Methods for Studying the Water-Holding Capacity of Proteins in MeatMETHODS FOR S T U D Y I N G THE W A T E R - H O I D I N G C A P A C I T Y OF P R O T E I N S I N M E A T
Rather than speak spec i f ica l ly on new techniques on protein as i n
A t the the printed program, I would l l k e t o spend a few minutes indicating t o you some of the methods we have found sa t i s fac tory i n our meat program. outset I should l ike t o say that as you observed the data and t h e curves which I shoved t h i s morning very l i t t l e was said on the methods f o r obtaining these data. It is only fair t o say that i n order to get good methods which are re- producible is not always easy, and t h i s is doubly so fo r some of our meat work. For example, Dr. Wierbicki spent almost two years working on our method f o r determining hydroxyproline. isfactory technique f o r measuring actomyosin i n meat. the point that sometimes the methods we use are not en t i r e ly original with us, but may be modifications of methods found by workers i n other f ields. Methods have been developed by muscle biochemists and physiologists who are interested i n understanding the action of muscle -_L i n s i t u , Methods by such workers are not d i r ec t ly adaptable t o meat. the or ig ina l w a s suggested by man, yet w e found it very d i f f i c u l t t o apply i n meat.
Almost a year was spent i n developing a sat- We should also make
Again I.eferring t o the hydroxyproline method,
In the newer methods which have been developed f o r the studying of proteins, such as electrophoresis, u l t r a centrimgation, index of refraction, and d ie l ec t r i c constant method, a l l require work i n re la t ive ly d i lu t e solu- t ions and are not par t icu lar ly applicable t o meat. All of these have been used i n one way o r another f o r t he study of protein hydration, but I think we must admit tha t hydration i n very d i lu t e solutions is quite d i f f i c u l t f o r hydration in meat, which Tuns better than 20% protein. We are, therefore, faced with the problem of developing methods which i n one way may be con- sidered empirical, but which may give usef'ul in formt ion nevertheless.
I would l i k e t o show you two slides of the type of information which we have been able t o obtain and which have made possible the interpre- ta t ions we gave t h i s morn-. In the case of the second slide I would l ike t o c a l l your a t ten t ion t o the radical changes i n tenderness and water-holding capacity which have been brought on by modification of the ionic atmosphere i n muscle. First I would l i k e t o say that one method which we have found most sa t i s fac tory which we have indicated in this slide is the modification of meat by IIWibnS of the infusion of the vascular system. do without D r . Cahi l l who has been so helpful i n th i s part of our program. We have been using Inf'usfon techniques f o r alumst eight years, have been studying the e f f ec t of cer ta in ions by using matched rounds of beef. The infusion technique I s very simple when one talks about it, yet I hasten t o add that it does take a b i t of pract ice in order t o apply ef f ic ien t ly . For whole animals w e modify by bleeding by the jugular vein and carot id arteries
I do not know w h a t we would
More recently w e
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and infused by way of the carotid artery. by pumping the available arteries. dis t r ibut ion may be obtained by using pressures equivalent t o normal blood pressure, i n the excised portions above blood pressure nay be necessary, per- haps f i f teen pounds o r more.
For excised par ts we infuse simply Whereas i n the in tac t animal fairly even
In studying the water-holding capacity of meat and i ts relat ion t o tenderness and shrinkage we gave considerable thought t o developing satis- factory methods which would give us suff ic ient data i n a reproducible manner t o permit the drawing of va l id conclusions. Our first evidence t o the re- lationship of shrinkage t o tenderness was obtained using fairly large samples of meat i n centrifuge bo t t l e s . This could be done, but it was cumbersome and time consuming. gram meat samples t o be used. You w i l l note that i n the upper portion of the tube w e put the sample resting on a loose f r i t ted glass disc , part of the tube is graduated so t ha t d i rec t visual measurement of the l iquid may be made. manner of heating t o assure reproducible r e su l t s . not prove t o be a great stumbling block. o r unground meat is placed i n the top portion of the tube, stoppered with Wlnsen valve o r capi l lary and heated i n a water bath at the temperature under study. The tube is cooled t o about 30-35' C. and then centrifiged at 170 tihlea gravity f o r ten minutes. using a magnifying glass and estimating any fat i n the liquid, juice expressed can be determined with an error of * o r - one-tenth aLllimeter. We have found that f o r studying the d r ip of fresh meat o r frozen ana defrosted meat the sample should be equilibrated t o 400 C. This i s t o pmvent so l id fat from interfering w i t h the determination. The tubes that I am showing you here w e r e made f o r us by the Corning Glass Works. We pay approximately Ten Dollars each. From the number of requests that w e have had, it appears that other laboratories am using them and I would hope that the price would drop, but I have no information on that.
We develQped these tubes which I show here which permit 25
The smaller
It was necessary t o establish the proper centrifugal rate, In this instance t h i s did
A weighed portion of e i the r ground
Heating is continued f o r t h i r t y minutes. While
A simplified method f o r determining water-holding capacity of
The method was developed by Drs. Hamm and Grau and has been The method uses filter paper.
meat proteins has been developed i n the German Ins t i t u t e of Meat Research a t Kulmbach, studied a l so i n England. t o six hundred milligrams of meat sample is placed on tbe f i l t e r paper which is then placed between two luc i t e plates, Five hundred pounds per square inch pressure is applied f o r one minute. The mount of water released i s approximately s ix ty milligrams per each square inch of wetted surface. authors have applied t h i s technique t o frozen beef, fresh beef and some pork products. f ind excellent agreement on fresh mat. German method do not agree on cooked meat. dicate how the method works. the moisture ring and a l so of the protein ring. a planimeter. juice expressed. protein f i lm does not contribute t o the juice figure. not know. I would l i ke t o share with you and I have drawn on t h e board a sketch of our resu l t s using the method of Hamm and Grau. before Dr . Wierbicki l e f t our laboratory and I am grateful t o him and the
Approximately four
These
We have compared t h i s with the tube method i n our laboratory and However, the tube method and the
I have two samples here which in- By using a pencil one outlines the periphery of
These then are measured by This difference between the two r i n g s is a measure of water o r
It is very interest ing that the area of the paper under the Why t h i s i s so we do
This work was begun
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Rath Packing Company f o r l e t t i n g him complete t h i s work and share these re- s u l t s with you, tween the area of free rnoisCure and t he grama of free moisture. This f i l t e r paper method has the advantage that it can be applied t o a very large number of samples and m3quIres less time t&an the tube method. Consequently it msy be of great value i n studying r a w and frozen meat. Since, however, it does not correUte well with the tube method f o r cooked meat we feel that we are obliged to uee only the tube method. The tube method has the f i r t he r ad- vantage that two usable f rac t ions are obtained for fu ture study. as you have seen, we have done considerabl4 study of the various ions i n the ju ice f rac t ion and a s 0 of the meat or protein coagulum f ract ion. these tubes show great promise ia studying the chemistry of meat during cooking.
f would like t o have you observe the l inea r relationship be-
In our work,
We feel that
You have noticed t h i s morning and again this afternoon tha t we have mde many references t o the sodium, potabsium, calcium and magnesium contents of meat o r fraction$ thereof. their water-holt3ing capacity is a f’unction of pH and the ions on the protein o r in t he protein solution. it became necessary t o develop a technique for measuring them with some de- gree of rapidity. t o embark on a study of these ions because of the d i f f i c u l t y of their deter- mination, However, with the advent of chromatography this has not proved d i f f i c u l t . Of hi6 stu&nts, Dr. W i l l i a m Sutton, modified several techniques of other workers in to a simplified scheme. The basis of their method is the chroma- tographing of the chlorides on ion-exchange resins and elut ing with varying strengths of hydrochloric acid.. By cal ibrat ing the column it became possible t o take aliquot8 of cer ta in f ract ions, evaporate t o dryness, and titrate the residual chloride. From this the cations could be computed. We have adpated t h i s t o meat and i ts fract ions. We f ind th i s more sa t i s fac tory then flame photometry because the r a t io s of sodium and potassium are more optimal f o r th i s technique.
The pmpert ies of proteins and par t icu lar ly
In order t o study the e f fec t of cer ta in ions
Twenty years ago one would be considered almost fool-hardy
In studying the ion balance i n milk, Professor E. F. Almy and one
Before concluding, I should l ike t o say that is no way do X want In- t o discount some of the more elegant techniques f o r studying protein.
deed they have very great potent ia l i n studying meat,
I put i n your hands of those.of you who are interested reprints of
If any of you are interested in any of the other methods we our work whYch gives the details of the development of these procedures I have discussed. are using i n our laboratory we w i l l be most happy t o share them with you,
DR. SCHWEIGERT: We certainly appEcia te your sharing with US some of these newer approaches and newer technics applicable t o protein and i t s interrelat ionship with other v i t a l consti tuents of meat.
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We are indebted t o our good friend, Dr. Hall, at Kansas State, for suggesting the next speaker t o the Research Methods Comlttee, and in circulating the coxnittee f o r t h e i r comnaenta they were very much in- terested i n what Dr. Clew would have t o share with us today. He all describe some of their recent protein research. I am sure that 80311~ of the approaches that he has will be of interest to us.
L.t is my real pleasure will review for similar period
to present Dr. Clegg at th i s time who their research. (Applause)
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