I S O L A T I O N AND l D E N T I F l C A T l O N OF CO#PONENTS R E S P O N S I B L E FOR H E A T F L A V O R BY U.SE OF

E L E C T R O P H O R E S I S A N D I N F R A R E D S P E C T R O S C O P Y

MILTON E. B A I L E Y

Future progress i n the f i e l d of meat technology w i l l become in- creasingly dependent on physical or physiochemical technics and t h i s i s par t icular ly t rue fo r the investigations of flavors and odors or similar problems. t i a l u t i l i za t ion of electrophoresis and infrared spectroscopy t o the studies of flavor and odor molecules from meat and meat products.

The purpose of t h i s paper is t o give a broad view of the poten-

Three important steps are involved i n the study and ident i f icat ion of flavor and odor compounds. o r fractionation and 3) identification, study of flavor molecules is largely a method of separating flavor compounds or t he i r precursors. Infrared spectroscopy, on the other hand, is primarily a physical procedure which can be used t o determine molecular structure and f o r chemical group analysis and best results are usually obtained w i t h puri- f i ed compounds.

These are: 1) concentration, 2) separation EZectrophoresis as a too l i n the

Electrophoresis, o r as it was once called, cataphoresis, involves

It is simply the mrtgration of charged par t ic les Many different technics have been

movement of a sol id phase in respect t o a l iqu id under the influence of an external e l ec t r i ca l f ie ld . i n a f l u i d between two e l ec t r i ca l poles. used t o separate par t ic les from mixtures according t o t h e i r e l ec t r i ca l properties, but the ones concerning us here m e moving boundary electrophor- esis and zone electrophoresis.

In the moving boundry method (slide 1 ) the experimenter measures the movement of the boundary of a mas8 of par t ic les . studied i s poured into the bottom of a U-tube; on top of t h i s is placed an electrolyte (buffer) solution and t h i s i s laid on carefully so t ha t there is a sharp boundary between the two solutions. passed through the solution by means of a posit ive electrode inserted i n the top of one arm of the tube and E negative electrode i n the other. object i s t o study the movement of boundaries under the urge of e lec t r ic i ty . IWst developments in this f i e l d were primarily due t o the e f fo r t s of Arne Wilhelm Kaurin Tiselius, whose technics employed i n measuring the m0vh-q boundary are so refined and important that most mdern types of moving boundary equipment axe commonly cal led the Tiselius apparatus. A photo- graph of the Tiselius-Lmgsworfh electrophoresis ce l l is shown i n s l ide 2.

The material t o be

An e lec t r i c current i s then

The

Both sol id and l iqu id anticovectants are used i n zone electrophor- esis t o s tab i l ize solutes. the individual components of a colloid mixture completely without incurring

This expedient makes it possible t o separate

77 . the r i s k of convection and hence of boundary instabi l i ty . frequently used sol id car r ie r i s f i l t e r paper, although other solids as starch, s i l i c a gel, sand, powdered glass, etc. have also been used, ous technics and arrangements have been used i n paper electrophoresis and many instruments are available. tance t o the separation and study of meat components i s the continuous paper electrophoresis of D u r n r m (s l ide 3).

By far the most

Numer-

One type which may be of extreme impor-

This i s one of the most promising two-dimensional methods devel- oped during the last $ew years. The solution of the materials t o be sep- arated is applied t o a suitable location on a ver t ica l paper sheet i n the form of a slow tr ickle . A uniform stream of buffer solution i s slowly allowed t o flow down the sheet o r curtain a t the same time. An e lec t r i c f ie ld i s applied t o the system a t r ight angles t o the direction of buffer flow. from t he i r or iginal vertical direction depending on the i r own charge and other environment conditions. t ions a re collected fo r further analysis.

As the solute par t ic les enter the e l ec t r i ca l f i e l d they are deflected

For preparation purposes, individual frac-

Sill another approach t o the separation of re la t ive ly large quan- t i t i es of material is the use of packed columns from which zones are eluted a f t e r separation under a potential gradient. Such a column i s depicted i n the next slide (s l ide 4). Jacketed Pyrex tube rest ing on a perforated tef lon disk. The lower end of the column i s immersed in an electrode vessel f i l l e d with electrolyte solu- tion. Contact with the other electrode vessel is obtained as shown i n the f igures .

The cellulose column is contained i n a water-

Electrophoresis has been widely used i n the separation of compo-

These include many naturally occurring constituents as well as t h e i r nents which might be of importance t o the study of flavors and odors i n meats. degradation and recombination products. amino acids, nucleic acids, choline derivatives, t r i s t e a r i n and similar esters, f a t t y acids, amino compounds, cer ta in carbohydrates and sterols.

Examples are proteins, peptides,

Certain of the f r ee amino acids bave characterist ic flavors which a re important as well as certain of t h e i r degradation products as amines, aldehydes, amides and f a t t y acids. studied by the various types of electrophoresis. meat such as various sugars carry no charge on their surface and remain stationary i n an e lec t r ic f i e l d and it is therefore useless t o attempt t o separate them unless charged complexes can be formed.

Some of these can be separated and Many other components of

Probably the most important use of electrophoresis i n the study of meat flavors and odors will be as an a i d i n the elucidation of the bio- logical mechanisms by which flavors are generated. Undoubtedly, some of the flavor compounds i n meats are produced from flavor-precursors by the catalyt ic effect of enzymes. which are constantly catalyzing chemical changes which may be important t o the production of flavor and odor compounds. dases, lipases, decarboxylases and deaminases may also produce characteris- t i c f lavors during the aging and storage of meat products.

I?umerous autolytic enzymes are present i n meat

Bacterial proteases, pepti-

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Electrophoresis can serve to help purify these proteinaceous catalysts and determine their purity so their importance in the formation of flavor from various precursors can be determined. concentrate and purify these enzymes so that they might be directly added to meat to enhance production of desirable flavors.

It may be possible to

An early use of electrophoresis to purify a flavor compound was that of Ilseda and S.Suzuki, who obtained a patent for "a method of makkg a nutritional and flavoring substance". These investigators hydrolyzed vegetable protein with acid and after removing the excess acid electrolyzed the amino acid mixture in a three compartment cell. The material collecting at the anode comgartment proved to have a very desirable flavor when added to meats and other foods; it was sodium glutamate,

The use of electrophoresis as a fractionation procedure along with other methods of fractionation such as chromatography, counter-current distribution, preferential solvent extraction, sublimation, fractional crystallization, mlecular distillation, dialysis, centrifugation and freeze drying have permitted the examination of the infrared absorption character- istics of almost every essential group of compounds found in meat products. These include nucleic acids, purines, and pyrimidines, proteins, polypep- tides, amino acids, lipides, fatty acids, carbohydrates, porphyrins and vitamins.

The infrared region as usually defined, lies between the visible and the radio portion of the spectrum, i.e., wave length6 between 0,001 mm to 1 mm. The range most useful in the studies of biological materials is between 0.0025 mrm and 0.015 mm. !l?he conventional Frequency unit in infra- red spectroscopy is the wave number waves In 1 cm. For example 0.01 300331 (waves per centimeter, wave number)

cm-l), which expresses the number of length) is equivalent to 1000 cm-l

When infrared radiation is passed through a compound, the com- pound characterizes itself by the wavelength it absorbs and those it trans- mits. The bonds within the compound have a natural f'requency of vibration determined by the masses of the two atoms held by the bond and the restoring forces of the bonds. A light wave with thie frequency of oscillation will have most effect on the bond, its energy w i l l greatly increase the natural vibration of the atoms. The molecule will absorb part of the energy of the light at this resonant frequency and an absorption detector w i l l show an absorption peak for that wavelength. The infrared spectrum is simply a dis- play of the resonant frequencies of the sample, but not all resonant fre- quencies give rise to absorption.

In general there are enough active resonant frequencies in a mole- cule so that the infrared spectrum is characteristic of the atom, the bonds and the geometrical arrangement of the molecule--and here we see the reason for the great value of infrared, i t s specificity. The infrared spectrum is the most nearly unique property of a substance. which have the same bonds but differ In arrangement can be distinguished by infrared.

Even geometrical isomers,

79.

An example of the type of information given by an infrared spec- trum i s shown on the next two s l ides (sl ide 5 and 6 ) , which i s the spectrum fo r acrylonitri le. by a sample of the material as the wavelength increases (from l e f t t o r ight) . ing that a natural frequency of molecular vibration has been reached. bration causes minima as indicated.

The curve represents the amount of energy transmitted

Dips indicate frequency at which energy is strongly absorbed, show- V i -

In the study of flavor compounds from meat we a re interested in using infrared spectroscopy primarily as a means t o establish the ident i ty of organic substances, The only serious disadvantage of infrared analysis i s t h a t the use of water as a solvent i s generally not desirable because of i t s intense absorption over much of the working range. One great advan- tage is that liquids, solids or gases can be exmined equally well by these technics and usually on a few milligrams of sample, o r with care with a few micrograms. very s l igh t ly and temporarily; samples may be recovered unchanged.

This method of measurement a l t e r s the sample being measured

The general l i n e of attack is t o compare the spectrum of the com- pound whose structure i s unknown with the spectra of compounds of known structure containing groups of atoms identical with and i n a similar envi- ronment t o those suspected of occurring i n the unknown. Extensive collec- t ions of infrared spectra have now been compiled by various laboratories and organizations, and the comparison can be made re lat ively rapidly by various mechanized sorting devices. spectra an hour from 5000 t o 625 cm-l may be obtained with present commer- c i a l instruments makes infrared a rapid and convenient way t o scan fractions of unknowns frommeat extracts.

This, plus the f ac t that three of four

One good i l l u s t r a t ion of the use of infrared as a means of identi- fying flavor compounds was the work of Jackson and Morgan who identified the substance causing a malty aroma i n milk--cultures of streptococcus Lactis - Var. Maltiaenes. hydrazones these investigators separated them by chromatography and analyzed the individual derivatives by infrared. the unknown phenylhydrazones with 2-methylbutanal and 3-methylbutanal. was concluded that the unknown odoriferous compound was 3-methylbutanal.

After isolat ing neutral carbonyls as the i r phenyl-

Slide 7 is a comparison of one of It

Further studies by these workers on t h i s problem showed that the organism involved or enzymes produced by t h i s organism could convert leucine and isoleucine t o t h e i r corresponding pentanals.

Another example of a degradation product of amino acids which has been ident i f ied as a flavor compound producing a broth-like flavor is methional (3-methylthiapropanal) . milk by infrared as i t s phenylhydrazone a f t e r purification by gas and paper chromatography. degradation from methfonine. the f ac t that light, heat and bacteria are all capable of inducing Strecker- l i ke degradations of amino acids t o f lavorful aldehydes indicates the impor- tance of t h i s general pathway t o flavors i n meat and meat products.

This compound has been identified i n

It i s formed by oxidative deamination through a Strecker The widespread occurrence of methionine and

80.

A further example o f the use of infrared in the study of flavor extracts, t h i s time from a product close t o the hearts of a l l present, is t ha t of Hornstein (private communication), who compared the infrared spectra of vo la t i le fractions of aqueous extracts f’rom beef and pork. Both fractions had a ”meaty”-type odor and produced practically the same infrared spectrum (slide 8 ) There apparently wa6 no qualitative differ- ence in the fractions involved.

Further fractionation of these vola t i les by chromatography yielded two very similar, but different compounds as determined by the i r infrared spectra (s l ide 9).

Rmerous other workers, particularly i n the dairy and horticul- tu re fields have used infrared spectroscopy as an a id in identifying flavor components. Many of these compounds were produced from molecules common t o meat and cer tainly technics ut i l ized by these investigators w i l l require special scrutiny on the par t of workers interested i n flavors from meat.

In conclusion I might say that the use of the various forms of electrophoresis may be somewhst limited t o most approaches t o flavor and odor studies of meat but should be extremely useful as a means of fraction- a t ing many flavor precursors i n meate; infrared, supplemented by other forms of spectroscopy and the classical methods of organic analysis w i l l be an important phase in the finel meat flavor s t ructural analysis by those fortunate enough t o haw It svailable. lche infrared spectrum is certainly not a magic c rys ta l ball in which one reads the s t ructural formula of an un- known compound but through i ts use and those technics offered by electro- phoresis we can shorten tremendouslythe time required t o complete cer ta in Jobs on problems concerning meat flavors.

sEuECI!ED BIBLIOGRAPHY

1. Bauman, R. P. and Clark, Ct (co-chairman), Biological applications of infrared spectrosc6py, in Annals of the New York Acadeq - of Sciences, 69, 1-254 (1957).

---- 2. Bellamy, L. J., The Infra-red Spectra of Complex Molecules, London;

Meuthuen and Co. Ltd. (1959).

3, Block, R. J., Durrum, E. L. and Zweig, G., A Wnual of Paper Chromatog- raphy and Paper- Electrophoresis,- Academic Press Inc .) New York

-

(1955) . 4. Clark, C., Infrared Spectrophotometry, In, Physical Techniques i n

Biological Research - Ed by Gerald Oster and Arthur W. P o m s t e r , Vol. I, pp. 206-316, Academic Press Inc., Mew York (1955).

5. Crawford, Bryce, Jr., Chemical Analyses by Infrared, Scientific American, October (1953) pp . 42-48 .

6. Gray G. W., Electrophoresis, Scientific American, December (1951) p. 45.

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7. Jbckson, H. W. and Mrgan, MI E., Ident i ty and origin of the malty aroma substance from-milk cultures of streptococcus Lactis Maltigenes, - 37, 1316-1324 (1954).

8. Stern, Kurt G., Electrophoresis and ionophoresis, in, Physical Techniques i n Biological Research - Ed by Gerald Oster and Arthur W, P x l i s t e r , Vol. 11, pp. 243-300, Academic Press Inc., New York (1956).

9. Sutherland. G. B., B. M.. Infrared analysis of the structure of amino acids; polypbtidesland proteins, i n Advances -r i n Protein Chemis- tyy, 8, 291-312 (1952).

10. Sutherland, G. B., B. M., Application of infrared spectroscopy t o biological problems, Reviews - of Elodern Physics, -. 31, 118-122, (1959) .

11. Svensson, Harry, Preparative Electrophoresis and ionophoresis, Advances - i n Protein Chemistry, 4, 251-292 (1948).

12. Tiselium, Arne, Electrophoresis, Methods i n Rizymology, - 4, 3-20 (1957)

13. Wolstenholme, GI E. W. and Millar, E. C. P. - Eds. Paper Electrophor- es is , Li t t le , Brown and Co., Boston (1956).

CHAIRMAN PEARSON: Thank you, Milton.

Our next topic t h i s afiernoon deals with Paper Chromatography, This paper w i l l be handled by Dr. Wendell A* Landmann of the American Meat Ins t i t u t e Foundation.

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