K. J. Carpenter

Mechanisms of heat damage in proteins

1. Studies have been made with solvent-extracted chicken muscle, bovine plasma albumin (BPA) and other proteins, all severely heated in the absence of carbohydrates so as to cause a large decrease in their fluorodinitrobenzene (FDNB)-reactive lysine contents. 2. epsilon-N-(beta-L-aspartyl)-L-lysine and epsilon-N-(gamma-L-glutamyl)-L-lysine isopeptides were determined after enzymic digestion of heated chicken muscle, and their content was found to increase as the material was subjected to more heat treatment. Heated chicken muscle was not found to contain lanthionine. Heated BPA, on the other hand, was found to contain lanthionine but not the isopeptides. Both lanthionine and isopeptide cross-linkages were detected in most of the other heated proteins. There was some difficulty in quantifying the amounts of isopeptides formed on heat treatment, because the enzymic digestion procedure used in their isolation appeared to be incomplete. Neither lysinoalanine nor ornithinoalanine was detected in any of the test materials.

Mechanisms of heat damage in proteins. 2. Chemical changes in pure proteins.

1. Bovine plasma albumin (BPA) containing approximately 14% moisture, when heated for 27 h at 115° suffered an appreciable loss of cystine and a small loss of lysine; at 145° all the amino acids except glutamic acid and those with paraffin side-chains, showed considerable losses. Isoleucine also showed some loss through racemization to alloisoleucine. 2. BPA heated at 115° evolved H 2 S; at 145° other sulphur compounds were released as well, all coming from the breakdown of cystine. Possible mechanisms for this are discussed. 3. Ammonia was also liberated from BPA heated at 115°. The degree of correlation of lysine binding in different proteins with ammonia liberation and amide changes has led us to suggest that the main reaction of e-amino lysine groups is with amide groups of asparagine and glutamine. Reaction of e-amino groups with carboxylic groups is thought to be less important. 4. Model experiments have shown that a reaction between amide groups and the e-amino group of lysine in proteins can occur at practical drying temperatures. 5. Reactions of the e-amino group of lysine with destruction products of cystine is also considered to be partially responsible for the lysine binding in heated proteins.

The digestion of heat-damaged protein

I. The apparent digestibilities for chicks, operated on so as to allow separate collection of urine and faeces, of the nitrogen in a heat-damaged cod flour (C35) and of a control, freezedried cod muscle (Cz3) were 77 and 90 yo respectively. 2. The differences are similar to those found for rats in earlier work and considerably smaller than the differences found in nutritional value of the materials as sources of either lysine or methionine for chicks. 3. Chicks killed 3 h after a test meal containing C23 showed little more N in their small intestine than did those on a N-free diet; other chicks receiving C35 showed much more N remaining in the gut. 4. It is hypothesized that significant quantities of heat-damaged protein may remain undigested in the small intestine, but may then be de-aminated by fermentation in the caecum so that values for the digestibility of N and of individual amino acids may be misleadingly high. 5. This hypothesis is supported by the finding that in caecectomized chicks the apparent digestibility of the N of C35 was only 68 %, whereas the digestibility of Cz3 remained the same as in intact chicks.

Mechanisms of heat damage in proteins: 1. Models with acylated lysine units *

1. ě-N -Acetyl-L-lysine showed a growth-promoting value for the young rat receiving a lysine-deficient diet approximately half that of the equivalent quantity of L-lysine; ě-N -propionyl-L-lysine showed negligible activity. 2. Considerable quantities of lysine were recovered from acid-hydrolysis of the urine of rats receiving these two compounds. Qualitative chromatography suggested that the compounds themselves were appearing in the urine. 3. Similar results were obtained from giving proteins that had the ě-NH 2 , groups of their lysine units either acetylated or propionylated. 4. Giving a pure protein in which the nutritional availability of the lysine had been reduced heat treatment resulted in greatly increased faecal lysine but little urinary lysine.

Mechanisms of heat damage in proteins. 6. The digestibility of individual amino acids in heated and propionylated proteins.

1. The digestibilities of protein and amino acids have been estimated by two different techniques: the analysis of faeces (conventional method) and the analysis of ileal contents (ileal technique). 2. Freeze-dried muscle protein was found by both techniques to be almost completely digested. After autoclaving, the digestibility for the same protein was estimated by the conventional and ileal techniques to be 0.65 and 0.57 respectively. 3. Unmodified lactalbumin was found by both techniques to have digestibility of about 0.90. Propionylation of the lactalbumin reduced digestibility to 0.82 and 0.79 as indicated by faecal analysis and ileal content analysis respectively. 4. In general, the digestibilities of individual amino acids in any one protein sample were rather uniform, and reflected over-all protein digestibility. For each amino acid, digestibility, as determined by both methods, was lower for the modified protein than for the corresponding control protein: estimates based on ileal content analyses were consistently lower than those obtained by conventional analyses. The ileal technique was considered to be both more convenient and meaningful. 5. From the results obtained by the ileal technique it appears that reduced digestibility is an adequate explanation for the reduction found in nutritional value of the autoclaved protein. In contrast, for the propionylated protein, reduced digestibility of lysine is only a partial explanation of the low availability of this amino acid as estimated by chick growth assay. 6. In our experiments we found that the type of dietary protein used did influence the amino acid composition of the ileal contents. This was most marked with the least-digestible protein. These findings do not support the views of Nasset (1962).

A GROWTH ASSAY WITH CHICKS FOR THE LYSINE CONTENT OF PROTEIN CONCENTRATES.

The amino acids that can be determined in a food by chemical analysis are sometimes not all nutritionally available; it is known that the discrepancy between ‘ total’ and ‘available’ lysine, in particular, can be large in processed foods and feeding-stuffs. Procedures have been suggested for obtaining by chemical means values more likely to approximate to the levels of lysine nutritionally available, but the usefulness of such values must be checked empirically against the direct results of biological tests. In this paper we report the use of weight gains of young chicks as a measure of their response to dietary sources of lysine. Such an assay was first described by Tsien, Johnson & Liener (1957) in their study of Torula yeast and by Kratzer & Green (1957), who were interested in blood meals; both these groups used basal diets containing natural feeding-stuffs of low lysine content. More recently, Ousterhout, Grau & Lundholm (1959) have conducted assays for a range of amino acids, including lysine, using a mixture of synthetic amino acids as the only nitrogen source in the basal diets. From studies on the rat (Gupta, Dakroury, Harper & Elvehjem, 1958; Calhoun, Hepburn & Bradley, 1960) and on the chick (Fisher, Griminger, Leveille & Shapiro, 1960; Fisher & Shapiro, 1961), it appears that, in assays of this kind, the relationship between live-weight gain and lysine content of the diet can be distorted by the nature of the supplement, in part through an effect on the appetite of the test animal. The intention of the work reported here was to find an assay procedure with chicks that was not liable to distortion. We also wished to design a growth assay that would provide results suitable for statistical analysis, as was done for the B vitamins by Coates, Kon & Shepheard (1950); one condition is that each test material should be given at more than one level. Owing to the high cost of synthetic diets, we took advantage of the primary deficiency in lysine that sesame seed has for the chick (Grau, 1948).

The availability of lysine in groundnut biscuits used in the treatment of kwashiorkor. 2

Malnourished children under treatment in the wards of the Infantile Malnutrition Research Unit, Kampala, have for several years received biscuit meals made largely from groundnuts, with smaller quantities of maize and wheat flours, cottonseed oil, sugar and skim-milk powder. The meals have been of two chief kinds. In the first, 8 or 15 yo skim-milk powder was cooked with the other ingredients, but in the second the powder was added after the other ingredients had been cooked and milled. The recipes were adjusted so that the total protein of the mixtures was always about 20 yo. The value of the meals has been compared with that of a diet providing the same amount of protein entirely from milk. The milk diet appeared to be excellent in every way; its ability to produce changes in clinical condition and composition of serum, and to promote nitrogen retention, provided a useful set of standards. In a long series of experiments (R. F. A. Dean, to be published) it was found that the biscuit containing 15 yo skim-milk powder added before cooking, although fairly satisfactory, failed to produce the standard changes. On the other hand, biscuit of the other kind-with 1 5 % powder added after cooking-was the equal of the milk diet in almost every way. The most likely explanation of the differences in the results was that cooking the biscuit reduced the value of the protein. It has been known for many years that proteins from vegetable and animal sources may be damaged by heat. One of the suggested causes is a change in amino acids that makes them unavailable for metabolic processes, and there is evidence that lysine is easily susceptible to the change, probably by the blocking of its €-amino groups. From a series of feeding trials with rats, Block, Cannon, Wissler, Steffee, Straube, Frazier & Woolridge (1946) reported that cooking the mixed ingredients of a cake mixture reduced the protein efficiency, which was further decreased by toasting slices of the cake. The protein efficiency was restored by supplementing the baked and toasted cake with lysine. Carpenter, Ellinger, Munro & Rolfe (1957) found that the biological values of various proteins were closely correlated with the numbers of free €-amino groups of lysine present. The possibility of damage to the lysine of our biscuit meals was of particular interest because the

Protein quality of feeding-stuffs. 5. Collaborative studies on the biological assay of available methionine using chicks.

1. The reproducibility of an assay for methionine based on live-weight gains in young chicks has been tested in a collaborative study in six laboratories. 2. Results were analysed by the slope-ratio procedure and, in general, the assays were statistically valid. The variability between laboratories was similar to that found in previous studies of variablity within a single assay in one laboratory. 3. With the combined estimates from five or six laboratories the standard error of the estimates was apporoximately 10% of the mean. Expressing response as‘g gain/g food eaten’ gave no more precision than using ‘weight gain’ alone, but is nevertheless thought to be less open to error due to apetite effects. 4. The experiments have shown that materials can be ranked consistently, but that the absolute estimates of potency varied between assays and further improvements are desirable if potency estimates are to be used for the calibration of in vitro procedures of protein evaluation.

Nutritional effects of autoxidized fats in animal diets

1. In two experiments, 100 pigs were fed from age 4 or 5 weeks to age 16–19 weeks on practical-type diets containing 10% of meat meal (15–17% lipid) of either low (3–17) or high (114–150 μmoles/g lipid) peroxide value. The cereal basis of the diet in the first experiment was wheat and maize and in the second barley. Other variants were the inclusion of a stabilized vitamin E supplement in the diet of one of the groups in Expt 1, the presence of 100 (Expt1) or 250(Expt 2)ppm of copper, and storage of a diet containing oxidized meat meal for 6–21 weeks before feeding to one of the groups in Expt 2. 2. Apart from an unusually high incidence of stomach ulcers which, however, were almost equally distributed between the control and oxidized fat groups and did not appear to affect the health and nutrition of the animals, the pigs grew well, with no significant difference between the groups in weight gain, food conversion ratio, liver weight, vitamin A storage in the liver, and aspartate aminotransferase and alanine aminotransferase activities in the serum. 3. Changes in the lipids of the diets during storage were studied and samples of back fat from the carcasses examined for fatty acid composition, tocopherol content and susceptibility to autoxidation. Moisture content appeared to be a major factor influencing the oxidative stability of the meat meals used. Tocopherol content and oxidative stability of the pig fat were highest in the group given a vitamin E supplement, and generally lower in Expt 2 than in Expt 1.

Mechanisms of heat damage in proteins. 8. The role of sucrose in the susceptibility of protein foods to heat damage

1. A high-protein yeast cake, based on ovalbumin and lactalbumin, and containing 200 g sucrose/kg was baked and toasted as previously described by Block, Cannon, Wissler, Steffe, Straube, Frazier & Woolridge (1946). This caused a severe reduction in its protein quality for rats, and in its fluorodinitrobenzene (FDNB)-reactive lysine content. The damage appeared to be caused by inversion of sucrose to glucose and fructose during fermentation by the yeast followed by Maillard reactions. Processing an albumin-sucrose mixture in a similar way but without fermentation caused no loss in nutritional value for rats and a small reduction in FDNB-reactive lysine. 2. Sucroselysine solutions were prepared and heated as previously described by El-Nockrashy & Frampton (1967). Contrary to their findings, we detected no loss of lysine after storage for 16 h at 35° or after heating for 2 h at 100°, although after heating for 4 h at 121° about 0.7 of the original lysine remained. At an alkaline pH, sucrose hydrolyses only slowly even at 121°, and this is catalysed to some extent by the presence of lysine. 3. A ‘dry’ albumin-sucrose mixture (10-200 mg moisture/g) was heated in sealed glass ampoules under a range of conditions. The loss of FDNB-reactive lysine was stongly dependent on the processing conditions; in particular it was decreased by an increase in pH. After 2 h at 121°, lysine damage caused by sucrose was similar to that caused by glucose. Lysine damage due to sucrose was believed to follow on from its breakdown to glucose and fructose. 4. Although the presence of sucrose does not make proteins highly sensitive to Maillard reactions and loss of nutritive value, it must not be considered entirely inert. Our results confirm earlier work indicating that its presence at relatively high levels in oil seeds may be largely responsible for the damage to protein quality observed when they are severely processed.