Optimum conditions for enzymatic degradation of some oilseed proteins

Proteínas de haba de soja, semilla de sésamo y harina de gérmen de arroz se hidrolizaron con dos enzimas, denominadas, papaina y bromelaina. Se han llevado a cabo experimentos para determinar las condiciones óptimas de cada enzima cuando actuan separadamente sobre cada sustrato. Los resultados mostraron que las mayores actividades relativas para la papaina se consiguieron con una E/S 0,06, 0,29, 0,19 y un pH 7.2, 7.0, 7.0 para las proteínas de haba de soja, sésamo y harina de gérmen de arroz, respectivamente. La temperatura óptima para la papaina durante la hidrólisis de los tres sustratos fue de 50C. Cuando se usa bromelaina las relaciones E/S óptimas que proporcionaron mayor actividad relativa fueron 0.067, 0.058 y 0.21 para las proteínas de habas de soja, sésamo y harina de gérmen de arroz respectivamente. El pH óptimo fue 6.0 y la temperatura óptima 45C para la bromelaina cuando se hidroliza la proteína de los tres sustratos. Con estos datos se hizo una correlación numérica del comportamiento enzimático para los diferentes sustratos.

Protein hydrolysates used in nutritional formulations are generally categorized into two broad categories, partially hydrolyzed and extensively hydrolyzed proteins.Each category possess different properties that influence their utilization in the final product.Presently there are no precise, widely accepted specifications to differentiate the protein hydrolysates on chemical basis.Extensively hydrolyzed proteins have substantially reduced immunological reactivities and are primarily used in hypoallergenic formulas, because of the need to almost completely eliminate their allerginicity to avoid sensitization of individuals consuming them (12) .These protein hydrolysates are usually comprised of amino acids and very short peptides (di and tri peptides).
The aim of the present investigation was to elucidate the optimum conditions for the enzymatic hydrolysis of soybean, sesame seed and rice bran meal protein, with two enzymes one of plant origin papain, and bromelain .The numerical correlation of enzymatic behaviour for different substrates was also elucidated.The results of this investigation will be the key to further work on partial degradation of these proteins.

Materials
2.1.1.Oilseed meals Soybean (glycine max) and sesame (Sesanum indicum) were supplied by the Research Institute of Field Crops, Ministry of Agriculture, Cairo, Egypt.
Rice bran (Oryza sativa ) was obtained from a local rice mill.
Soybeans and sesame seeds were dehulled and ground then subjected to several extractions with n-hexane to extract the oil.After two extractions, the meats are regrounded and extraction continued until residual oil in the meals did not exceed 1%.Rice bran meal was prepared in the same manner but excluding the dehulling step.The defatted meals were spread to dry at room temperature and then ground and seived to pass an 80 mesh screen.

Determination of relative activity
To a 250 ml beaker that was placed in a thermostatic water bath,were added 100 ml water and a weight of meal to containing 5g protein.The mixture was continuously stirred with an electric stirrer and the pH and temperature adjusted to desired values.The reaction was carried on for 30 minutes while stirring and the pH and temperature maintained at the at previously adjusted values.The reaction was stopped by the addition of 0.5M TCA, and the hydrolysate filtered through Whatman no. 4, the residue washed with distilled water until an approximate volume of 250ml was collected.Aliquots of the filtrate were analyzed for total protein using the kjeldahl method and the relative activity was calculated as follows: Relative activity (RA) =Cp -Hp x 100 Cp Where: Hp is total protein obtained in enzyme assay Cp is total protein in original meal ( control).

Determination of the optimum conditions for the proteolytic activity of the enzymes
This was accomplished through a series of experiments.
In the first set of experiments the enzyme papain was used together with sodium sulphite ( which acts as an activator for papain ) on the three oilseed meals.The relative activity was determined by kjeldahl as mentioned above.
The first investigated criteria was the E/S ratio which is the ratio of concentration of enzyme to concentration of substrate.The E/S investigated ranged between 0.01 to 1.83.The temperature and pH were those recommended by the manufacturer, and the experiment was carried as for determination of relative activity.
The pH was the second investigated criteria, the E/S ratio was the best resulting from the former experiment, the temperature was that of the manufacture.The investigated pH were 6.8, 7.0, 7.2, 7.4, 7.6, the experiment proceeded as described for the relative activity determination.
In the third experiment the temperature was the variable ranging from 40 to 80 o C, best pH and best E/S as previously elucidated, and experiment proceeded as for determination of relative activity.
In the second set of experiments the enzyme bromelain was used on the three oilseed substrates.The sequence of experiments followed the same route as those carried with papain.The E/S investigated were 0.021 to 0.078, pH 5.8, 6.0, 6.2, 6.4, 6.6, and 6.8, the temperature 35 to 50 o C.

Analysis
Moisture, oil, ash, fiber and nitrogen were determined according to AOCS (13) standard methods.Protein calculated as N x 6.25 for sesame and rice bran, and N x 5.7 for soybean.

RESULTS AND DISCUSSION
In order to carry the enzymatic degradation of the proteins in the most effective manner, optimum conditions of E/S ratio, pH and temperature of the reaction were examined .Two enzymes papain and bromelain were investigated and three substrates including soybean, sesame seed and rice bran meal proteins.
Table I gives the chemical composition of the three investigated oilseed meals.All values in table are given on moisture free basis.

Optimum conditions for the two enzymes studied
The optimum conditions for the enzymes are expressed in terms of highest relative activity of the enzyme at different values of E/S ratios, pH values, and temperatures.The time of reaction was fixed at 30 minutes to be investigated in a continuation of this study.The optimum conditions for each enzyme was determined on each substrate separately.
Figures 1-3 represent the relation between the relative activity of the enzyme (papain) and the investigated parameters including E/S ratio, pH, temperature.
•    14), hydrolysed rapeseed protein concentrate with papain using the following conditions: pH 6.9, temperature 65 o C and an enzyme concentration of 0.30%.Childs (15), reported optimum pH for papain to be 7.5, while Sekul and Ory (3), working with peanut flour protein and papain recommended a temperature of 45 o C and enzyme concentration 0.5%.Arzu et al. (1) determining the relative activity of ten commercially available proteolytic enzymes reported relative activity of papain concentrate to be 129.1 at pH 7.2, and the relative activity of two bacterial proteinase to be 49.5 and 58.1 at pH 6.8.

Numerical correlation of enzymatic
behavior for different substrates 3.2.1.Papain   Emperical formulae, which correlate the influencing parameters with the relative activity of papain are obtained by plotting E/S and temperature against the relative activity on ordinary scale and conducting of regressions, the following were the results.
The relative activity is directly proportional to the pH of the reaction media, and is proportional exponentially with approximately 2.8 E/S, in case of soybean and sesame while with approximately -0.4 E/S in case of rice bran, and is also proportional exponentially with approximately -0.03 temperature.Emperical formulae, which correlate the influencing parameters with the relative activity of bromelain were obtained for each substrate at certain specific ranges of those parameters.
Plotting of E/S, pH and temperature against relative activity on ordinary scale, and by conducting linear regressions,one can find that the relative activity of bromelain for each substrate ( soybean, sesame an rice bran ) is directly proportional to E/S, pH, and temperature.Therefore in case of soybean RA = 31.776e 0.05 ( E/S .pH .T ) (5) In the ranges of The relative activity of papain and bromelain calculated from the above emperical formulae were in fair agreement with those obtained from experimental work, and the mean standard errors for each enzyme were within the ± 10% as shown in (Figures 7 and 8).

Figure 2 Figure 3 Figure 4
Figure 2Effect of pH on relative activity of papain enzyme.

Figure 5
Figure 5Effect of pH on relative activity of bromelain enzyme.

Figure 8
Figure 8The relationship between experimentally calculated RA & emperically calculated RA of bromelain at different substrates.

Figure 7
Figure 7The relationship between experimentally calculated RA & emperically calculated RA of papain at different substrates.