THE

EFFECT AMINO
BY

OF SEVERE HEAT TREATMENT ACIDS OF FRESH AND CURED

UPON PORK*

THE

J. F. BEUK,
the

E. E. RICE Chicago)

(From

Research Laboratories,

Swift and

Company,

(Receivedfor publication, March 15, 1948) On the basis of animal experimentation, heat has been shown to have a deleterious effect on proteins (1-13). Some investigators have intimated that dry heat destroys certain amino acids because the addition of these amino acids to the heated protein improves the biological value; others have claimed heat damage to be due to decreaseddigestibility of the heated proteins. Fairbanks and Mitchell (1) showed that the addition of lysine to scorched skim milk powder increased the biological value, but that the addition of extra cystine did not. Additions of lysine and histidine, but not cystine, tyrosine, or tryptophan, increased the biological value of the heated casein studied by Greaves, Morgan, and Loveen (2). Similarly, Clandinin et al. (3) found that supplementing an overheated soy bean meal with lysine and methionine increased the growth rates of chicks. Hodson and Krueger (4) and Eldred and Rodney (5) reported lossesof lysine in heated evaporated milk and heated casein, respectively. That the digestibility of protein is impaired by dry heat was reported by Chick (6), who found that heating lactalbumin lowered its biological value and significantly decreased its digestibility. Heating casein at a higher temperature decreased both the biological value and the digestibility. Using t,otal protein or sulfur, cystine, or methionine in the undigested protein as criteria, Evans (7) reported raw soy bean meal and meal which had been autoclaved for 30 minutes at 100-130 to be more digestible than meal which had been autoclaved at 130 for 60 minutes as determined either by the Chick method or in vitro with trypsin and erepsin. Seegersand Mattill (8) found a marked decrease in the digestibility of the protein of dried liver which had been heated for 72 hours at 120. However, acid hydrolysates of fresh liver and heated liver gave approximately equal growth rates, biological values, and digesbibilities when supplemented by tryptophan. These writers stated: It is believed that the low biological value . . . is the result of a decreased digestibility such that the resulting amino acid proportions are not representative of the original protein. Block el al. (9) and Zittle and Eldred (10) have shown that raw and
* Presented before the Division Society at New York, September of Biological 17, 1947. 291 Chemistry of the American Chemical

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EFFECT OF HEAT

ON AMINO dCIDS

heated casein yielded the same amounts of lysine on acid hydrolysis determined by chemical isolation and by lysine decarboxylase methods, respectively. Riesen et al. (II), using microbiological assaysof soy bean oil meals for individual amino acids, found that autoclaving 4 hours decreased the amounts of lysine, arginine, and tryptophan which could be liberated by 2 x HCl. Enzymatic digestion, measured in terms of amino acids liberated, improved during proper heat treatmcnt but decreased after prolonged heating. There have been few investigations of the conditions normally existing in the preparation of edible meats. These have indicated that heating causes slight, possibly insignificant, reductions in amino acid contents. Thus Schweigert. et,al. (12) showed retention of 86 to 106 per cent for leutine, isoleucine, and valine during the curing and cooking of various cuts of meat, and Greenhut et al. (13) found the retention of tryptophan and phenylalanine to be 73 to 96 per cent and 91 to 110 per cent, respectively, for several types of meat. No lossesof lysine were found by Wilder and Kraybill (14) during t,he cooking of fresh pork, but, a 12 per cent loss was shown for an autoclaved pork luncheon meat,. The small losses always posethe question as to whether amino acids have been destroyed or whether experimental conditions have prevented measurements sufficiently exact to detect the amount of loss. The use of extreme conditions similar to those which have been shown to reduce the biological values of dry proteins offers a means of detecting possible losses and of estimating the significance of losses which may occur in normal procThis technique has been applied in the present. study of the effect essing. of heat upon the amino acid content of fresh and aut-oclaved pork.

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Preparation of Rumples---A 50 pound lot of lean pork trimmings was comminuted in a high speed chopper (silent cutter), mixed several times to assure uniformity, and divided into two portions. One portion was mixed with a standard commercial curing formula; the other remained untreated. Both were held in a cooler at 2-3 for 48 hours. This holding period was necessary to permit. normal c+uringof the meat.. The two lots of meat were mixed separately and then packed in 5 ounce cans and sealed under a vacuum in a semiautomatic can closer. Several cans of each portion were stored at -29 as controls. The remaining cans were autoclaved for 24 hours at 112, cooled immediately with cold water, and stored at -29. Preparation of Hydrolysates-Acid hydrolysates of each of the four sam1 This formula contained salt, sugar, nitrate, and nitrite in proportions conforming to the regulations of the United States Department of Agriculture, Ruroau of Animal Industry.

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ples were prepared by refluxing 10 gm. portions with 100 ml. quantities of 8 N HCl for 8 hours. Previous experiments had indicated t#hese conditions to be adequate for complete liberation of the amino acids and that the treatment destroyed no detectable amount of any amino acid other than tryptophan. Enzyme digests of the samples were prepared by treat.ing 10 gm. portions suspended in 100 ml. quantities of water with 100 mg. of commercial trypsin powder. The suspensions were adjusted wit,h NaOH to pH 7.5 and incubated for 24 hours at 37. Then 100 mg. quantities of erepsin were added to t,he suspensions which, after readjustment to pH 7.5, mere incubated for an additional 24 hours. The pH values of the digests mere checked periodically during the 48 hour incubation and readjustments were made whenever necessary. Toluene was used as a preservative throughout the incubation periods. At the completion of the trypsin-erepsin treatment, the digests mere brought to pH 4.5 and heated in a steam bath to inactivate the enzymes and to remove the toluene. Acid and enzyme hydrolysates were diluted to 250 ml. volumes, filtered, were neutralized during subsequent and stored at 2. The hydrolysates dilutions. The amino acid contents of enzymatic digests of raw pork made in this may were generally slightly louver than those of the acid hydroly&es (see the second column in Tables I and II). This may have been due to losses during filtration, since filtered enzyme digests of raw pork contained 5 to 10 per cent less nitrogen (corrected for nitrogen of the enzyme) than did the acid hydrolysates. Assay Procedures-Except for confirmatory tests with the chemical method for histidine (15), microbiological assay methods were used exclusively in this series of experiments. Leucine, isoleucine, valine, methionine, and tryptophan were determined by use of Lactobacillus arabinosus 17-5 with the medium described by Shankman (16). Cystine, methionine, and lysine were determined with Leuconosfoc mesenteroides P-60 with Medium C of Dunn et al. (17), modified by a S-fold increase of arginine (Kirch (18)). Histidine was determined with Streptococcus lactis R and arginine with Lactobacillus delbrueclcii LD5 with the media described by Stokes et techniques were essentially al. (19). The media and the manipulative those reported except that final volumes in the assay tubes were 5 ml. and that 0.05 r\ sodium hydroxide was used for titrations. Determination oj Eflect of Autoclaving-To determine the effect of severe autoclaving, enzyme digests and acid hydrolysates of fresh, autoclaved, fresh cured, and autoclaved cured samples were prepared. All solutions were assayed simultaneously for each amino acid. Check analyses made several months later confirmed the initial values. Average values and retentions are listed in Tables I and II. For simplicity, the values are given

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294

EFFECT

OF

HEAT

ON

AMINO

ACIDS

per gm. of sample, this being a valid basis since there w w for change in n-eight. during nutoclaving.
DISCUSSION

no opportunity

Analysis of the acid hydrolysates of the autoclaved samples indicated Cyst& almost 100 per cent retention of the amino acids except cystine. showed a 56 per cent retention in both fresh and cured samples after autoclaving (Table I). A sulfide odor from the autoclaved samples indicated the possible breakdown of a sulfur-containing amino acid. Since tryptophan is acid-labile, analyses for that amino acid were not
TABLE

I in Fresh and Cured Pork qfter

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Acid

Hydrolysis;

Retention of Amino Acids Autoclauing at 112O for

24 ROUTS
Cured pork -..per cenl 100

utoclaved g. per gm.

Retained )W cent

Raw

Autoclaved

g. fier gm. mg. per p.

Threonine Leucine Isoleucine Valine. Methionine Phenylalaninc Cystine Lysine Arginine. Histidine Tryptophan* * Alkaline hydrolysis

I ::I I !

9.0 13.5 9.0 9.5 3.8 6.5 2.7 14.0 6:: 1.90 (possibly

9.0 13.5 0.3 9.7 3.9 6.6 1.5 13.2 7.5 6.6 1.90 slight

100 100 103 102 102 101 56 95 96 101 100 destruction

8.7 13.3 8.9 z 6.6 2.7 13.4 7.5 6.5 -___ during

8.7 i 13.3 1 9.1 I i:; 1 6.8 / 1.5 i 12.8 / 7.4 6.0 ___.-refluxing I
-L

100 102 101 100 103 56 96 99 93

(20)).

made on the acid hydrolysates. Assays of alkaline hydrolysates (5 N NaOH for 8 hours, under a reflux) gave similar values for fresh and autoclaved samples. The 1.9 mg. of tryptophan found per gm. of sample correspond to 1.1 per cent tryptophan in the protein, a value slightly lower than the 1.4 per cent reported by Greenhut et al. (20). Apparently, partial destruction of tryptophan occurred during refluxing, an occurrence which would be expected in view of the report of the lat,ter authors that alkali partially destroys and racemizes the try.ptophan. However, if there were equal rates of loss during hydrolysis of the raw and autoclaved samples, the 100 per cent retention value for the autoclaved sample should be valid. The apparent amino acid retention based on enzyme hydrolysates varied from 27 per cent for cystine to 56 per cent for leucine in the autoclaved

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295

fresh sample and from 33 per cent for cystine to 65 per cent for isoleucine in the autoclaved cured samples (Table II). This finding is in accord with the marked reduction in the biological values2 of these raw and cured pork samples during autoclaving, as found by Lockhart.3 Loss of cystine was not in itself sufficient cause for the decreases in biological value, as indicated by failure of cystine to improve markedly the rate of gain when added to the diet in place of a small amount of the protein. Replacement of autoclaved protein with raw protein permitted rapid gain. The low apparent retention cannot be due to the destruction of the amino acids during autoclaving, since the acid hydrolysates showed a definite loss of cystine only. It may be caused by a decrease in the digestibility of the protein in the autoclaved
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TABLE II Retention of Amino Acids in Fresh and Cured Pork after Autoclavins at lid0 for 9.4 Hours
-

Enzyme Hgdpolysis;

Fresh pork Lutoclavec

i Retained . #61 cart

T
--?I

Cured RiiW .-

pork Retained per cent

A ,utoclaved nsg. )CI pm.

y. 9er gm.

ng. +cr gm

Kg. )ergm<

Threonine....... Leucine.........
Isoleucine ....... Valine ........... Methionine. ..... Phenylalanine... Cystine.......... Lysine........... Arginine. ........ Histidine. ....... Tryptophan .....
-

8.4 13.1 8.7

3.4 7.4 4.3 5.0

41 56
49 54 48

8.0 11.6 7.6

2.8 6.6
4.9

35 57
65 58 49 54

5.4

1.8
3.2 0.7 6.4 3.8 2.8 0.6

0.8 2.6 13.7 7.4 5.3 1.7

47 27 47 52
53

35
-

3.7 6.5 2.1 13.0 7.0 4.6 1.6

1.8
3.4 0.7 5.0 3.8
2.9

33
39

0.6

56 63 38

samples. The curing mixture does not seemto affect the retention of the amino acids whether measured on acid or enzyme hydrolysates. Enzyme digestion of raw samples yielded clear solutions in which small amounts of finely divided material were suspended. Similar treatment of autoclaved meat resulted in turbid solutions containing a larger quantity of insoluble particles. Micro-Kjeldahl analyses showed precipitates from enzyme-digested raw meat to contain 3 to 5 per cent of the nitrogen of the sample; those from autoclaved samplescontained 9 to 16 per cent. Hence, part of the low apparent retention is due to the removal of insoluble material during filtration of the digests of cooked samples. This, however,
* Rat growth s Unpublished comparisons. data, Lockhart, H. B., Research Laboratories, Swift and Company.

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EFFECT

OF

HEAT

ON

AMINO

ACIDS

cannot account for the entire apparent loss, since calculation of the values for the filtrates on a nitrogen basis did not greatly improve recovery values. Furthermore, the nitrogen content of the precipitates removed from enzyme hydrolysates of the cooked samples amounted to only 9 to 16 per cent of the tot,al nitrogen, but the lowest apparent amino acid loss was 35 per cent. This would indicate the presence in the filtrates of some amino acids in a form not available to the bacteria used in making the assays. To determine whether the amino acids in the enzyme digests could be recovered by acid hydrolysis, unfiltered enzyme digests of the four samples were treated with 8 N HCl for 8 hours as described under Preparation of
TABLE

III Retention at 1120

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Enzyme

Hydrolysis Fresh and

Followed by Acid Cured Pork after

Hydrolysis; Autoclaving
pork

of Amino for 24 HOUIX

Acids

in

Woclaved mng.per

II Retained -Igm. per cent

ng. jer gm.

Lutoclaved __ ng. per gm.

Retained -__ zh7 cent 98 99

Threonine

.............

Leucine ............... Isoleucine .............

8.1 13.6 9.3 9.6 3.7 6.3 2.6 14.5 7.7 6.3

8.1 14.8 9.9 9.5 3.9 6.5 1.4 14.5 7.4 6.8

7.7 13.1 8.1 9.7 3.6 5.3 2.3 14.5 6.9 6.6

7.5 12.9 8.5 9.6 3.6 5.1 1.3 12.8 7.0 6.5

105 99 loo 97 57 90 101 99

hydrolysates. This procedure did not appreciably affect the values for raw samples, but it did increase the amino acid levels found for the autoclaved samples to such an extent that no marked losses were indicated except for cystine (Table III). These values agree with those in Table I within the range of experimental error. Furthermore, preliminary analyses of acid-hydrolyzed filt,rates of enzyme-digested, cooked samples indicated increases in the quantities of amino acids available to the test organisms. Definite increases in total free amino acids measured by the method of Folin, as modified by Sahyun (21)) are obt,ained under such circumstances. Since the amino acids, except cystine, survive autoclaving for 24 hours at 112, it is to be expected t,hat the much less severe household and commercial processes would likewise cause no losses other than possibly that of cystine. Whether or not reductions in biological values or in enzyme di-

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297 cooking

gestibility would occur under the mild schedules remains to be determined.

editions

of normal

Determinations of the effects of severe heat treatment upon the amino acids of fresh and cured pork mere made by comparing the amino acid contents of the raw and cured pork with those of samples which had been autoclaved at 112 for 24 hours. Analyses of acid- or alkali-hydrolyzed autoclaved samples indicated complete retention of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The retention of cystine was 56 per cent. Low apparent retention (27 to 65 per cent) was observed when the autoclaved samples were hydrolyzed with enzymes. However, treatment of these enzyme hydrolysates with acid permitted recovery of the initial amounts of all of the amino acids except cystine. The low apparent retention was probably due t,o the failure of the enzymes to liberate completely all of the amino acids of the autoclaved pork. Part of the bound amino acid fraction is soluble but unavailable both to bacteria and t,o rats. The failure of severely treated pork to show losses of amino acids other than cystine makes it seem improbable that ordinary cooking or processing operations destroy any significant amounts of amino acids except cystine. Decreases in the enzyme digestibility suggest the possibility that the milder processes of home or commercial cooking may likewise decrease digestibility, but that the changes may not be detectable under normal conditions.
BIBLIOGRAPHY

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1. Fairbanks, B. W., and Mitchell, H. II., J. Agr. Res., 61, 1107 (1935). 2. Greaves, R. O., Morgan, A. F., and Loveen, hf. K., J. Nutr., 16, 115 (1938). 3. Clandinin, D. R., Cravens, W. W., and Elvehjem, C. A., Poultry SC., 26, 399 (1946). 4. Hodson, A. %., and Krueger, G. M., Arch. Biochem., 10, 55 (1946). 5. Eldred, N. R., and Rodney, G., J. Biol. Chem., 162,261 (1946). 6. Chick, H., Hutchinson, J. C. D., and Jackson, H. M., Biochem. J., 29,1712 (1935). 7. Evans, R. J., McGinnis, J., and St. John, J. L., J. Nutr., 33, 661 (1947). 8. Seegers, W. II., and Mattill, H. A., J. Biol. Chem., 110,531 (1935). 9. Block, It. J., Jones, D. B., and Gerdsdorff, C. E. F., J. Biol. Chem., 106,667 (1934). 10. Zittle, C. A., and Eldred, N. It., J. BioZ. Chem., 166, 401 (1944). 11. Riescn, W. H., Clandinin, D. R., Elvehjem, C. A., and Cravens, W. W., J. Biol. Chem., 167, 143 (1947). 12. Schweigert, B. S., Tatman, I. E., and Elvehjem, C. A., Avxh. Biochena., 6, 177 (1945). 13. Greenhut, I. T., Potter, R. L., and Elvehjem, C. A., Arch. Biochem., 16,469 (1947).

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14. 15. 16. 17. 18. 19. 20. 21.

EFFECT

OF

HEAT

ON

AMINO

ACIDS

Wilder, 0. H. M., and Kraybill, H. It., J. Nutr., 33, 235 (1947). Albanese, A. A., Frankston, J. E., and Irby, V., J. Biol. Chem., 160, 441 (1945). Shankman, S., J. Biol. Chem., 160, 305 (1943). Dunn, M. S., Camien, M. N., Shankmen, S., Frankl, W., and Rockland, L. B., J. Biol. Chem., 166, 715 (1944). Kirch, E. R., J. Am. Charm. Assn., SC. Ed., 36, 345 (1947). Stokes, J. L., Gunness, M., Dwyer, I. M., and Caswell, M. C., J. Biol. Chem., 160, 35 (1945). Greenhut, I. T., Schweigert, B. S., and Elvehjem, C. A., J. Biot. Ckam., 166, 325 (1946) 1 Sahyun, M., Outline of the amino acids and proteins, New York (1944).

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