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The effects of twin-screw extrusion cooking on cereal enzymes
Journal
0/ Cereal Science 5 (1987) 73-82
The Effects of Twin-screw Extrusion Cooking on Cereal Enzymes* BARBARA FRETZDORFF and KURT SEILERt
Federal Research Centre afGrain and Potato Processing, P.O. Box 23, D-4930 Detmold, Federal Republic of Germany Received 4 December 1985 and in revised/orm 11 July 1986 The effects of extrusion cooking on the residual activities of six enzymes in wheat, rye and oats and several milling products were investigated. In general,enzyme inactivation increased as extrusion temperature increased. ex-Amylase activity in oats, however, was higher in some extrusion products than in the original grain. The thermostable enzymes (lipolytic acyl hydrolase, a-amylase, and peroxidase) differed consistently from the thermolabile enzymes (ex-benzoyl-L-arginine·p-nitroanilidehydrolase, catalase and lipoxygenase). Wheat bran, broken and unbroken oat kernels were extruded at two moisture levels (18 and 30%). Whereas lipolytic acyl hydrolase activity was lower in material extruded at 30 %moisture, residual a-amylase activities were higher at this moisture level. Peroxidase activity in oats, unlike that in wheat bran, was inactivated to a greater extent at the 30 % than at the 18 %moisture level.
Introduction In addition to the effect of technology of production in extrusion cooking, modifications of the constituents during extrusion are important for the quality of the food products. A recently published review by Bjorck and Asp! on the nutritional value of extruded products covers enzyme inhibitor denaturation, lysine losses, texturisation, lipids, carbohydrates, dietary fibre and vitamins. Enzymes, especially lipase, lipoxygenase and peroxidase, which might survive food processing, are partly responsible for the development of off-flavours during food storage. Thermolabile enzymes, such as catalase and BAPAase, are determined to indicate heat treatment of grains; e.g. the extent to which heat damage during drying might affect baking quality 2. In addition to characterising cereal enzymes according to their heat stability in buffers, thermal inactivation has been investigated in dough systems, bread and pastas to evaluate the influence of residual enzyme activities on the end products, e.g. bread staling. These studies concentrated on ex-amylase in dough and bread 3 and pasta4 ; some concerned the lipolytic activity in cookies· or peroxidase in Abbreviations used: BAPAase = (X·benzoyl·L·arginine-p-nitroanilide hydrolase; LAH = lipolytic acyl hydrolase; ds = dry substance. * Contribution No. 5330 from Federal Research Centre of Grain and Potato Processing, D-4930 Detmold, Federal Republic of Germany. t To whom correspondence should be addressed. 0733-5210/87/010073+ 10 $03.00/0
© 1987 Academic Press Inc. (London) Limited
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B. FRETZDORFF AND K. SEILER
pasta6 • Little information is available on the influence of extrusion cooking on enzyme activities. Lorenz et aU described the inactivation of urease by extrusion cooking at temperatures of 121-149 DC. Gardner et at. B stated that extrusion cooking of corn grits resulted in a complete inactivation of peroxidase. Linko et at. 9 found residual enzyme activities of a-amylase in cereal products after extrusion cooking under relatively mild processing conditions. It is well established that several factors, including moisture content of heat-treated raw materials, play an important role in enzymatic inactivation. The combination of factors which govern enzyme inactivation has been the subject of several, in part controversial, studies. Generally, inactivation increases as moisture content of the material increases 1o ,11. Rothe 5 stated, however, that the water content during heat treatment was inconsequential at temperatures above 100°C. More recent extrusion investigations by Linko et at. 9 showed that a-amylase was less damaged as moisture content of the raw material increased. Similarly, Thomann and Schoene12 demonstrated, with model wheat drying experiments, that an increase in water content reduced the heat inactivation of a-amylase and peroxidase. The objectives of this investigation were (1) to investigate the influence of two extrusion cooking parameters (temperature and moisture) on six enzymes, which differ in stability and in significance for processing in wheat, rye and oats, and (2), to compare and evaluate the stability of the enzymes in the grains in their native environment at a relatively low moisture content. Experimental Materials Grain samples, grown in the Federal Republic of Germany, the Netherlands and Sweden, harvested in 1982 and 1983 were used. German spring wheat - cv. Schirokko, 1983 (ash 1'85% ; protein 13,6%, falling number 336 s, sedimentation value 44 units)* - and German rye, Type R47, 1983 (ash 1,88%, protein 11·1 %, maltose number 2'6% ,fallingnumber291 s, 880 amylogram units at max. 72 0C)* were ground in a hammer mill, provided with a screen having 4 mm round holes. Untreated wheat bran was included in the experiments (ash 6,42%, protein 14,87%, fat 5,07%, starch 13·6 %)*. In addition, soft wheat grits from roller milling a blend of selected varieties (Okapi, 1983: 60% ; Goetz, 1983: 13% ; Kanzler, 1983: 12% ; Schirokko, 1982: 15%)were used (ash 0·58% , protein 10,7%, wet gluten 24,8%, maltose number 1'6%)*. The wheat grits were ground to a homogenous wheat flour which was processed in the same manner as the grits (ash 0·47%, protein 10'9%, wet gluten 26'4%, falling number 348 s, maltose number 1'2%)*. Oats, grown and harvested in Sweden 1982 (ash 2'20%, protein 15'5%, fat 6'24%)* and in the Netherlands in 1982 (ash 2,02%, protein 14·3 %, fat 5·88%)*, were used untreated and dehulled. The Swedish oats were milled to give broken kernels.
Extrusion technology The extruder used was a co-rotating, intermeshing twin-screw machine type Continua 58 (Werner & Pfleiderer Corporation). The actual screw diameter (D) of the Continua extruder is 58 mm, the length of cylinder, consisting offive barrel segments, in total equals 20 x D. The maximum feeding rate is 100 kg/h raw material. Screw elements consist of kneading blades as well as intermeshing shear elements (Figs. I and 2). ... Proximate analysis: ash content, protein content, maltose number, fat content, all on a dry substance basis, falling number, sedimentation value, amylogram data, wet gluten 13 , and starch content on a dry solids basis14 •
EXTRUSION COOKING AND CEREAL ENZYMES
75
FIGURE 1. Twin screw of the extruder with kneading elements, producing mild processing conditions.
FIGURE 2. Twin screw of the extruder with shear elements, producing intensive extrusion conditions. Extrusion parameters for processing the starchy raw materials; pressure, temperature and moisture content, are given in Table I.
Enzyme activity assays The 'Phadebas-Test'lii was used to determine a-amylase activity; amounts of buffer and reaction times were adapted to the different activity levels. BAPAase activity was determined according to Breyer and Hertel 16 , optimal enzyme assay conditions for each cereal were investigated in preliminary experiments. An artificial substrate, p-nitrophenylpalmitate, was used to measure lipolytic acyl hydrolase according to Galliard 17 after optimising the assay conditions for each cereal. Lipoxygenase activity was determined using linoleic acid as substrate and an oxygen electrode according to Grossman and Zakut18 and Fretzdorffl 9 • The peroxidase assay was published previously20. Catalase activity was measured with an Orion oxygen electrode; the increase in oxygen concentration caused by the reaction of catalase in the cereal extract (phosphate buffer pH 7'5) on hydrogen peroxidase was recorded.
Results and Discussion
Extrusion conditions (Table I)
Processing wheat and rye wholemeal required both low mass temperatures (80-100 0c) and low screw speeds. Increasing the temperatures to 170°C required higher speeds, a different screw geometry, and increasing feed rate of raw material.
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B. FRETZDORFF AND K. SEILER
TABLE 1. Extrusion conditions
Sample
Mass temperature
eq
Total Mass feed Mass Die Screw moisture pressure dia. Processing rate turn content (mm) elements (speed/min.) (bar) (g/min) (%)
Wheat wholemeal
80 90 98 173
150 150 150 250
42 42 63 95
8 8 7 5
Mild Mild Mild Intensive
770 775 703 1100
24 27 23 18
Rye wholemeal
81 90 99 172
150 150 150 250
46 53 68 85
8 8 7 5
Mild Mild Mild Intensive
772 770 720 1100
29 27 25 18
Soft wheat grits
44 50 60 80 90 98
100 125 150 150 150 150
28 28 28 44 56 87
8 8 8 8 8 7
Mild Mild Mild Mild Mild Mild
500 625 750 727 760 710
35 35 35 28 26 24
Soft wheat flour
40 50 60
100 125 150
25 32 33
8 8 8
Mild Mild Mild
535 625 750
39 35 35
Untreated wheat bran
80 90 98
70/100 100/100 100/100
53/10 42/7 42/7
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Oat kernels (Dutch, 1983)
80 90
98
70/100 90/100 100/100
87/63 77/47 70/46
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Oat kernels (Swedish, 1982)
80 90 98
80/100 100/100 100/100
88/18 77/14 63/11
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Broken oat kernels (Swedish, 1982)
80 90 98
70/100 70/100 100/100
95/31 105/25 74/14
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Extrusion conditions of processed soft wheat grits and soft wheat flour demonstrate the typical characteristics of extrusion cooking of starchy foodstuffs. Increasing mass temperatures, together with moderate screw speed, considerably increased the mass pressures within the' gap' of the barrel, even under mild screw geometries including low shear and modest kneading forces. Decreasing mass pressure was caused by increasing screw speed, which increased rate of energy dissipation and output of extruder products. Wheat bran, untreated oat kernels and broken kernels were extruded at two moisture levels (18 and 30%). The pressure data reflect the fact that extrusion processing of whole and broken oat kernels with an unchanged screw geometry decreased the pressure in the
EXTRUSION COOKING AND CEREAL ENZYMES
77
TABLE II. Enzyme activities a of cereal products used for the extrusion cooking experiments
Wheat bran Wheat wholemeal Wheat grits Rye wholemeal Oat kernels (Dutch, 1983) Oat kernels (Swedish, 1982) Broken oat kernels (Swedish, 1982)
Wheat bran Wheat wholemeal Wheat grits Rye wholemeal Oat kernels (Dutch, 1983) Oat kernels (Swedish, 1982) Broken oat kernels (Swedish, 1982) a
LAHb (A.Am/min/g)
ex-Amylase (A o23 103 /min/g)
Peroxidase (A.A 44n /min/g)
0·87 0·32 Traces 1·78 5·84
60'3 22·5 39·5 22·1 5·9
1177-3 1166·6 681·0 414·5 471·1
5·19
10·1
430·5
4'03
9·0
244·7
BAPAase (A.A 400 /min/g)
Catalase (A.ppmOdmin/g)
Lipoxygenase (A. %p02/min/ g)
2-61 1'22 1·37 2·23 2-88
104·2 17·2 8·0 79·2 35·0
2757-8 1582·} 522·0 3182·2 Traces
2'51
•
2-35
16'1 8·1
Activities calculated on a dry solids basis.
bLAH = Lipolytic acyl hydrolase.
last section of the barrel with a simultaneous increase of the twin screw speed and a rise in mass temperatures. This characteristic behaviour of extrusion cooking of oats is due to the high lipid content, which is seven to eight times higher in oats than in wheat and rye. The effect of lubrication within the barrel of the extruder is the main reason: for this decrease in pressure.
Effects of extrusion cooking on enzyme activities Enzyme activities of the raw materials given in Table II represent the 100% basis of the inactivation studies. Differences in the enzyme activities are due to the cereals (wheat, rye, oats), to the wheat milling fractions (bran, wholemeal, grits), and to variety, year of harvest, and processing (oats).
Wheat wholemeal (Table III). Wheat wholemeal was extruded at four temperatures. The residual percentage activities of the six enzymes are listed in Table III. A large difference
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B. FRETZDORFF AND K. SEILER
TABLE III. Residual enzyme activities in wheat wholemeal after extrusion cooking at various temperatures (expressed as % of original activity)a Temperature eC) Enzyme
80
90
98
173
LAHb ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase
75·3 51·7 41·5 5·1 1·0
59·9 39·8 15·2 3·2 1·7 0·6
60·2 8·0 0·9 0 0·7 0
47·7 0 0
0·9
See Table I for moisture contents of samples. bLAH = Lypolytic acyl hydrolase.
a
TABLE IV. Residual enzyme activities in the wheat bran after extrusion cooking at various temperatures and two moisture levels (expressed as % of original activity) Temperature (0C) 80 Moisture (%): LAW' ex-Amylase Peroxidase BAPAase Catalase a
90
98
18
30
18
30
18
30
63·2 10·5 4·7 0·5 0·19
60·9 27·7 16·0 0 14·1
57·4 0 0·17 0 0·08
55·1 0 0·73 0 1·3
51·7 0 0·01 0 0·04
42·5 0 0-02 0 6·23
LAH = Lipolytic acyl hydrolase.
was found between the thermostable (lipolytic acyl hydrolase, a-amylase, peroxidase) and the thermolabile enzymes (BAPAase, catalase, lipoxygenase). At least 95% of the activities of thermolabile enzymes were eliminated by processing at 80°C. The activities of the thermostable enzymes decreased with increasing temperature to a smaller extent. Peroxidase was more thermolabile than a-amylase in the 80 and 100°C range. Only lipolytic acyl hydrolase activity partly (c. 50%) survived extrusion cooking up to 170°C mass temperature. Wheat bran (Table IV). The enzymes in the bran were more inactivated than in wheat wholemeal, except for catalase at the high moisture level (Table IV). Lipolytic acyl hydrolase in the 30% -moisture bran was more affected than in the 18 % -moisture bran. This confirms findings of Williams et al.n, who stated that the lower the internal moisture, the higher the temperature required to inactivate lipase during extrusion cooking. In contrast, a-amylase, peroxidase, and catalase were destroyed less at the high
79
EXTRUSION COOKING AND CEREAL ENZYMES
TABLE V. Residual enzyme activities in wheat grits and wheat flour after extrusion cooking at various temperatures (expressed as % of original activity)& Mass temperature (oq 40 Wheat grits: ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase Wheat flour: a-Amylase Peroxidase BAPAase Catalase Lipoxygenase
44
50
60
80
90
98
20·8 92·2 21·1 2·0
24·6 91·5 8·9 1·6 4·0
20·8 92·8 4·2 1·4 3·2
26·1 12·2 0 0 0
\3·2 5-6 0 0 0
0 0·07 0 0 0
24·7 89·0 3·9 1·0 5·2
87·4 traces 0·6 2·5
H
20'7 91·9 30·9 2·4
7-3
21-6
&See Table I for moisture contents of samples.
moisture level. These findings, concerning a-amylase, are in agreement with results of Linko et al. 9 In wheat bran at 80°C a-amylase was more thermostable than peroxidase, but at 90 °C and 98°C peroxidase was still detectable while a-amylase showed zero activity. This discrepancy is explained, in part at least, by the more sensitive peroxidase activity assay (1/1000 of the original peroxidase activity, but only 1/50 of the original a-amylase activity can be detected at the enzymatic levels given in Table II). Preliminary experiments established that peroxidase activity in two bran samples was well detected at 30% moisture at 130 DC (0,1 % residual activity) and 140°C (0'06 % residual activity), in contrast to samples processed at 18 % moisture level (zero activity in both cases). Wheat grits and flour (Table V). Enzyme inactivation in grits and flour was similar. At the low temperatures (40-60 DC) catalase was more sensitive than lipoxygenase and BAPAase. Loss ofBAPAase activity between 40 and 50°C was 10% in grits and about 13% in flour, probably due to the smaller particle sizes in the flour. The thermostable enzymes were influenced in a similar way: peroxidase was almost unaffected and a-amylase was about 80% inactivated. While peroxidase was much more stable than a-amylase at temperatures between 40 and 60 DC, at 80 and 90°C a-amylase was less damaged. Rye wholemeal (Table VI). The thermosensitive enzymes in rye wholemeal were affected in a similar manner to those in wheat wholemeal. In rye wholemeal, a-amylase was more stable than peroxidase. Peroxidase, usually considered thermostable, showed a rather thermosensitive pattern. The lipolytic acyl hydrolase in rye wholemeal was very heat-resistant.
80
B. FRETZDORFF AND K. SEILER
TABLE VI. Residual enzyme activities in rye wholemeal after extrusion cooking at various temperatures (expressed as % of original activity)a Mass temperature COC)
LARa ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase
81
90
99
172
54·7 88·0 24-9 7·0 4·5 1·2
59·4 62·1 1-2 2·1 0·6 0
59·5 11·5 0·4 0 0·3 0
28·2 0 0
See Table I for moisture contents of samples. bLAH = Lipolytic acyl hydrolase.
a
TABLE VII. Residual enzyme activities in oat kernels and broken oat kernels after extrusion cooking at various temperatures and two moisture levels (expressed as % of original activity) Temperature (0C) 80
Moisture (%):
90
98
18
30
18
30
18
30
Oat kernels (Dutch, 1983): LARa a-Amylase Peroxidase BAPAase Catalase
81·4 90-2 71·4 5·7 H
78·8 105·6 63·2 11·0 5·1
76·4 106·7 60·2 3·2
H
74-4 126-0 48·7 5·3 3-8
63-6 53·7 28·4 2-3 1·3
63-6 73·1 36'5 4·0 2·2
Oat kernels (Swedish, 1982): LARa a-Amylase Peroxidase BAPAase Catalase
82·7 84·3 53·2 6·7 3·7
65·5 168·3 35·2 6·5 1·5
67-8 54·1 37·4 4·5 1·2
43·6 78-2 21·5 4·8 1·1
69·2 41·5 23-8 5·3 0·9
26·6 27·3 10·4 2·1 0·8
Broken oat kernels (Swedish, 1982): LAHa 100·3 a-Amylase 102·5 Peroxidase 104·1 BAPAase 7·7 Catalase 19·2
86·1 137·2 76·8 15·2 7·7
99·3 73'6 99·3 8·0 17·0
81-6 116·2 67·1 7·9 3·0
75'2 38·4 39·9 2·3 2·5
58·3 76·8 30·4 1·3 1·2
• LAH = Lipolytic acyl hydrolase.
Whole oat kernels and broken oat kernels (Table VII). The enzymes in broken oat kernels were less inactivated than in wheat and rye wholemeal. The thermostable enzymes in broken oat kernels were unaffected at 80°C and 18% moisture. It remains to be determined, whether the apparently greater stability of the oat enzymes was due to milder extrusion conditions (e.g. shorter residence times).
EXTRUSION COOKING AND CEREAL ENZYMES
81
BAPAase and catalase in oats were thermolabile, as in the other cereals. Less than 15% residual BAPAase activity and less than 20% catalase activity in broken oat kernels or 5% in oat kernels were found in the extrusion products, but up to 98°C at both moisture levels these activities were detected. In the whole kernels, the differences in these activities at the various temperatures and moisture levels were small and no consistent effect of moisture could be established. Catalase in broken kernels was more damaged at the higher moisture level. Peroxidase was more stable in oats than in wheat and rye. With one exception, peroxidase was inactivated more at 30% than at 18% moisture. In our investigation, a-amylase was the only enzyme which showed higher activities after extrusion cooking. At the 30% moisture level, activity was, in general, higher than at the 18 % moisture level. It is unlikely that activation or de novo synthesis takes place during or after extrusion cooking especially in broken kernels. One might speculate, therefore, that a thermosensitive inhibitor might have been destroyed to a higher degree than a-amylase itself. If that assumption is correct, this inhibitor might have been more damaged at 30 % moisture than at 18 % moisture. If that assumption is incorrect, and an activation has taken place, a-amylase was more activated and/or less destroyed at the 30% moisture level. While both Swedish oats (broken and unbroken kernels) showed the highest a-amylase activities after 80°C extrusion cooking, the a-amylase activities of the Dutch oats were highest after 90 °C cooking. As in the other cereals, lipolytic acyl hydrolase was the most stable enzyme. Whereas in the broken and unbroken Swedish oats, lipolytic acyl hydrolase was inactivated more at the higher moisture level, moisture had no consistent effect in the Dutch oats. Conclusions
Generally, enzyme inactivation increased as extrusion temperature increased. The patterns ofinactivation of the thermostable and thermolabile enzymes differed. Similarly, inactivation patterns of the thermostable enzymes in the cereals differed. While lipolytic acyl hydrolase was inactivated more at the higher moisture, a-amylase activities were higher at the high moisture level. Further investigations on the thermostable enzymes in products extruded at 120-170°C and different moisture levels are needed to establish the effects of processing at high temperatures as used in industry. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Bjorck, J. and Asp, N. G. J. Food Eng. 2 (1983) 248-308. Doerfer, J. and Berthold, H. Baecker Konditor 28 (1980) 313-315. Walden, C. C. Cereal Chern. 32 (1955) 421--431. Kruger, J. E. and Matsuo, R. R. Cereal Chern. 59 (1982) 26-31. Rothe, M. Ernaehrungsforscll. 4 (1959) 67-71. Laignelet, B. GetI'. Melli Brot 30 (1976) 277-280. Lorenz, K., Jansen, G. R. and Harper, J. Cereal Foods World 25 (1980) 161-162,171-172. Gardner, H. W., Inglett, G. E. and Anderson, R. A. Cereal Chern. 46 (1969) 626-634. Linko, P., Colonna, P. and Mercier, C. in 'Advances in Cereal Science and Technology', Vol. 4 (Y. Pomeranz, ed.), AACC, St Paul, MN (1982) pp 204-207.
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B. FRETZDORFF AND K. SEILER
10. Svensson S. in 'Physical, Chemical, and Biological Changes in Food Caused by Thennal Processing' (T. H0YeOl and O. Kraete, eds.), Applied Science Publishers, London (1977) pp 202-217. 11. Williams, M. A., Hom, R. E. and Rugala, R. P. Food Eng. Int. (1977) 57, 59, 61, 62. 12. Thomann, R. and Schoene, K. Getreidewirtschaft (1982) 59-62. 13. Standard-Methoden fUr Getreide, Mehl und Brat, Moritz Schaefer, Detmold (1978). 14. Seiler, K., Rabe, E. and Goedecke, U. Zucker- u. Suesswaremvirtschaft 33 (1980) 5, 156-162, 175. IS. Drews, E., Fretzdorff, B. and Ocker, H.-D. Getr. Mehl Brot 30 (1976) 320-323. 16. Breyer, D. and Hertel, W. Getr. Mehl Brat 29 (1975) 185-188. 17. GalIiard, T. Biochem. J. 121 (1971) 379-390. 18. Grossmann, S. and Zakut, R. Methods Biochem. Anal. 25 (1978) 303-309. 19. Fretzdorff, B. Bericht 35. Tagung Getreidechemie, Granum-, Detroold (1984) pp 179-181. 20. Fretzdorff, B. Z. Lebensmittelunters u. Forsch. 170 (1980) 187-193.
0/ Cereal Science 5 (1987) 73-82
The Effects of Twin-screw Extrusion Cooking on Cereal Enzymes* BARBARA FRETZDORFF and KURT SEILERt
Federal Research Centre afGrain and Potato Processing, P.O. Box 23, D-4930 Detmold, Federal Republic of Germany Received 4 December 1985 and in revised/orm 11 July 1986 The effects of extrusion cooking on the residual activities of six enzymes in wheat, rye and oats and several milling products were investigated. In general,enzyme inactivation increased as extrusion temperature increased. ex-Amylase activity in oats, however, was higher in some extrusion products than in the original grain. The thermostable enzymes (lipolytic acyl hydrolase, a-amylase, and peroxidase) differed consistently from the thermolabile enzymes (ex-benzoyl-L-arginine·p-nitroanilidehydrolase, catalase and lipoxygenase). Wheat bran, broken and unbroken oat kernels were extruded at two moisture levels (18 and 30%). Whereas lipolytic acyl hydrolase activity was lower in material extruded at 30 %moisture, residual a-amylase activities were higher at this moisture level. Peroxidase activity in oats, unlike that in wheat bran, was inactivated to a greater extent at the 30 % than at the 18 %moisture level.
Introduction In addition to the effect of technology of production in extrusion cooking, modifications of the constituents during extrusion are important for the quality of the food products. A recently published review by Bjorck and Asp! on the nutritional value of extruded products covers enzyme inhibitor denaturation, lysine losses, texturisation, lipids, carbohydrates, dietary fibre and vitamins. Enzymes, especially lipase, lipoxygenase and peroxidase, which might survive food processing, are partly responsible for the development of off-flavours during food storage. Thermolabile enzymes, such as catalase and BAPAase, are determined to indicate heat treatment of grains; e.g. the extent to which heat damage during drying might affect baking quality 2. In addition to characterising cereal enzymes according to their heat stability in buffers, thermal inactivation has been investigated in dough systems, bread and pastas to evaluate the influence of residual enzyme activities on the end products, e.g. bread staling. These studies concentrated on ex-amylase in dough and bread 3 and pasta4 ; some concerned the lipolytic activity in cookies· or peroxidase in Abbreviations used: BAPAase = (X·benzoyl·L·arginine-p-nitroanilide hydrolase; LAH = lipolytic acyl hydrolase; ds = dry substance. * Contribution No. 5330 from Federal Research Centre of Grain and Potato Processing, D-4930 Detmold, Federal Republic of Germany. t To whom correspondence should be addressed. 0733-5210/87/010073+ 10 $03.00/0
© 1987 Academic Press Inc. (London) Limited
74
B. FRETZDORFF AND K. SEILER
pasta6 • Little information is available on the influence of extrusion cooking on enzyme activities. Lorenz et aU described the inactivation of urease by extrusion cooking at temperatures of 121-149 DC. Gardner et at. B stated that extrusion cooking of corn grits resulted in a complete inactivation of peroxidase. Linko et at. 9 found residual enzyme activities of a-amylase in cereal products after extrusion cooking under relatively mild processing conditions. It is well established that several factors, including moisture content of heat-treated raw materials, play an important role in enzymatic inactivation. The combination of factors which govern enzyme inactivation has been the subject of several, in part controversial, studies. Generally, inactivation increases as moisture content of the material increases 1o ,11. Rothe 5 stated, however, that the water content during heat treatment was inconsequential at temperatures above 100°C. More recent extrusion investigations by Linko et at. 9 showed that a-amylase was less damaged as moisture content of the raw material increased. Similarly, Thomann and Schoene12 demonstrated, with model wheat drying experiments, that an increase in water content reduced the heat inactivation of a-amylase and peroxidase. The objectives of this investigation were (1) to investigate the influence of two extrusion cooking parameters (temperature and moisture) on six enzymes, which differ in stability and in significance for processing in wheat, rye and oats, and (2), to compare and evaluate the stability of the enzymes in the grains in their native environment at a relatively low moisture content. Experimental Materials Grain samples, grown in the Federal Republic of Germany, the Netherlands and Sweden, harvested in 1982 and 1983 were used. German spring wheat - cv. Schirokko, 1983 (ash 1'85% ; protein 13,6%, falling number 336 s, sedimentation value 44 units)* - and German rye, Type R47, 1983 (ash 1,88%, protein 11·1 %, maltose number 2'6% ,fallingnumber291 s, 880 amylogram units at max. 72 0C)* were ground in a hammer mill, provided with a screen having 4 mm round holes. Untreated wheat bran was included in the experiments (ash 6,42%, protein 14,87%, fat 5,07%, starch 13·6 %)*. In addition, soft wheat grits from roller milling a blend of selected varieties (Okapi, 1983: 60% ; Goetz, 1983: 13% ; Kanzler, 1983: 12% ; Schirokko, 1982: 15%)were used (ash 0·58% , protein 10,7%, wet gluten 24,8%, maltose number 1'6%)*. The wheat grits were ground to a homogenous wheat flour which was processed in the same manner as the grits (ash 0·47%, protein 10'9%, wet gluten 26'4%, falling number 348 s, maltose number 1'2%)*. Oats, grown and harvested in Sweden 1982 (ash 2'20%, protein 15'5%, fat 6'24%)* and in the Netherlands in 1982 (ash 2,02%, protein 14·3 %, fat 5·88%)*, were used untreated and dehulled. The Swedish oats were milled to give broken kernels.
Extrusion technology The extruder used was a co-rotating, intermeshing twin-screw machine type Continua 58 (Werner & Pfleiderer Corporation). The actual screw diameter (D) of the Continua extruder is 58 mm, the length of cylinder, consisting offive barrel segments, in total equals 20 x D. The maximum feeding rate is 100 kg/h raw material. Screw elements consist of kneading blades as well as intermeshing shear elements (Figs. I and 2). ... Proximate analysis: ash content, protein content, maltose number, fat content, all on a dry substance basis, falling number, sedimentation value, amylogram data, wet gluten 13 , and starch content on a dry solids basis14 •
EXTRUSION COOKING AND CEREAL ENZYMES
75
FIGURE 1. Twin screw of the extruder with kneading elements, producing mild processing conditions.
FIGURE 2. Twin screw of the extruder with shear elements, producing intensive extrusion conditions. Extrusion parameters for processing the starchy raw materials; pressure, temperature and moisture content, are given in Table I.
Enzyme activity assays The 'Phadebas-Test'lii was used to determine a-amylase activity; amounts of buffer and reaction times were adapted to the different activity levels. BAPAase activity was determined according to Breyer and Hertel 16 , optimal enzyme assay conditions for each cereal were investigated in preliminary experiments. An artificial substrate, p-nitrophenylpalmitate, was used to measure lipolytic acyl hydrolase according to Galliard 17 after optimising the assay conditions for each cereal. Lipoxygenase activity was determined using linoleic acid as substrate and an oxygen electrode according to Grossman and Zakut18 and Fretzdorffl 9 • The peroxidase assay was published previously20. Catalase activity was measured with an Orion oxygen electrode; the increase in oxygen concentration caused by the reaction of catalase in the cereal extract (phosphate buffer pH 7'5) on hydrogen peroxidase was recorded.
Results and Discussion
Extrusion conditions (Table I)
Processing wheat and rye wholemeal required both low mass temperatures (80-100 0c) and low screw speeds. Increasing the temperatures to 170°C required higher speeds, a different screw geometry, and increasing feed rate of raw material.
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B. FRETZDORFF AND K. SEILER
TABLE 1. Extrusion conditions
Sample
Mass temperature
eq
Total Mass feed Mass Die Screw moisture pressure dia. Processing rate turn content (mm) elements (speed/min.) (bar) (g/min) (%)
Wheat wholemeal
80 90 98 173
150 150 150 250
42 42 63 95
8 8 7 5
Mild Mild Mild Intensive
770 775 703 1100
24 27 23 18
Rye wholemeal
81 90 99 172
150 150 150 250
46 53 68 85
8 8 7 5
Mild Mild Mild Intensive
772 770 720 1100
29 27 25 18
Soft wheat grits
44 50 60 80 90 98
100 125 150 150 150 150
28 28 28 44 56 87
8 8 8 8 8 7
Mild Mild Mild Mild Mild Mild
500 625 750 727 760 710
35 35 35 28 26 24
Soft wheat flour
40 50 60
100 125 150
25 32 33
8 8 8
Mild Mild Mild
535 625 750
39 35 35
Untreated wheat bran
80 90 98
70/100 100/100 100/100
53/10 42/7 42/7
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Oat kernels (Dutch, 1983)
80 90
98
70/100 90/100 100/100
87/63 77/47 70/46
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Oat kernels (Swedish, 1982)
80 90 98
80/100 100/100 100/100
88/18 77/14 63/11
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Broken oat kernels (Swedish, 1982)
80 90 98
70/100 70/100 100/100
95/31 105/25 74/14
8 8 8
Mild Mild Mild
700 700 700
18/30 18/30 18/30
Extrusion conditions of processed soft wheat grits and soft wheat flour demonstrate the typical characteristics of extrusion cooking of starchy foodstuffs. Increasing mass temperatures, together with moderate screw speed, considerably increased the mass pressures within the' gap' of the barrel, even under mild screw geometries including low shear and modest kneading forces. Decreasing mass pressure was caused by increasing screw speed, which increased rate of energy dissipation and output of extruder products. Wheat bran, untreated oat kernels and broken kernels were extruded at two moisture levels (18 and 30%). The pressure data reflect the fact that extrusion processing of whole and broken oat kernels with an unchanged screw geometry decreased the pressure in the
EXTRUSION COOKING AND CEREAL ENZYMES
77
TABLE II. Enzyme activities a of cereal products used for the extrusion cooking experiments
Wheat bran Wheat wholemeal Wheat grits Rye wholemeal Oat kernels (Dutch, 1983) Oat kernels (Swedish, 1982) Broken oat kernels (Swedish, 1982)
Wheat bran Wheat wholemeal Wheat grits Rye wholemeal Oat kernels (Dutch, 1983) Oat kernels (Swedish, 1982) Broken oat kernels (Swedish, 1982) a
LAHb (A.Am/min/g)
ex-Amylase (A o23 103 /min/g)
Peroxidase (A.A 44n /min/g)
0·87 0·32 Traces 1·78 5·84
60'3 22·5 39·5 22·1 5·9
1177-3 1166·6 681·0 414·5 471·1
5·19
10·1
430·5
4'03
9·0
244·7
BAPAase (A.A 400 /min/g)
Catalase (A.ppmOdmin/g)
Lipoxygenase (A. %p02/min/ g)
2-61 1'22 1·37 2·23 2-88
104·2 17·2 8·0 79·2 35·0
2757-8 1582·} 522·0 3182·2 Traces
2'51
•
2-35
16'1 8·1
Activities calculated on a dry solids basis.
bLAH = Lipolytic acyl hydrolase.
last section of the barrel with a simultaneous increase of the twin screw speed and a rise in mass temperatures. This characteristic behaviour of extrusion cooking of oats is due to the high lipid content, which is seven to eight times higher in oats than in wheat and rye. The effect of lubrication within the barrel of the extruder is the main reason: for this decrease in pressure.
Effects of extrusion cooking on enzyme activities Enzyme activities of the raw materials given in Table II represent the 100% basis of the inactivation studies. Differences in the enzyme activities are due to the cereals (wheat, rye, oats), to the wheat milling fractions (bran, wholemeal, grits), and to variety, year of harvest, and processing (oats).
Wheat wholemeal (Table III). Wheat wholemeal was extruded at four temperatures. The residual percentage activities of the six enzymes are listed in Table III. A large difference
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B. FRETZDORFF AND K. SEILER
TABLE III. Residual enzyme activities in wheat wholemeal after extrusion cooking at various temperatures (expressed as % of original activity)a Temperature eC) Enzyme
80
90
98
173
LAHb ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase
75·3 51·7 41·5 5·1 1·0
59·9 39·8 15·2 3·2 1·7 0·6
60·2 8·0 0·9 0 0·7 0
47·7 0 0
0·9
See Table I for moisture contents of samples. bLAH = Lypolytic acyl hydrolase.
a
TABLE IV. Residual enzyme activities in the wheat bran after extrusion cooking at various temperatures and two moisture levels (expressed as % of original activity) Temperature (0C) 80 Moisture (%): LAW' ex-Amylase Peroxidase BAPAase Catalase a
90
98
18
30
18
30
18
30
63·2 10·5 4·7 0·5 0·19
60·9 27·7 16·0 0 14·1
57·4 0 0·17 0 0·08
55·1 0 0·73 0 1·3
51·7 0 0·01 0 0·04
42·5 0 0-02 0 6·23
LAH = Lipolytic acyl hydrolase.
was found between the thermostable (lipolytic acyl hydrolase, a-amylase, peroxidase) and the thermolabile enzymes (BAPAase, catalase, lipoxygenase). At least 95% of the activities of thermolabile enzymes were eliminated by processing at 80°C. The activities of the thermostable enzymes decreased with increasing temperature to a smaller extent. Peroxidase was more thermolabile than a-amylase in the 80 and 100°C range. Only lipolytic acyl hydrolase activity partly (c. 50%) survived extrusion cooking up to 170°C mass temperature. Wheat bran (Table IV). The enzymes in the bran were more inactivated than in wheat wholemeal, except for catalase at the high moisture level (Table IV). Lipolytic acyl hydrolase in the 30% -moisture bran was more affected than in the 18 % -moisture bran. This confirms findings of Williams et al.n, who stated that the lower the internal moisture, the higher the temperature required to inactivate lipase during extrusion cooking. In contrast, a-amylase, peroxidase, and catalase were destroyed less at the high
79
EXTRUSION COOKING AND CEREAL ENZYMES
TABLE V. Residual enzyme activities in wheat grits and wheat flour after extrusion cooking at various temperatures (expressed as % of original activity)& Mass temperature (oq 40 Wheat grits: ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase Wheat flour: a-Amylase Peroxidase BAPAase Catalase Lipoxygenase
44
50
60
80
90
98
20·8 92·2 21·1 2·0
24·6 91·5 8·9 1·6 4·0
20·8 92·8 4·2 1·4 3·2
26·1 12·2 0 0 0
\3·2 5-6 0 0 0
0 0·07 0 0 0
24·7 89·0 3·9 1·0 5·2
87·4 traces 0·6 2·5
H
20'7 91·9 30·9 2·4
7-3
21-6
&See Table I for moisture contents of samples.
moisture level. These findings, concerning a-amylase, are in agreement with results of Linko et al. 9 In wheat bran at 80°C a-amylase was more thermostable than peroxidase, but at 90 °C and 98°C peroxidase was still detectable while a-amylase showed zero activity. This discrepancy is explained, in part at least, by the more sensitive peroxidase activity assay (1/1000 of the original peroxidase activity, but only 1/50 of the original a-amylase activity can be detected at the enzymatic levels given in Table II). Preliminary experiments established that peroxidase activity in two bran samples was well detected at 30% moisture at 130 DC (0,1 % residual activity) and 140°C (0'06 % residual activity), in contrast to samples processed at 18 % moisture level (zero activity in both cases). Wheat grits and flour (Table V). Enzyme inactivation in grits and flour was similar. At the low temperatures (40-60 DC) catalase was more sensitive than lipoxygenase and BAPAase. Loss ofBAPAase activity between 40 and 50°C was 10% in grits and about 13% in flour, probably due to the smaller particle sizes in the flour. The thermostable enzymes were influenced in a similar way: peroxidase was almost unaffected and a-amylase was about 80% inactivated. While peroxidase was much more stable than a-amylase at temperatures between 40 and 60 DC, at 80 and 90°C a-amylase was less damaged. Rye wholemeal (Table VI). The thermosensitive enzymes in rye wholemeal were affected in a similar manner to those in wheat wholemeal. In rye wholemeal, a-amylase was more stable than peroxidase. Peroxidase, usually considered thermostable, showed a rather thermosensitive pattern. The lipolytic acyl hydrolase in rye wholemeal was very heat-resistant.
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B. FRETZDORFF AND K. SEILER
TABLE VI. Residual enzyme activities in rye wholemeal after extrusion cooking at various temperatures (expressed as % of original activity)a Mass temperature COC)
LARa ex-Amylase Peroxidase BAPAase Catalase Lipoxygenase
81
90
99
172
54·7 88·0 24-9 7·0 4·5 1·2
59·4 62·1 1-2 2·1 0·6 0
59·5 11·5 0·4 0 0·3 0
28·2 0 0
See Table I for moisture contents of samples. bLAH = Lipolytic acyl hydrolase.
a
TABLE VII. Residual enzyme activities in oat kernels and broken oat kernels after extrusion cooking at various temperatures and two moisture levels (expressed as % of original activity) Temperature (0C) 80
Moisture (%):
90
98
18
30
18
30
18
30
Oat kernels (Dutch, 1983): LARa a-Amylase Peroxidase BAPAase Catalase
81·4 90-2 71·4 5·7 H
78·8 105·6 63·2 11·0 5·1
76·4 106·7 60·2 3·2
H
74-4 126-0 48·7 5·3 3-8
63-6 53·7 28·4 2-3 1·3
63-6 73·1 36'5 4·0 2·2
Oat kernels (Swedish, 1982): LARa a-Amylase Peroxidase BAPAase Catalase
82·7 84·3 53·2 6·7 3·7
65·5 168·3 35·2 6·5 1·5
67-8 54·1 37·4 4·5 1·2
43·6 78-2 21·5 4·8 1·1
69·2 41·5 23-8 5·3 0·9
26·6 27·3 10·4 2·1 0·8
Broken oat kernels (Swedish, 1982): LAHa 100·3 a-Amylase 102·5 Peroxidase 104·1 BAPAase 7·7 Catalase 19·2
86·1 137·2 76·8 15·2 7·7
99·3 73'6 99·3 8·0 17·0
81-6 116·2 67·1 7·9 3·0
75'2 38·4 39·9 2·3 2·5
58·3 76·8 30·4 1·3 1·2
• LAH = Lipolytic acyl hydrolase.
Whole oat kernels and broken oat kernels (Table VII). The enzymes in broken oat kernels were less inactivated than in wheat and rye wholemeal. The thermostable enzymes in broken oat kernels were unaffected at 80°C and 18% moisture. It remains to be determined, whether the apparently greater stability of the oat enzymes was due to milder extrusion conditions (e.g. shorter residence times).
EXTRUSION COOKING AND CEREAL ENZYMES
81
BAPAase and catalase in oats were thermolabile, as in the other cereals. Less than 15% residual BAPAase activity and less than 20% catalase activity in broken oat kernels or 5% in oat kernels were found in the extrusion products, but up to 98°C at both moisture levels these activities were detected. In the whole kernels, the differences in these activities at the various temperatures and moisture levels were small and no consistent effect of moisture could be established. Catalase in broken kernels was more damaged at the higher moisture level. Peroxidase was more stable in oats than in wheat and rye. With one exception, peroxidase was inactivated more at 30% than at 18% moisture. In our investigation, a-amylase was the only enzyme which showed higher activities after extrusion cooking. At the 30% moisture level, activity was, in general, higher than at the 18 % moisture level. It is unlikely that activation or de novo synthesis takes place during or after extrusion cooking especially in broken kernels. One might speculate, therefore, that a thermosensitive inhibitor might have been destroyed to a higher degree than a-amylase itself. If that assumption is correct, this inhibitor might have been more damaged at 30 % moisture than at 18 % moisture. If that assumption is incorrect, and an activation has taken place, a-amylase was more activated and/or less destroyed at the 30% moisture level. While both Swedish oats (broken and unbroken kernels) showed the highest a-amylase activities after 80°C extrusion cooking, the a-amylase activities of the Dutch oats were highest after 90 °C cooking. As in the other cereals, lipolytic acyl hydrolase was the most stable enzyme. Whereas in the broken and unbroken Swedish oats, lipolytic acyl hydrolase was inactivated more at the higher moisture level, moisture had no consistent effect in the Dutch oats. Conclusions
Generally, enzyme inactivation increased as extrusion temperature increased. The patterns ofinactivation of the thermostable and thermolabile enzymes differed. Similarly, inactivation patterns of the thermostable enzymes in the cereals differed. While lipolytic acyl hydrolase was inactivated more at the higher moisture, a-amylase activities were higher at the high moisture level. Further investigations on the thermostable enzymes in products extruded at 120-170°C and different moisture levels are needed to establish the effects of processing at high temperatures as used in industry. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Bjorck, J. and Asp, N. G. J. Food Eng. 2 (1983) 248-308. Doerfer, J. and Berthold, H. Baecker Konditor 28 (1980) 313-315. Walden, C. C. Cereal Chern. 32 (1955) 421--431. Kruger, J. E. and Matsuo, R. R. Cereal Chern. 59 (1982) 26-31. Rothe, M. Ernaehrungsforscll. 4 (1959) 67-71. Laignelet, B. GetI'. Melli Brot 30 (1976) 277-280. Lorenz, K., Jansen, G. R. and Harper, J. Cereal Foods World 25 (1980) 161-162,171-172. Gardner, H. W., Inglett, G. E. and Anderson, R. A. Cereal Chern. 46 (1969) 626-634. Linko, P., Colonna, P. and Mercier, C. in 'Advances in Cereal Science and Technology', Vol. 4 (Y. Pomeranz, ed.), AACC, St Paul, MN (1982) pp 204-207.
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B. FRETZDORFF AND K. SEILER
10. Svensson S. in 'Physical, Chemical, and Biological Changes in Food Caused by Thennal Processing' (T. H0YeOl and O. Kraete, eds.), Applied Science Publishers, London (1977) pp 202-217. 11. Williams, M. A., Hom, R. E. and Rugala, R. P. Food Eng. Int. (1977) 57, 59, 61, 62. 12. Thomann, R. and Schoene, K. Getreidewirtschaft (1982) 59-62. 13. Standard-Methoden fUr Getreide, Mehl und Brat, Moritz Schaefer, Detmold (1978). 14. Seiler, K., Rabe, E. and Goedecke, U. Zucker- u. Suesswaremvirtschaft 33 (1980) 5, 156-162, 175. IS. Drews, E., Fretzdorff, B. and Ocker, H.-D. Getr. Mehl Brot 30 (1976) 320-323. 16. Breyer, D. and Hertel, W. Getr. Mehl Brat 29 (1975) 185-188. 17. GalIiard, T. Biochem. J. 121 (1971) 379-390. 18. Grossmann, S. and Zakut, R. Methods Biochem. Anal. 25 (1978) 303-309. 19. Fretzdorff, B. Bericht 35. Tagung Getreidechemie, Granum-, Detroold (1984) pp 179-181. 20. Fretzdorff, B. Z. Lebensmittelunters u. Forsch. 170 (1980) 187-193.