Enzymatic treatment of sunflower kernels before oil extraction

Food Research International,Vol. 28, No. 6, pp. 531-545, 1996 Copyright 0 1996 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd Printed in Great Britain 0963-9969/96 $15.00 +O.OO

ELSEVIER 0963-9969(95)00044-S

Enzymatic treatment of sunflower kernels before oil extraction H. Dominguez, J. Sineiro, M. J. Ntiiiez* & J. M. Lema Department of Chemical Engineering, University of Santiago de Compostela, Avda. de las Ciencias s/n, 15706 Santiago de Compostela, Spain

Sunflower kernels were enzymatically treated before pressing, with the aim of enhancing oil extractability. Following the response surface methodology, the combined effects of moisture, enzyme/kernel ratio and treatment time were examined. The effect of these variables on the pressing efficiency, the protein digestibility, the fiber content and the meal color was assessed. In a wide range of conditions, it was found that the pressing efficiency was higher for treated kernels, obtaining 13% additional oil compared with untreated samples. Also, the in vitro apparent digestibility coefficient of the meal was improved and the total fiber content was reduced. A slight darkening of the meal was observed as a result of the operational conditions during the treatment. The solvent extractability of the enzymatically treated pressed cakes was enhanced compared with that of the untreated sample. Copyright 0 1996 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. Keywordr: sunflower kernels, enzymatic treatment, digestibility.

apparent

With sunflower having a high oil-content seed, three oil extraction procedures can be used: (i) full pressing, (ii) combined pre-pressing and solvent extraction of the cake, and (iii) direct solvent extraction (Bernardini, 1983; Ward, 1984). Pressing and solvent extraction is the most widely used technique for the processing of high oil-bearing seeds in the industry, owing to its high efficiency in oil recovery. The enzymatic treatment could be incorporated with some modifications in the current system. Its application would require additional steps of incubation at intermediate moisture (1540%) and further drying before pressing and solvent extraction. Since the costs of enzyme treatment and subsequent drying would constitute the major costs of the treatment, both moisture and enzyme should be optimized (Sosulski et al., 1988). The beneficial effects of the enzymatic treatment on sunflower oil extractability by hexane were previously observed (Dominguez et al., 1993). Considering the high efficiency of the solvent extraction step, it would be of greater interest to attain improved performance on the pressing stage, that eventually would avoid the solvent extraction. This investigation was undertaken to further study the performance of the treatment under

INTRODUCTION Vegetable oil is found inside plant cells, linked with proteins and a wide variety of carbohydrates (cellulose,

hemicellulose, pectin, starch, etc.). In order to facilitate its extraction from seeds, it is necessary to degrade the cell walls to increase the permeability for oil (Olsen, 1988). This can be achieved by mechanical and thermal treatments. Oil extraction can also be favoured upon partial hydrolysis of the vegetable cell walls by means of appropriate enzymes. Enzyme treatment with carbohydrases and proteases was reported to enhance the oil extractability of seeds (Lanzani et al., 1975; Fullbrook, 1983; Dominguez et al., 1994). The enzymatic treatment was successfully performed either during aqueous processing for oil and protein extraction (Fullbrook, 1984; Marek et al., 1990) or during conventional oil extraction by pressing (Sosulski & Sosulski, 1990a). These latter authors also observed that the enhanced extraction yield and rate for canola oil was accompanied by an increase in the protein quality. *To whom correspondence

oil extractability,

should be addressed. 531

538

H. Dominguez,

J. Sineiro, M. J. Ntin’ez, J. M. Lema

conditions more similar to those used during the first step in the industrial operation. The objective of the present work was to establish empirical models describing the influence of the operational variables (moisture, enzyme/substrate ratio and length of treatment time) on the efficiency of the enzymatic hydrolysis, in order to find the optimum conditions for the treatment. In addition, the effect of the treatment on the oil and meal quality was evaluated.

MATERIALS AND METHODS Sunflower kernels Dehulled sunflower seeds (obtained from commercial suppliers), were stored at 4°C wrapped in plastic bags until use. Proximate analysis indicated that the whole kernels contained 63.0% oil, 20.0% protein, 6.0% neutral detergent fiber, 3.6% ADF, 2.3% ash, and 1.3% chlorogenic acid (dry basis). Enzymes Commercial food-grade enzymes adequate to enhance sunflower oil extractability were employed (Dominguez et al., 1991). A mixture (2:l) of a cellulase (Celluclast 1.5 1) and a pectinase (Pectinex ULTRA SP), both kindly supplied by Novo Nordisk A/S, and a multiactivity complex (MultifectTM), a gift from Finnish Sugars Co. Ltd, were used in the experiments. Analytical methods Moisture, ash, crude fat, neutral detergent fiber and ADF were determined by standard procedures (AOAC, 1990). Protein was calculated as Kjeldahl Nitrogen x 5.3 (Moss&, 1990). The oil extracted by pressing was calculated as the difference between the total oil and the residual in the cake, analysed by Soxhlet. Estimation of sugars, as glucose equivalents, extracted from the defatted meal with 80% ethanol, was carried out by using the DNSA reagent to determine reducing sugars and by using hydracine sulphate in sulphuric acid to measure total sugars. Available lysine was determined by the TNBS method (James & Ryley, 1986). In vitro digestibility was measured by an enzymatic assay proposed by Hsu et al. (1977). The free fatty acid content in the oil, expressed as percent of oleic acid was determined by the calorimetric method of Lowry & Tinsley (1976). The phosphorus content, measured as phospholipids in the oil, was measured by the method of Raheja et al. (1973). Peroxide value was measured as the iodine liberated from the cadmium-iodide complex, by the peroxides in the oil (Sosulski & Sosulski, 199Ob). The meal colour was visually observed, using an

arbitrary scale from 0 to 4; the higher values corresponded with the darker colours and the zero corresponded to the natural colour of the untreated samples. This parameter has been suggested as a guide for the oxidation of the phenolics and, consequently, for the availability of protein (Elias et al., 1979). Treatment The enzyme solution was added to the whole kernels, and the enzymatic treatment was performed at 50°C at the unmodified pH of the seeds (6.6-6.8) with different enzyme/kernel ratios, moisture content in the range 2040% and treatment time ranging from 2 to 10 h. Pressing assays After drying in oven (6&7O”C), samples were pressed at room temperature. Neither thermal nor mechanical conditioning previous to pressing was used, although the efficiency and rate of extraction could be improved by so doing (Vadke 8c Sosulski, 1988). However, since assays were pressed only to assess the effect of the enzymatic treatment and to select the optimum conditions to perform the treatment, maximum oil recoveries were unnecessary. For this purpose the batch press was a suitable tool. A laboratory batch press of 50 g kernels capacity, with canals in the bottom to drain the extracted oil and reaching pressures ranging from 1.5 x lo7 to 3.5 x lo7 N/m*, was used. Time of pressing and pressure of operation, both favouring the removal of oil from the seeds, were fixed for all analyses at 20 min and 3 x lo7 N/m*, respectively. Sample moisture was reduced to 5-6% before pressing as moisture hinders pressing. On the other hand, drying for prolonged periods or at high temperatures causes toasting of the samples, and the subsequent hardening inhibits pressing, reducing the oil yield; a compromise solution was established and samples were air dried at 60-70°C.

RESULTS AND DISCUSSION Factorial design for selecting operational conditions during enzymatic treatment In the study of the enzymatic treatment of sunflower kernels, three independent variables were considered: the moisture content during the enzymatic treatment, m (g water/100 g kernels), the enzyme/kernel ratio, r (g enzyme/100 g dry kernels, using the 2: 1 mixture of cellulase/pectinase) and the treatment time, I (h). The appropriate range for each operational variable was selected based on previous experience (Dominguez et al., 1993), m = 20-40, r = 0.1-3.0, t = 2-10. Because the factorial design employed two levels (+ 1, -l), coded variables needed to be defined so that m, T and t

Enzymatic treatment of sunfower kernels before oil extraction

acquired the values + 1 and - 1, 0 being the midpoint (high, low and central values, respectively). Hence, the coded variables were M = (m - 30)/10, R = (r - 1.55)/ 1.45 and T = (t - 6)/4. To evaluate the reproducibility of the experiment, a 23 factorial design with four replicates at the central point (Akhnazarova & Kafarov, 1982) was used in the modelling of the enzymatic treatment. The structure of the experimental design allowed the development of empirical equations where each objective function, F, can be calculated as the sum of a constant, three first-order effects (BM, BR, BT) and the interaction terms (BICIR,BRT, BMUTand BMRT): Fi(M,R,T)=Bo+BM.H+BR.R+BT.T +B~MR.M.R+B~~.M.T+BRT.R.T -tBMRT.M.R.

(1)

T

To measure both the extent and efficiency of the enzymatic reaction, the following objective functions were taken into consideration: the pressing efficiency, F, (% total oil extracted after pressing), the in vitro apparent digestibility coefficient (ADC), F2, and the total fiber content of the defatted cake, F4. The colour of the kernels (F3) was observed as a parameter indicating the darkening caused by oxidation of phenolic compounds. Table 1 shows the experimental matrix corresponding to the coded independent variables and the experimental values obtained for the objective functions under study. An additional experiment pressing untreated kernels was performed. This provided a reference against which to measure the effect of the enzymatic treatment on the oil extractability, apparent digestibility, fiber content

539

and colour of the meal. The favourable action of the enzymatic treatment on the pressing efficiency and, to a minor degree, on the in vitro digestibility was noticeable; the darkening effect was also seen in the treated samples. It can be observed from Table 1 that the maximum FI value was registered for prolonged treatment time and high enzyme/kernel ratio and moisture (Experiment 2). Other high values were attained with high enzyme/kernel ratio, low moisture and prolonged treatment time (Experiment 6). For short treatment periods, if the enzyme concentration was high (Experiments 4 and S), good results were also obtained whatever the moisture value. Table 2 lists the set of coefficients obtained by multiple regression of data. As only some of the contributions to each effect were statistically significant at the 95% confidence level by applying a t-test, the nonsignificant coefficients were neglected in the original model and the new regression analysis provided the parameters measuring both the correlation and statistical significance (Table 3). It can be inferred from the coefficients in Tables 2 and 3 that the pressing efficiency (F,) was mainly affected by the enzyme/kernel ratio and the length of treatment time, the favourable influence of the moisture content, in the range studied, being statistically non-significant. The digestibility model indicated that the three factors influenced the meal digestibility (Fz), the moisture content during the treatment having the greatest effect; this factor showed a significant and negative first-order effect (Table 1). The enzyme/kernel ratio also presented a significant influence, whereas the lower non-significant coefficient of treatment time revealed this variable had only a minor influence. In contrast to the behaviour

Table 1. Operational conditions of the different experiments of the factorial design corresponding to the coded variables, and experimental results obtained for the objective functions under study Experiment

1 2 3 4 5 6 7 8 9 10 11 12 13 “Coded moisture. bCoded enzyme/kernel ‘Coded time. dApparent digestibility

+1 +1 +1 +1 -1 -1 -1 -1 0 0 0 0

-1 +1 -1 +1 -1 +1 -1 +1 0 0 0 0 Untreated

ratio. coefficient.

T”

Pressing

ADCd (%) efficiency (% total)

+1 +1 -1 -1 +1 +1 -1 -1 0 0 0 0

75.35 78.25 72.06 76.68 73.49 77.94 70.95 76.46 75.72 76.36 76.67 75.08 63.9;

81.37 81.56 80.84 81.74 84.56 83.82 82.57 85.24 82.73 82.90 82.81 82.43 80.36

Colour

3 3 2 1 1 I 0 1 2 2 2 2 0

Total fiber

13.24 12.16 13.67 12.46 13.75 12.99 15.10 13.78 13.13 13.43 11.97 14.21 16.21

H. Dominguez, J. Sineiro, M. J. NtiEez, J. M. L&ma

540

Table 2. Regression coefficients for the empirical models Coefficient

Bo B‘U BR BT &UR BRT BMT BMRT

Pressing efficiency

ADC”

Colour

Total fiber

75.42 0.44 2.18 1.11 -0.30 0.10 -0.35 -0.08

82.71 -1.33 0.38 0.11 -0.10 -0.02 -0.52 0.33

1.67 0.75 0.00 0.50 -0.25 0.25 0.00 0.25

13.32 -0.51 -0.55 -0.36 -0.03 0.17 0.08 -0.05

OApparent digestibility coefficient. Significant coefficients at 95% confidence are indicated in bold.

Table 3. Statistical parameters for the empirical models including only the significant terms (P 6 0.05) Objective function

Fl F2

4 F4

R2

Corr R2

0.8788 0.9826 0.7500 0.5246

0.8519 0.9726 0.6944 0.4190

found for the pressing efficiency, the coefficients of the crossed effects involving the moisture content during treatment (BMr and BMIRT)are significant, indicating a negative double effect of A4 with T and a positive one of the three variables. The highest digestibility values corresponded to Experiment 8 (high R and low 7) and Experiment 5 (low R and high 7), both with the lowest M value. The response of the ADC to the different variables can be understood by considering several facts; the action of carbohydrases frequently results in freeing proteins from the polysaccharide matrix in which they may be entrapped, improving the susceptibility of the seed protein to the digestive enzymes. An efficient enzymatic attack would increase the availability and/or nutritional quality of proteins. For this purpose, prolonged treatment times and a high enzyme/kernel ratio are beneficial. The moisture content favours the wall degradation, because water plays an important role in hydrolytic reactions, favouring the diffusion and mobility of both the enzymes and the products (prone to inhibiting the enzymatic reaction). However, high moisture during the treatment reduces the ADC. The expression obtained to define the darkening of the meal is only suitable for qualitatively interpreting the responses, but the values of the coefficients are not valid since an arbitrary scale has been used. The most important effects on F3 were associated with variations in the moisture content and treatment time; the enzyme/ kernel ratio had no effect, as expected, because darkening is caused by the presence of substances which tend to react with the proteins.

F=P

32.61 98.64 13.50 4.96

Probability Lp ’ FstI

< 0.01 < 0.01 < 0.01 < 0.04

The total fiber content of the defatted meal (F4) varied in a narrow range. Values lower than that of untreated samples were obtained for any of the conditions used, this effect being favoured by the three considered variables. The resulting analysis of variance for the experimental data corresponding to F4 shows that the equation, but not the coefficients, is statistically significant. This indicates that there is not an absolute minimum fiber content (caused by a maximum enzymatic attack to degrade the cell walls), but under a series of conditions the enzymatic action was favoured. The cell wall degradation, considered as a reduction in fiber content, was more marked for hydrolysis with high moisture content, high enzyme/kernel ratio and prolonged treatment time. The total demolition of the cell wall is unnecessary to allow an attack of the polymeric structures of the wall and subsequently increase the oil extractability; therefore, below optimal conditions for reducing the fiber content are desirable because the ADC and oil extractability can be favoured. The enzymatic attack of the cell walls caused an enhanced pressing efficiency, as well as a reduction in fiber content. Fiber may prevent access of the enzyme to the protein; thus, after enzymatic hydrolysis, easier access to the protein by proteases can be achieved, improving the meal digestibility. Higher efficiency could probably be achieved if, instead of natural pH, a buffer of the optimum pH was used, although the effects in the meal are not known and the water activity could be reduced. In order to improve the enzymatic treatment, a second series of experiments based on the indicated results was carried out to investigate particular parameter influences. The moisture content was kept con-

Enzymatic treatment of sunfower kernels before oil extraction Table 4. Opedo~I

541

conditions of the ditferent experimenti of the second factorid ddgn CorrespolldIaetothecodedvarirMcs,aml experkntal reeds obtdned for tbe objective fuoetks tlader study

Experiment

F

7+

Pressing efficiency

1 2 3 4 5 6 7 8 9

-1 +1 -1 +1 0 0 0 0

-1 -1 +1 +1 0 0 0 0

75.72 76.13 76.39 77.38 76.03 76.57 76.19 76.75 63.97

Untreated

ADC’(%) 84.88 84.57 83.75 83.46 83.99 84.18 83.48 84.26 80.36

Colour 1 3 2 4 3 2 3 3 0

‘Xhded enzyme/kernel ratio. bCoded treatment time. ‘Apparent digestibility coefficient. Table 5. Regression caefficients for the empirical models Coefficient

BO BR BT BRT

Pressing efficiency 76.39 0.35 0.48 0.14

ADC”

Colour

84.11 -0.15 -0.56 0.00

2.65 l.Qo 0.50 0.00

“Apparent digestibility coefficient. Significant coefficients at 95% are indicated in bold.

stant in its minimum value, owing to its negative effect on in vitro digestibility and because it produced low darkening effects. A 22 factorial design was devised to study further the contributions of the enzyme/kernel ratio and hydrolysis time on the treatment performance. The central point for R was the optimum found in the previous experiments for improving the pressing efficiency, the ADC and reducing the fiber content (3 g enzyme/100 g dry kernels), and the interval of variation of r was 1 g enzyme/l00 g dry kernels. The treatment time was reduced with the aims of finding more operative times and, owing to the negative combined effect of R and T on the ADC, reducing the variation limits in the range 2.5-7.5 h (Table 4). The new coded variables were: R = (r - 3)/l, and T = (t - 5)/2.5. As in the previous design, first-order equations were used to describe the effect of the variables on the objective functions fitting the experimental values. The real effect of the enzymatic treatment on the oil extraction of sunflower kernels is mainly described by the pressing

efficiency and the meal digestibility. The fiber content, although valid to measure the extent of the hydrolysis, does not need to be optimized in order to enhance the efficiency of the process; whereas it is desirable to maximize Fi and F2, and to minimize darkening. Therefore, the evaluated objective functions in this design are the pressing efficiency, the in vitro digestibility and the colour of the meal. The resulting coefficients are shown in Table 5. The statistical parameters, once the coefficients that were non-significant at 95% confidence were dropped from the model, are indicated in Table 6. It can be noticed that there was not a well defined maximum, since there was a range of conditions that presented optimal response. In these conditions, pressing efficiencies as high as 77.4% were achieved. Both R and T exerted a beneficial effect on oil extractability by pressing. The respective individual coefficients, although presenting a low value, were statistically significant at 95% (R) and 99% (7) probability. For the treatment time range 2.5-7.5 h, oil extraction yield increased only slightly from 75% to 77%, the performance for the treated samples being 12% higher than for untreated samples. The enzyme/kernel ratio also showed a similar, although less pronounced, effect. Any value in the range would be suitable to enhance the oil extractability. The pressing efficiencies obtained in the first experimental design were as high or higher than those found in the second. In spite of the higher R values, extractability was not enhanced as a result of the reduced moisture and treatment time. The higher enzyme/kernel ratio requirements at lower moisture levels were, however, observed by Sosulski et al. (1988) in the oil extractability with solvents of flaked canola.

Table 6. Statistical parameters for tbe empirical models including only the significant terms (P < 0.05) Objective function

4 F2 F3

R2

Corr R2

FexP

Probability

0.7222 0.9139 0.8510

0.6811 0.8795 0.7915

84.75 26.55 14.28

< 0.03 < 0.01 < 0.01

542

H. Dominguez, J. Sineiro, M. J. NtiZez, J. M. L.ema

-1

-0.6

-0.2

R

0.2

0.6

1

Fig. 1.

Dependence of the pressing efficiency (F,) on the enzyme/kernel ratio (R) and the treatment time (T).

A non-significant negative influence of the enzyme/ kernel ratio on the in vitro digestibility of the cake was observed. Also, the treatnient time exerted an unfavourable effect, significant at 99% probability. The colour of the samples (F3) was strongly dependent on both variables, the coefficients indicating that the effect of R was more significant (99O/ probability) and more important than that of T (93% probability). The variation range of T did not affect the value of the coefficient obtained for this variable with respect to the experimental design of Table 1, whereas the effect of R, nonexistent in the previous experimental design, was revealed to be the most influential. The response surfaces for the pressing efficiency and digestibility in the examined intervals enabled modelling of their dependence (Figures 1 and 2, respectively) on the coded variables R and T. Keeping the moisture content at 20%, the oil yield from treated samples increased compared with untreated samples for increased enzyme/ kernel ratios and treatment times. The ADC showed a slight tendency towards increasing values for the lowest R and T in the studied interval. Owing to the opposite effects of R and T (mainly T, as the influence of R in the studied range is lower) on the pressing efficiency and on the in vitro digestibility, as well as the absence of a

Fig. 2. Dependence of the meal in vitro apparent_digestibility coefficient (&) on the enzyme/kernel ratio (R) and the treatment time (7).

definite optimum, the conditions near to the central point could be suitable for enzymatic treatment. Product

quality

Crude oil Table 7 summarizes some of the characteristics of the crude oil obtained by pressing kernels treated with either a mixture of cellulase and pectinase (already employed in the experimental designs) or a multiactivity complex. It can be seen that both enzymes behave similarly. The free fatty acid content, as a percentage of oleic acid, was slightly higher in the oil from treated samples as compared with that from untreated samples, owing to the high temperature and moisture during the treatment and because no previous tempering was performed to inactivate endogenous enzymes. Sunflower oil contains a low fraction of phospholipids, and only slightly more phospholipids as phosphorus was removed in the oil from treated samples. Sosulski & Sosulski (1990b) verified this effect for canola oil obtained by

Table 7. Quality of crude oil from untreated and enzymatically treated sunfIower kernels

Characteristics

Free fatty acid (% oleic) Phosphorus kg P/g) Peroxide (meq Oz/kg) Absorbance

420 nm 453 nm

Untreated

1.2 13.8 4.5 0.215 0.266

Treated Cellulase/pectinase

Multiactivity complex

1.4 16.8 4.3

1.3 14.3 4.1

0.280 0.309

0.266 0.290

Enzymatic treatment of sunflower kernels before oil extraction

543

Table 8. Composition and characteristics of the cake for untreated and enzymatically treated samples [Untreated]

Treated Cellulase/pectinase

Multiactivity complex

Composition Residual oil (%, total oil) NDF” (g/l00 g dry defatted meal) ADFb (g/100 g dry defatted meal) Ash (g/100 g dry defatted meal) Reducing sugars (g/l00 g dry defatted meal) Total sugars (g/100 g dry defatted meal)

‘36.03 16.21 9.82 6.29 1.03 5.18

22.92 14.70 7.93 6.81 1.87 6.64

24.21 14.03 7.75 6.72 2.01 6.21

Characteristics ADC’ (%) Available lysine (mg/16 mg N)

80.36 3.25

84.32 3.28

83.95 3.19

“Neutral detergent fiber. bAcid detergent fiber. ‘Apparent digestibility coefficient.

pressing from enzymatically treated flaked seeds. In both treated and untreated samples, peroxide content was under the maximum allowed value of 10. The colour of crude oils from treated kernels (measured as absorbance of a 1:l oil/hexane solution with hexane as blank at 420 and 453 nm) was slightly darker than that from untreated kernels. Meal The cakes from enzymatically treated kernels contained ll-13% less total oil than untreated samples (Table 8). For treated samples, the reduction in neutral detergent fiber content in the defatted meal (1.5-2.2 g/100 g defatted meal below that of untreated meals) represents a 9.3-13.6% reduction. This value is lower than that reported by Sosulski & Sosulski (1990a) for canola treated with multiactivity enzymes and employing the optimal enzyme/seed ratio and could be attributed to the use of a smaller particle size (flaked canola seeds), which facilitates the accessibility of the enzyme to the substrate. Both total and reducing sugars were slightly increased as a result of the degradative action on the cell walls. With meal quality being determined by factors as palatability, digestibility, nutritional balance of amino acids and antinutritional or toxic factors, in vitro digestibility and available lysine (the most deficient essential amino acid and easily affected by processing conditions) were chosen as indicative of the nutritive value. The ADC of enzymatically treated samples was 4% higher than that for untreated samples but available lysine levels were not significantly affected by the treatment. The reduction in fiber content of the meal facilitates the accessibility of protein to digestive enzymes, improving the meal digestibility. During the continuous pressing process, an increase in the available lysine and protein digestibility would be expected, caused by

the fiber content reduction and also the reduction in exposure time and temperature for the cake (Sosulski & Sosulski, 1990a). Solvent extractability

of the cakes

In industry, after pressing to l&20% residual oil content in the cake, high oil-content seeds are extracted with hexane. In order to evaluate the effect of the enzymatic treatment in this second stage, the pressed kernels were submitted to extraction in Soxhlet with hexane. Figure 3 presents the extraction kinetics, measuring the oil extraction yield as a percentage of the oil contained in the cakes (22.7 g/100 g pressed kernels for untreated kernels, 14.44 g/100 g for those treated with the mixture of cellulase and pectinase and 15.25 g/100 g for those treated with the multiactivity complex). The extractability of cakes from untreated kernels and from kernels treated with either the mixture Celluclast/Pectinex (2: 1)

Fig. 3. Hexane extractability of the residual sunflower cakes after batch pressing.

544

H. Dominguez, J. Sineiro, M. J. NdrTez.J. M. Lema OIL EXTRACTION YIELD (g/l00 @kornek)

52

59

60

61

62

62

01234SB

HYDROLYSISTIME (h)

55c

of different temperature programmes on the solvent oil extractability of the enzymatically treated whole kernels.

Fig. 4. Effect

or the multiactivity complex MultifectTM was compared. Significant improvement in oil extraction was observed after 4 to 6 h extraction (at which time the maximum difference between samples was 3%) and a maximum difference of 10% more total residual oil could be extracted from treated than untreated samples. The mixture of cellulase and pectinase was observed to be slightly more efficient in enhancing oil extractability than the multiactivity complex in both stages (pressing and solvents). The beneficial effect of the mixture of these activities was nevertheless observed in the selection studies with different commercial enzymes using whole kernels and kernels cut in half transversally (Dominguez et af., 1991). Also, Lanzani et al. (1975) observed this favourable effect for proteases and cellulases and their mixtures. However, contradictory effects were reported when using only one of these activities. Although Badr & Sitohy (1992) found cellulases to be more efficient with ground kernels, pectinases were revealed as more favourable in the previously cited works. Temperature programme Assuming that each enzyme, a cellulase and a pectinase, of the mixture has a different optimum temperature (40 and 55”C, respectively), this variable should be changed along the enzymatic treatment in order to progressively favour both activities. Also, the cellulolytic activity (measured as the production of reducing sugars on filter paper) was observed to be higher when an increasing temperature programme was used. A sequence towards higher values of temperature appears to be advantageous as no inactivation occurs during

short term hydrolysis, and activity can be favoured at higher temperatures, reducing the deactivating effect during the first periods of hydrolysis. For these optimal operational conditions, the effect of a sequence of increasing temperatures was studied to improve the enzymatic treatment performance. Three sequences of temperature were accomplished for the mixture Celluclast/Pectinex (2:l) at9 g enzyme/ 100 g kernel. In Procedure A, the enzyme treatment was carried out at 50°C for 6 h. In Procedure B, three periods of 2 h each were performed: the temperature for the first 2 h was 40°C (the optimal range for pectinase activity is 40-45”(Z), for the second period it was 50°C and for the final period it was 55°C. With Procedure C, the same temperatures (40, 50 and 55°C) were used during 1, 3 and 2 h respectively. Dried and ground samples were extracted in Soxhlet for 3 h at 60 drops/ min. Figure 4 shows the effect of temperature programme on the oil extractability with hexane of the enzymatitally treated samples. N.T. corresponds to untreated samples. With any of the sequences of increasing temperatures, enzyme stability was probably improved as high temperatures are only used in the last stages, avoiding early enzyme deactivation. Of Procedures B and C, the latter appears to be the more efficient in enhancing sunflower oil extractability.

CONCLUSIONS The application of the response surface methodology to the enzymatic treatment of sunflower kernels provided empirical correlations for the variables moisture, enzyme/kernel ratio and treatment time, indicating their influence on the efficiency of the treatment. The importance of the moisture content during treatment was highlighted, with an opposite effect in the meal apparent digestibility and in the enzyme efficiency (measured as pressing yield and fiber content reduction). Since the most significant effects on the oil extractability and protein digestibility were in relation to the enzyme/kernel ratio and length of treatment time, the influence of these variables was evaluated in a second experimental design. Best results correspond with 20% moisture and values near 3 g enzyme/l00 g kernels and 5 h of treatment time. The use of an enzymatic treatment for high oilcontent seeds not only enhanced the oil extractability in the pressing stage, but also made the residual oil in the cake more easily extractable by solvents, achieving increases of up to 10%. More investigation is required to incorporate this enzymatic treatment in the real industrial operation by means of either reducing the moisture content to avoid additional drying costs or reducing the enzyme/kernel ratio to diminish the expenses that would represent the incorporation of the treatment. It does not seem

Enzymatic treatment of sunflower kernels before oil extraction possible to minimize both aspects at the same time, since both influence the enzyme efficiency.

ACKNOWLEDGEMENTS This work was financed by the Spanish Commission of Science and Technology (CICYT) (Project BI092-0568)

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Hsu, H. W., Vavak, D., Satterlee, L. D. & Miller, G. A. (1977). A multienzyme technique for estimating protein digestibility. J. Food Sci., 42, 1269-73. James, N. A. JL Ryley, J. (1986). The rapid determination of chemically reactive lysine in the presence of carbohydrates by modified trinitrobenzensulphonic acid procedure. J. Sci. Food Agric., 37, 1516. Lanzani, A., Petrini, M. C., Cozzoli, O., Gallavresi, P., Carola, C. & Jacini, G. (1975). On the use of enzymes for vegetable-oil extraction. A preliminary report. La Riv. Ital. delle Sostanze Grasse, 52, 226-9.

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(Received

27 May 1994; accepted

1 May 1995)