i s t i c of the f a t, or by chemical eetimation of the products of reaction. It is extremely important with a l l accelerated methods t o c a l i b r a t e the method with the p a r t i c u l a r type of f a t studied."... The accelerated methods which are commonly used are: (1)Schaal Oven Method; ( 2 ) Swift Active Oxygen Method; and (3) Barcroft-Warburg Method. Time does not allow t o discuss the r e l a t i v e merits of each but a good discussion by B. W. Beadle can be found i n OIL AND SOAP, 23, (1946). I n addition t o these methoda, Mador and Watts report a simple accelerated method which consists of incubating the samples a t 30%. f o r 60 hours. Chloroform is used w i t h the ground meat t o prevent bacteriological decomposition.

4 102. There are a number of methods f o r determining peroxides i n c l u d i w e have used Stanaby's almost exclusivethose of h a, Wheeler and Stamby. W ly. I n general, all of the methods c o n s i s t of using f a t i n a highly a c i d i f l e d solvent, adding potassium iodide and t i t r a t i n g the liberated iodine with sodium t h i o s u l f a t e using s t a r c h as an indicator. The Kreie test has also been used aa a method for determining fat s t a b i l i t y, It has a number of limitation8 since fate which are not rancid may give the test. C w e 8, hwever, may be obtained which are useful i n studying s t a b i l i t y. Watts and MaJor have ehown t h a t peak values obtained by B e i s t e s t s v w i e d greatly while peroxide numbre vere quite constant. Both peroxide and &eis values go t o a maximum during prolonged oxidation of the fat. The peroxide peak, however, camea long after the Kreis peak. High temperature a l s o has a depressing effect on Ereis values when compared with the p e r a i d e values. Methods f o r Nitrogen Compounds "here a r e a number of methods available i n connection with proteins and other nitrogenous products. These include s p e c i f i c t e s t s such a s t h e Millon reaction f o r tyrosine; t h e color i s specific f o r t h e phenol group. There a r e a l s o t e s t s f o r the aromatic nuclei which a r e e a s i l y n i t r a t e d such as i n tryptohpane and tyrosine and a l s o f o r other groups. Unfortunately, these t e s t s are not generally quantitative although a few have been adapted f o r quantitative determination of such amino acids a s tryptophane and tyrosine. Perhaps the best quantitative method f o r amino acids is t h e Van Slyke method. By the apparatus of the same name it i s possible t o determine the amino and carboxyl groups i n amino acids or groups of amino acids. By Sorenson's Method carboxyl and amino a c i d groups can be t i t r a t e d. There a r e also methods f o r group analyses such a s Hausmann's f o r ammonia nitrogen, basic nitrogen and non-basic nitrogen. Methods which have been especially adapted t o meat studies include collagen and e l a s t i n determinations, These methods were developed o r i g i n a l l y a t I l l i n o i s t o study tenderness. D r. Hall of Kansas t e l l s me that h i s method for collagen i s t o be published shortly i n FOOD RESEARCH. There a r e several nitrogen determinations that a r e of value. These include water soluble nitrogen, heat coagulable water soluble nitrogen, nonp r o t e i n nitrogen and f r e e amino nitrogen. The l a t t e r i s p a r t i c u l a r l y valuab l e f o r studies gf autolysis. Increase i n sulfhydryl content a r e a l s o c h a r a c t e r i s t i c of t h e e a r l y stages of denaturization and can be determined quantitatively. Ab 6 orp t ion Spect r o sc opy Mea sur ement s One type of analysis that would appear t o be adapted t o c e r t a i n phases of meat work i s absorption spectroscopy. As you know, it i s concerned w i t h t h e amount of l i g h t absorbed or transmitted by a solution of t h e substance a t each wave length throughout t h e spectrum. It i s not only adapted to measuring color changes such a s occur i n t h e oxidation of hemoglobin t o methemoglobin but a l s o t o other changes such a s occur i n t h e oxidation of fats. So f a r no g r e a t adaptation has been rmde t o meats work. The accuracy and precision of' spectrophotometric quantitative methods is high a t various concentrations. For very low concentrations it i s r e a l l y more accurate than t h e ordinary a n a l y t i c a l methods. It s u f f e r s by comparison, honaver, a t high concentrations. One of t h e chief advantages it has i s t h e speed i n which t h e determinations can be made.

5 103, I should like to rmke one closirg observation and that is that a chemical method is but another tool. The effectiveness of this tool is in no smll way related to its adaptability to the task at hand and the accuracy of the worker who uses it. Unfortunately, there are many of us who do not have the time or the inclination to study these various methods, their adaptations and limitations. I believe the discussion on peroxide values should have brought out this point. There are many perhaps who feel that this method is not satisfactory for determining rancidity. They had hoped it could be used with the various types of fat handled by different methods or with various antioxidants in a straight forward manner. Certainly, no such claim6 have ever been made for the method. Given a rather standardized set of conditions it can, like other chemicalmethods, be very useful in giving reproducible results supplementing and correlating with other lees objective methods. CHAIRMAN BUTLER: Thank you very much, Dr. Brady. And now we will have a discussion of Dr. Brady's paper, with Professor Farwell of M-lchigan State College leading, PROF. FARWELL: I know that Dr. Brady's paper has reviewed some of these chemical methods in your minds. Many of you have worked with them, and some of the rest of you probably have read a good deal about them. I think that the?ragram Committee is to be complimented on the timing of these papers. I think that yesterday we began to get a few ideas in the chemistry of meats in our minds, and last night certainly, Dr. Newton's paper drove home a few things. The subject not covered in Dr. Brady's paper, that might stimulate a lot of work and a lot of thinking, would be the possibilities of using same means of chemistry in the tenderizing of meats, and there probably will be a lot of advance in that field in the future. Do any of you have any questions you would like to direct to Dr. Brady now? PROF. NELSON: Dr. Brady, you were mentionins a moment ago the protein decomposition products. You mentioned tryptophan and some of the amino acids. Have you tried the microbiological methods of assay for those? DR. BRADY: No, I have not. PROF. NELSON: They are quite sensitive and pretty adequate for about ten or twelve of the essential amino acids. DR. BRADY: Quantitative? PROF. NELSON: They are quite quantitative. They work very nicely. DR. BRADY: That is a thing we are just getting into, so we are very much interested in your remarks. PROF. NEEON: I have not worked any on meat recently, but I know it will work, very similar to your vitamin assay procedures. mof. FARWELT: Are there any other questions?

6 104. PROF. R. C. MILUR: I have a camment. I think D r. Brady has handled t h i s subject in a very creditable manner, A chemist is up against one more proposition i n his chemical methods, though they are very useful. The meat man hands over h i s material t o the chemist, and the chemiet is qualified t o apply the methods. Hmever, between those two points there akises another question, namely the question of sampling, which is a very serious one i n many c a m s arid may have a marked e f f e c t on the r e s u l t s a chemist obtains. I do not how whether that is a problem f o r the meat man or f o r t h e chemist. I merely suggest that it might be a suitable topic f o r discussion a t another time. PROF. FABWEIL: I think that goes back night, and a l s o Dr. Brady mentioned it. It i s a l y, i t w a s brought out i n Dr, Bradyls discussion ide t e a t, i n a l o t of caaes, is a matter of team t h e chemist needs w i t h what w e can give him. t o Dr. Newton's talk last matter of team work. Certainthat the value of the peroxwork and correlating what A r e there any other questions? PROF. BUTLER: I want t o comment. The quantitative ICreis Test, as developed by Watts and Major, should be more e f f e c t i v e i n estimating rancidity as it masures one of the end. products. I n some work on pork sau8a+je, I used that and ueed it along w i t h the peroxide test Wheeler's method f o r 32 samples on two separate occaaions, and the correlation between the quantitative &vis Test and t h e peroxide test was almost perfect i n both cases. I n one case i t was.96; i n the other case,.95. Apparently they both have a p r e t t y severe handicap, because n e i t h e r one of t h e m correlated too highly with the organoleptic t e s t on t h e same sample. -0 PROF. FARWELL: -- Are there any other question8 o r discussions? If not I w i l l t u r n t h e meeting back t o the chairman. CBAIRMAN BUTLEA: Thank you, Professor Farwell. Now w e axe going t o have a paper on "Methods Used i n Studying BacteS?icgenic Problems i n Curirg and C a n n i r g Meat", by Professor Willard 0. Nelson, who is now Assistant Professor of Dairy Bacteriology a t t h e University of Illinoid. He was with Wilaon & Co. f o r several years, and was a Captain i n the Medical Corps. Mr. Bull says that he i s a meat man who went t o the bad and went over i n t o the dairy department, ###

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