The Inhibitory Effect of Reducing Sugars on the Hydrolysis of Casein by Trypsin. A. Tu and N. A. M. Eskin Department of Foods and Nutrition Faculty of Home Economics University of Manitoba Winnipeg, Man.
Introduction The decrease in nutritive value associated with the processing and storage of many food products is the result of interactions between the proteins and sugars present (Bender, 1966; Eskin and Shenai, 1970; Reynolds, 1965). The essential amino acid lysine is particularly important through tht participation of the epsilon amino group in these reactions, since the majority of the alpha amino groups in the protein chain are chemically bound in peptide bonds (Patton et al. 1948). Losses in nutritive value associated with heat:d milk led to a number of studies on caseinglucose systems in which a decrease in lysine availability was reported (Henry et al., 1948; Mauron and IMottu 1958). Subsequent investigations confirmed these~arlier findings regarding the loss of lysine which was accompanied by a reduction in biological value (Finot et al., 1968; Rao et al., 1963). This paper describes experiments to determine the effect of the chemical nature of sugar on the susceptibility of casein to hydrolysis by trypsin in an attempt to define the mechanism of these reactions more fully.
Kjeldahl method (A.O.A.C., 1965). to ~etert;rtine ~he TCA-soluble nitrogen released durmg dIgestIOn wIth trypsin. All Kjeldahl titres were corrected for controls and converted to percent of casein hydrolysed by trypsin using the value of 14.5% for the nitrogen content of casein.
Results and Discussion After digestion with trypsin for 2 hours at 37°C, free casein was 77.4% hydrolysed while under identical conditions the same amount 'of casein incubated with xylose (0.275M) for 4 days was only 12.6% hydrolysed. This indicated that the presence of xylose inhibited the hydrolysis of casein by 83.7%. In contrast, the non-reducing sugar sucrose (0.275M) did not appear to exert any inhibitory effect. The latter observation agreed with earlier studies carried out in this laboratory using spectrophotometric methods for assaying trypsin activity and is probably related to the lack of sucrose breakdown during this period (Tu, 1971). The effect of different sugar concentrations on the 80
Method Casein-sugar mixtures were made up in phosphate buffer (O.lM Na2HP04/NaH2P04, pH7.6) so that the final concentrations were: casein 1 (vitamin-free) 1%; glucose, fructose and xylose 0.15, 0.30 and 0.55M and sucrose 0.55M. Individual casein-sugar mixtures were transferred to screw top vials in an air atmosphere, sealed, and then incubated at 55°C. Duplicate s.amples were removed initially and following 4 days mcubation. Enzymatic hydrolysis was carried out according to the method described by Rick (1963). Casein-sugar mixtures (4.0 ml) were added to O.lM Na2HP04/NaH2 P04 buffer (pH7.6; 2.8ml) and trypsin 2 (bovine pancreas) (1.2 ml; 0.10 mg/ml in 0.001 N HCI) to give a final concentration of 0.5% for casein and 0.075, 0.150 and 0.275M for glucose, fructose and xylose and O.275M for sucrose. Hydrolysis proceeded at 37°C and the reaction terminated after 2 hours by withdrawing a 1.0 ml sample and adding it to aqueous trichloroacetic acid (3.0 ml; 5.0% W Iv) ("TCA"). The solutions were mixed vigorously and centrifuged. The supernatants were analyzed by the MicroI Nutritional Biochemicals Corp., Cleveland, OhIo. 2 Miles Laboratories, Inc., Elkhart, Indiana.
Concentration of Monosaccharides (Molar)
Fig. 1. Percentage hydrolysis by trypsin at pH 7.6 (37°C for 2 hrs.) of casein and casein-monosaccharide systems incubated at 55°C for 4 days. Can. Inst. Food Sci. Techno!. J. Vo!. 6, No.1, 1973
enzymic hyd1'olysis of casein is illustrated for glucose, fructose and xylose in Figure 1. A marked decrease in casein hydrolysis was observed in all monosaccharide systems studied with inCl'ensing sugar concentration. 1'he pentose sugar, x,ylose, exerted the greatest inhi· bitory effect followed by fructose and glucose. This is not too unexpected since the rate of browning of xylose-glycine systems was reported to be extremely rapid compared to other monosaccharides (Spark, 1969) . Tltis study shows that reducin!!: sugars exert an inhibitory effect on the hydrolysis of casein by trypsin, which could explain the unavailability of certain essential amino adds as a result of nonenzymic browning reactions.
Acknowledgements This research was financially supported by the Defence Research Board of Canada, Grant No. 886508.
J. Inst. Can. Sci. Techno!. Aliment. Vo!. 6, No I, 1973
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