- FUNOAHElVTAl HISTOLOGICAL NE THODS CONSIDERATIONS

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1 175. HISTOLOGICAL NE THODS - FUNOAHElVTAl CONSIDERATIONS The purpose of' adopting histological methods in meat research is t o explore and establish relationships between the structure and quality of mat and its by-products. With a microtome, certain indispensable biologic a l stains and a microscope, the histologist prepares, exmines, and com~ 8 2 ~ 2the 6 detailed structure of muscles and other materials e i t h e r in t h e i r n o m s t a t e o r under experimental conditions. M s methods of analysis sometimes may be qualitative, but more often quantitative o r both, depending on the nature of the problem. For example, in an extensive study (1) undertaken by several divisions a t the Foundation not long ago, characteri s t i c structural differences aseociated with carcass grade and weight were both qualitatively and quantitatively appraised. Such histological entities as the m s c l e fiber dlamter, muscle fiber burydle size, fat, collagen and elaatin content from thausands of samples were detenained by methods (1,2), which were considered a t least no less reliable than corresponding chemical methods of determination. In fact, the histological techniques possess the unique attribute of being capable of preserving the distributional patterns of any meamred element whether it is fat, collagen o r elas t in. Similarly, we have studied beef and pork casings, pointing out t h e i r basic structural difference8 and disclosing the function of each of the operating processes in caspreparations (3,4). The hog dehairing process was experimentally studied and the adverse effect of overscalding was structurally demonstrated (5). The histological manifestation of polyoxyethylene monostearates (bread-eoftener) and of vitamin B12 deficiency on the v i t a l organs of the rat were determined (6,7, 8). Cooked beef was studied f o r the f a t e of fat with BOM interesting results (9). In short, t h e basic histological technique renders i t s e l f well t o any situation requiring the examination of the structure of tissues o r organs. A second basic histological technique involves the isolation of muscle tissue into i t 0 components and their subsequent individual experimental treatment. The isolation is accomplished by beating up instead of cuttin@up small pieces of meat in a Waring blendor a t a much reduced speed and with a completely dulled knife (12). I will devote the rest of t h i s t a l k t o the problem of tenderness and discuss with you some of the results obtained with both these histological methods. Abat two years q o we started a program of studying t h e effect of proteolflic enzymes on the structure of mat. Our primary assumption has been that the structural alterations brought about the action of enzymes might provide a basis for evaluating the potential tenderiziw properties of these enzymes, thereby enabling us t o b e t t e r understand the real nature of tenderness (u). was One of t h e first results obtained from the isolated muscle fibers a denonstration of the muscle fiber membrane, a compound structure

2 176. consisting of an inner ultra-thin sarcolemma and an outer fibrous envelope itmediately surrounding the former (13, 14). Furthermore, it waa shown that the true sarcolemma is obtainable only from raw tissue. But when meat i s cooked or/and chemically fixed, then the ~arcol~elmnct and the fibrous envelope become separated together from the macle f i b e r protein upon blendoripg. This separation is due t o a shrinkage of the actowosin a8 a result of moisture The significant thing is that the sarcolemma and the envelope both are not effected in any way by either cooking or aging. It may be inferred from t h i s that these structures might have something t o do with tenderness as w i l l be presently shown t o be so. We have developed a wprocedure f o r measuring "muscle fiber extens i b i l i t y " in isolated single m2scle fibers * This property represents a n additional distance t o an i n i t i a l 5 mm. length of the fiber, a t which the f i b e r breaks under the tension. It has been shown previou6ly by us (12) that t h i s property is not only related t o carcaas grade and weight, but i s actually correlated with tenderness ratlng of the coolced meat, i. e., the greater the macle fiber extensibility, the less tender i s the meat. mrthermore, it was conclusively shown that muscle fiber extensib i l i t y decreased during aging and increased on cooking. In order t o further establish a relationship between muscle f i b e r extensibility and tenderness, single muscle fiber6 were immersed in an enzyme solution and irrrrmediately afterwards, t h e i r extensibil-ity was determined, It was shown that the enzyme treatment had reduced t h e i r ext e n s i b i l i t y, Moreover, the degree of t h i s reduction appeared t o be visibly correlated with the extent of' structural degradation which the fibers had undergone, i. e. the more highly degraded a muscle f i b e r i s, the l e s s extensible it would be. Coupled with these results, our data show that frozen-dried beef steaks after being rehydrated i n a suitable concentrat i o n of an enzyme which is known t o affect the muscle f i b e r protein were rated more tender than control steaks nottreated with an enzyme. These findings have given ua a working hypothesis that tenderization m y be achieved through decreasing the extensibility of muscle fibers. For treating whole muscle tissue with an enzyme we have developed a technique f o r quick uniform penetration by rehydrating pieces of frozen-dried steaks i n an aqueous enzyme solution (10,ll). Enzymes o r enzyme preparations tested t h i s way have revealed other epecific effects on the muscle tissue components. For instance, Age-it, a preparation containing salt, -pain and a bydrolyzed vegetable protein i s capable of disintegrating the sarcolemma and the muscle fiber envelope and reducing the endomysia1 collagen i n t o a granular material; the muscle fibers, on the other hand, were not affected to aqy appreciable degree. Beef steaks treated with Age-it o r with enzymes known t o attack connective tissues of meat were consistently rated higher i n tenderness than the controls. Thus, it appears that another way of achieving tenderization i s through the breakdown of the muscle fiber membrane and the endomiysial collagen f i l l i a g the spaces betmen the m s c l e fibers. Other enzymes tested have indicated very potent action on the connective ti88ue6~collagen and elastin, and much less, if my, on the muscle fibers. Papain, Brawlin and Mcin, a31 of which are of' tropical plant origin, belong t o t h i s class. Any one of these, when applied t o steaks in exceedingly low concenltration, was shown t o be capable of

3 . 177 tenderizing the meat. However, mushineser can easily result from a slight over treatment. There i e no doubt t h a t a third way of achieving tenderizst i o n is through hydrolyaie or gelatization of the perimysial connective tissues, consisting of relatively larger amounts of collagen. W e plan, next, t o apply enzymes directly t o connective tissue elements as well as t o the muscle fiber envelopes i n the earn manner a B the isolated muscle fibers referred t o above, There can be no more direct way than t h i s approach of obtaininformtion with regard t o the specific effect o r effects of an enzyme on each cxf the definitive tissue components. f t is hoped that these results coupled with those obtained from whole tissues treated with these e n z p e will eventually lead us t o a better understanding of the lnechaniem of' tenderization an8 also provide b e t t e r practical man8 of tenderizing lower grades of mat t o met the rising demand. KEFERENCES Doty, D. M,, Vang, H. and Schweigert, B. S. e t al. The chemical, physical h i e t ological and organolept i c c h e r a ct e r ist i c 8 of beef as related t o carcs8s grade and carca8e weight. Unpublished data., A hiato-photoelectric method of collagen determinwang, H. ation in narscles. Anat. Rec., 15: 537 (1949). Wang, H. Histolcgy of beef casings. b r i c a n &at Institute Foundation Bulletin No. 17 (19%). Wang, H. H i e t o l w of pork casings. Amsrican k a t Institute Fbundation Bulletin No. 25 (1955). Wang, E., Debeukelaer, F. I,., Maynard, Nehema. Histological study of the hcg dehairing process. Jour. Am. Bather Chemists ABSO., 49: (1954). W a n g, H., &Bride, B. E. ~ n dschweigert, B, S. Histological manifestations OP feeding polycnsyethylene Monosfaaratee t o weanling haaasters. Roc, Soc. Exper. B i d. and b d. (7), 75: (1950) W m g, H., Scheid, E. E., and Schweigert, B. S. Hietological studies with rat6 fed diets containing iodinated casein and different level8 of vitamin B Proc. Soc. &per. Biol. and B d., 85: (dh). Wang, E., Histological manifestation of vitamin B12 deficiency in laboratory t e e t animals. A brief review of research a t the Foundstion. American k a t Xnstitute Foundation Circular No. 20 (1956 )

4 178. (9) Wan$, H., Rasch, Ellen, Bates, Virginia, Beard, Fa J. Pierce, J. C. and Eankine, 0. G. Histological observations on fa* l o c i and d i s t r i b u t i o n i n cooked beef. Food Research, 19: (10) Wang, (19%). H., Auerbach, E., Bates, Virginia, Doty, D. M. and Kraybill, H. R. A h i s t o l o g i c a l aad histochemical study of beef dehydration. IV, Properties of frozendried muscle t i s s u e s and the influence of prefreezing temperatures Food Research, 19 : (1954).. (11) Wang, H. and b y n a r d, &hema. Studies on enzymatic tenderi z a t i o n of meat. I. Basic technique and h i s t o l o g i c a l observations of enzynatic action. Food Research, 20: (1955) (12) Wang, H., Doty, D. Ma Beard, F, J. Pierce, J. C. and (13) Wang, H. The sarcolemma and fibrous envelope of s t r i a t e d muscles in beef Anat. Rec., 124: 378 (1956). (14) Wang, H. The sarcolennna and fibrous envelope of s t r i a t e d muscles i n beef. Wperimental Cell Research, i n press. (1956) Hankins, 0, G, E k t e n s i b i l i t y of s i n g l e beef muscle fibers. Jour. Ani. Sci., 15: (1956). CHAPEARSON: We want t o thank you, D r. Wang, f o r your very i n t e r e s t i n g presentation. D r. Breidenstein will lead the discussion. DR. BREIDENSTEIN: Thank you, Mr. Chairman. I should l i k e first of a l l t o thank our two speakers f o r their very excellent presentations of their respective pagers, and t o compliment our Chairman t h i s year on h i s recommendations of speakers. Taking the papers i n the order i n which they were presented, D r. Ayres' paper c e r t a i n l y should promote an awareness of sources of contamination of our producte. W e c e r t a i n l y have become more aware of the numerous sources of such contamination and t h e p o s s i b i l i t i e s f o r multiplication and growth of microbial f l o r a. I should like t o d i r e c t t o D r. Ayres a couple of subjects f o r commnt. I don't know whether I can put t h e m i n the form of

5 179. questions o r not. 1 should Ilk t o hear hi6 conrments on t h e use of a n t i b i o t i c s in the preservation of our n o m 1 meat animals, both i n t r a peritoneal induction and infusion. Also I would l i k e t o hear h i s comments on the c l a i m of the p o p l e who manufacture the Turbo-Chill cooler i n that they conterd t h a t they can keep meat f o r such an extended period of' time. DR. AYRES: Well, i n reverse order, I don't f e e l that I am equipped t o say much about he Turbo-Chill cooler as yet. But concerning the use of a n t i b i o t i c s on mat, they have one problem there that is a little dif'ferent from its use on chicken. With chickens you can destroy all of the a n t i b i o t i c s by heating the product afterwards, and with neat the way that some of us eat it and especially the way the group I wa6 w i t h last night were eating it it i s l i t t l e more than Harmed up. So that there would be t h e problem of' the residual a n t i b i o t i c that would be present i n t h e mat If it were in3ected i n t o the animal Some evidence is being accumulated t o indicate t h a t the breakdown of the t e t r a c y c l i e is such that these products are harmlees, and when t h a t material appears in the l i t e r a t u r e, if and when it does, I think that one might have a l i t t l e d i f f e r e n t view concerning its use * I would, however, wonder about the use of any preservat i v e or any a n t i b i o t i c unless it m6 necessary t o improve and prolong the storage l i f e of the product and otherwise it would be impossible. I would not in any sense condone the ube of an a n t i b i o t i c where it wae a substitute for cleanllnees. DR, BREDENSTEIN: Are there questions that anyone would like t o direct. t o Dr, Ayres? Dr. Wang's paper certafnly again demonstrates the possib i l i t y f o r a t least another approach t o some of the subjects t h a t were covered yesterday, XXXW~Y, the important one In the quality of meat and even perhaps the most important eingle f a c t o r c o n t r i b u t i w t o quality, namely, tenderness. It seem8 that these studies may eventually point the way t o major technological advances i n meat processing and, as he mentioned, may make so- of our lower grade products much more acceptable t o the consumer. Do you have any questions t o d i r e c t t o him? CHAIRMAN PEARSON: I should l i k e t o ask Dr. Wang concerning the enzyme preparations be used, whether he attempted t o standardize them 88 t o a c t i v i t y or j u s t exactly how he handled the situation, DR. WANG: So far we have been using per cent solutions. In other vards, enzymes supplied t o us by the d i f f e r e n t manufacturers. Each enzyme i e labeled and the u n i t s are given according t o the chemical sesay methods that they detennlned in their own plenrtrs and a t our awn laboratory we have not t r i e d t o standardize them.

6 . 180 For instance, I used, say.015 of a solution f o r those immersions of iaolated single fibers, and so on. Those concentration6 have been carefully tried. If you use too high a solution those m6cl.e f i b e r e w i l l go completely, A critical range is establiehed by the t r i a l and e r r o r method. W e have been spending time determining the range t h a t it3 usable f o r determination of the f i b e r exteneibility, and then you have t o determine the concentration range f o r actual use on t h e steaks. W e have not 80 f a r but we may i n the future attempt t o standardize the potency of those enzymes. DRB BREDENSTEIN: Are there other questione? MR. WAMlERSTOCK: What are your current recommendations r e l a t i v e t o the use of tenderizers? DR, WANG: W e have quite a b i t of data on these things, e have tried a dozen o r more d i f f e r e n t enzymea. but I don't know. W You noticed that 1 grouped the three t h a t are derived from proteol y t i c enzymes, Papain, Bromelin and Ficin, since t h e i r properties a r e very similar. I am speaking only of frozen dried steaks because the concentrate w i l l be very different i f you use it on raw neat, We have evidence that when you use the e n z p on frozen dried it get6 into the tissue because the steak loses thrsequarters by weight on dehydration. If' you want t o apply those enzyme values I w i l l give you a recommendation of,002 per cent f o r those tropical enzyxces. That is very d i l u t e. But on raw t i s s u e it i s an e n t i r e l y d i f f e r e n t problem, The reason we have not used raw t i s s u e i n t h i s program is simply the f a c t t h a t we do not get even penetration. If you cut; sections of a piece of r a w meat treated with the enzyme by immersion and you g e t emulsion on the surface, indicating t h a t the enzyme has over-acted on the surface but i n t h e middle you g e t nothing o r very l i t t l e, there is a gradfent of the enzyme penetration. So that f o r t h i s fundamntal study we d e f i n i t e l y wanted t o apply the enzymes t o frozen dried meat. We a l s o tried other sources of enzymes, largely, say, from rhozyme 11, r h o z p e 48 and protease, all derived from fungi, and t h e i r properties are dffferent from he Papain, Bromelin and Flcin. Their action neem6 t o be more on the muscle fiber protein and l i t t l e or nothing on the connective tissue. I n between you have some other enzynses t h a t are f a i r l y gradual, i n that they a c t r e l a t i v e l y more on the f i b e r s and less on the connective tissue and a t t h e opposite end you have enzymes that a c t more on the connective t i s s u e and much l e e s on the fibers and you have a l l the gradients i n between. t5 DR, BREIDENSTEIN: We have time f o r one more question, and we have two men with t h e i r hands up, Bob and Lyman. f t h i n k we w i l l take Bob. MR, HENRICKSON: I presume that it would be much more desirable if we had natural enzymes t o use rather than a r t i f i c i a l enzymes,

7 181 DR, WANC: By aature,l you wan of anims3. origin? DR. W m : Such as from the pancreas, MR, BENRICKSON: WhaC proareas have we made toward ieo- lating the enzymes from mat and using them? DR, WANO: We have made no attempt t o isolate enzymes from meat. You can obtain it in crystal form Prom wheat, but we have not u8ed wheat.!he only natural enzyme that we have used is a product called Viocaae, which is a whole pancreas gwder. We used it the 8way and we didn't find it as deslrable e8 some of the enzymes that are of paacreas orlgin. We have tried it but without any too f i n e results, MR. KENRICKSON: Eave you t r i e d actually injecting a c t i n o r lsyo6in? DR, WANG: W e have not done anything on it. These are a l l agiplied t o samples of meat. DR. BREIDENgPEIN: I think we will turn t h i s back t o our ChaiMPan a t this time, The,nk you. CHAIRMAN PEARSON: We wish t o thank you, Dr. Breidenstein, f o r leading the discussion, and t h e gentlemen who have participated In the Research Method6 Committee's pr0g;ram. Also we particularly thank t h e committee. They set up a very f i n e program which was enjoyable for all of US. o'clock. ) (hllowing announcements, the meeting recessed a t 12:10