Volume 7
Issue 2
Biotechnology
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Available Online: http://www.ejpau.media.pl/volume7/issue2/biotechnology/art-05.html
THE INFLUENCE OF ADDITIONAL ENZYMES TREATMENT OF CORN AND WHEAT STRCHES ON FILTRATION PROPERTIES OF HYDROLYSATES
Lucyna Słomińska, Danuta Wi¶niewska, Joanna Niedbach
The influence of additional enzymes: lysophospholipase, beta glucanase and pentosanase on corn and wheat starch were studied. Research was carried out with different starch concentration, enzyme dosages and time of enzyme action. The following analyses were made: dextrose equivalent, the iodine absorbance value, and colour measurement, viscosity and filtration rate. Simultaneous action of alpha amylase and lysophospholipase on corn starch influence on the increase of filtration rate of hydrolysates by 12% and on the decrease of their viscosity, colour indicator and iodine absorbance. Beta glucanase and pentosanase complex used together with lysophospholipase independently on way their introduction to starch increase 2.5 times of the filtration rate of wheat hydrolysates.
Key words:
wheat starch, corn starch, lysophospholipase, beta glucanase, pentosanase, filtration..
INTRODUCTION
Physico-chemical properties of corn and wheat starches create problems during starch hydrolysis namely the formation of precipitants, which are difficult to filter [4, 8, 13]. It is connected with the presence of amylose–lipids complexes and non-starch polysaccharide constituents mainly pentosans. Filtration speed of produced starch hydrolysates can be increased by the application of specific enzymes, such as lysophospholipase, beta-glucanase and pentosanase. They decreased viscosity of hydrolysates partly due to depolymerization of starch impurities:
The aim of research work was the estimation of the effect of the additional enzyme action introduction on improvement of technology of corn and wheat starch hydrolysates.
MATERIALS AND METHODS
Enzymes:
Bacillus licheniformis heat-stable a-amylase /Termamyl 120L/
(Novo Nordisk, Denmark). The enzyme activity was 120L KNU/g (KNU = Kilo Novo Units alpha amylase) – the amount of enzyme, which breaks down 5.26 g of starch per hour at Novo’s standard method for determination of alpha amylase.
Aspergillus niger lysophospholipase /Gammazym 154L/
(Gamma Chemie GmbH, Germany)
Humicola insolens beta-glucanase and pentosanase /SP 348/
Novo Nordisk, Denmark)
Substrate:
Corn starch – Avebe Company, Holland
Wheat starch – Factory of Potato Industry, Poland
|
Physico-chemical properties |
Corn starch |
Wheat starch |
|
Humidity [%] |
12.6 |
11.9 |
|
Proteins content |
0.5 |
0.4 |
|
Lipids content |
0.14 |
0.1 |
|
Ash content |
0.26 |
0.14 |
Experimental procedure
25, 30, 35 and 40% DS corn or wheat starch suspension was treated by a-amylase in amount of 0.2% and lysophospholipase in dosage of 0.015, 0.02, 0.025 and 0.05%. Process was affected at 85 and pH 6.0-6.5.°C and took 1-3 h.
Additionally action of mixture beta-glucanase and pentosanase was used for liquefied wheat starch in amount of 0.1, 0.15 and 0.2% at the temperature of 50°C and pH 6.0-6.5.
The following measurements were made in the samples:
the amounts of reducing groups by modified Schoorl-Rogenbogen method [12],
the viscosity measurement in 50°C using Rheotest RV2
the iodine absorbance value defined as the optical density at 500 nm using a 4 cm cell
the colour measurement by Spectrophotometer S.7501 [11]
the filtration rate using 7 cm filtration funnel equipped with Whatman filter paper under the pressure of 0.04 - 0.05 MPa
RESULTS AND DISCUSSION
Corn starch hydrolysates
Influence of starch concentration
Corn and wheat starch suspension at different concentrations (25,30, 35,40% DS) was treated 1h by alpha amylase Termamyl (0.2% w/w) and lysophospholipase Gammazym LPL (0.025% w/w) at 85-90°C and pH 6.0-6.5. Fig.1a presents the comparison of DE values of hydrolysates treated only by alpha amylase and by both enzymes: alpha amylase and lysophospholipase. DE value increased as the increase of starch concentration. Additional lysophospholipase action does not influence on DE value of hydrolysates. The growth of starch concentration reduces colour indicator and iodine absorbance value of hydrolysates (Fig.1b). In this case the effect of additional action of lysophospholipase can be observed. These measured values are higher in the case of simultaneous action of alpha amylase and lysophospholipase. Fig 1c presents the influence of starch concentration and additional lysophospholipase action on filtration rate and on viscosity of hydrolysates. The decr
ease of viscosity and the increase of filtration rate of hydrolysates (11-12% connected with additional action of lysophospholipase and the growth of starch concentration.
| Fig. 1. Effect of starch concentration on corn starch hydrolysis Reaction conditions: starch concentration: 25, 30, 35, 40 % DS, pH: 6.0 - 6.5, time hydrolysis: 1h, temperature: 85-90°C, enzyme dosage: a-amylase /T/: 0.2%w/w + lysophospholipase /G/: 0.025%w/w |
| A |
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| B |
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| C |
 |
Influence of reaction time
Similarly the effect of reaction time on the same indicators was checked. Action of lysophospholipase on corn starch suspension (40% DS) was prolonged to 3h (Fig.2). The increase of time to 3h does not influence on DE value of hydrolysates (Fig.2a) obtained by simultaneous action of enzymes, filtration rate and viscosity. Results indicate the decrease of colour indicator and iodine absorbance (Fig.2b), the decrease of viscosity and the increase of filtration rate of hydrolysates (Fig.2c) obtained by 1h action of both enzymes: alpha amylase and lysophospholipase in comparison to hydrolysate obtained by only alpha amylase action.
| Fig. 2. Effect of reaction time on corn starch hydrolysis Reaction conditions: starch concentration: 40% DS, pH: 6.0 - 6.5, temperature: 85-90°C, a-amylase dosage /T/: 0.2% w/w, time hydrolysis T-1h, a -amylase dosage /T/: 0.2% w/w + lysophospholipase dosage /G/: 0.025% w/w, time hydrolysis: T+G1-1h, T+G2-2h, T+G3-3h |
| A |
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| B |
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| C |
 |
Influence of enzyme dosage
Effect of lysophospholipase dosage on the tested indicators shows Fig.3. The increase of enzyme dosage from 0.015 –0.5% w/w does not influence on DE value of hydrolysates (Fig.3a). Application of lysophospholipase in amount of 0.025% w/w of starch decreases in the highest degree tested factors: colour indicator, iodine absorbance and viscosity but most of all increases the filtration rate of hydrolysates. The application of higher dosage of the lysophospholipase does not have any influence on the change of tested indicators.
| Fig. 3. Effect of enzyme dosage on corn starch hydrolysis Reaction conditions: starch concentration: 40% DS, pH: 6.0 - 6.5, temperature: 85-90°C, time hydrolysis: 1h, /T/ a - amylase dosage: 0.2% w/w, /T+G1/ a- amylase: 0.2% w/w + lysophospholipase dosage: 0.015%w/w, /T+G2/ a- amylase: 0.2% w/w + lysophospholipase dosage: 0.02% w/w, /T+G3/ a- amylase dosage: 0.2% w/w + lysophospholipase dosage: 0.025%, /T+G4/ a- amylase dosage: 0.2% w/w + lysophospholipase dosage: 0.05%w/w |
| A |
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| B |
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| C |
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Wheat starch hydrolysates
Influence of b-glucanase and pullulanase action
The influence of additional action of beta-glucanase and pentosanase (SP 348) on wheat starch suspension (40% DS) treated initially by alpha amylase and lysophospholipase is shown by Fig 4. The applied dosage of SP 348 was 0.15% w/w. The same dosage of SP 348 with simultaneous action of lysophospholipase (Gammazym LPL) was used for liquefied wheat starch by Termamyl. Action of SP348 and Gammazym LP was carried out for 24h. Results indicated that the application of both enzymes: SP 348 and Gammazym LPL, independently on the way of their introduction to substrate, give the highest decrease of colour indicator, iodine absorbance, viscosity of hydrolysates and the highest increase of filtration rate of hydrolysates after 3h of enzyme action.
| Fig. 4. Influence of additional action of beta-glucanase and pentosanase on wheat starch hydrolysis Reaction conditions: starch concentration: 40% DS, pH: 6.0 - 6.5, time hydrolysis: 1h, temperature: 85-90°C, a- amylase dosage /T/: 0.2 % w/w, a- amylase dosage: 0.2 % w/w +lysophospholipase dosage: 0.025 w/w % /T+G/, pH: 6-6.5, temperature: 50°C, time: b-glucanase and pentosanase dosage /S/: 0.15% w/w, 1 - T+G - 85-90°C, S - 50°C, 1, 2, 3, 24h, 2 – T- 85-90°C, G+S - 50°C, 1, 2, 3, 24h. |
| A |
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| B |
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| C |
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Influence of enzyme dosage
A joint action of different dosages SP 348 (0.1, 0.15 and 0.2% w/w) and Gammazym LPL (0.05, 0.1 and 0.15 % w/w) on liquefied wheat starch was applied by 1h at 50°C (Fig 5). The optimal dosages for SP 348 and Gammazym LPL are 0.025% w/w of SP 348 and 0.015% w/w Gammazym LPL. These dosages of enzymes give high filtration rate of the hydrolysates and low colour indicator, iodine absorbance and viscosity of the hydrolysates.
Application of additional enzyme, which improves filterability of hydrolysates influence in considerable degree on filtration rate of wheat hydrolysates than corn hydrolysates.
| Fig. 5. Influence of enzyme dosage on wheat starch hydrolysis Reaction conditions: starch concentration: 40% DS, temperature: 85-90°C, pH: 6.0 - 6.5, time hydrolysis: 1h, A - a- amylase dosage: 0.2% w/w, lysophospholipase dosage at 50°C, pH 6-6.5, 1h: B – lysophospholipase 0.015% w/w + beta-glucanase and pentosanase 0.15% w/w C – lysophospholipase 0.025% w/w + beta-glucanase and pentosanase 0.1% w/w D – lysophospholipase 0.025% w/w + beta-glucanase and pentosanase 0.15% w/w E – lysophospholipase 0.025% w/w + beta-glucanase and pentosanase 0.2% w/w F - lysophospholipase 0.05% w/w + beta-glucanase and pentosanase 0.15% w/w |
| A |
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| B |
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| C |
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The detectable quantities of nonstarchy polysaccharides, mainly pentosanes, which are found in wheat starch, make necessity of application of pentosanase in the process of enzymatic wheat starch conversion. Pentosanases degrade arabinoxylans, which are physically or covalently bound to proteins. They reduce the hydrolysates viscosity and increase their filtration rate. Additionally they decrease the risk of turbidity caused by precipitation of xylans at low temperatures [5].
Bowler, Toweseya and Gillarda [1] investigated the filtration characteristic of glucose wheat syrup. They compared the filterability of syrup obtained by glucoamylase action and complex of glucoamylase and pentosanase action. Results indicated that pentosanase treatment caused a decrease of in apparent viscosity, partially due to depolymerization of pentosan but, more importantly, to the solubilization of insoluble pentosan, which results in change in the nature of the insoluble residue from gelatinous, deformable particles, which clog the filter into rigid materials, which do not block the filter. Research showed that without pentosanase treatment of wheat starch the material is impossible to filter economically.
Profits influenced from pentosanase application in production of wheat hydrolysates were also indicated by Niemann, Ubehauma and Meuser [10]. They treated wheat starch suspension by alpha-amylase and combination of alpha-amylase and pentosanase isolated from Tr. Resesei. Degradation degree of starch obtained by alpha-amylase action was 39.2% but obtained by simultaneous action of alpha-amylase and pentosanase was 56.9%.
Matser and Steeneken [7] investigated the effects of poor filtration characteristics of wheat starch hydrolysates too. They tested the influence of different enzymes (glucoamylase, protease, xylanase, lipase, beta-glucanase, pentosanase and mixture more than six enzymes: arabinase, xylanase, beta-glucanase, hemicellulase, pectinase and cellulase) on filtration rate of wheat starch hydrolysates. They concluded that application of glucoamylase and mixture more than six enzymes increased 15 times and 4-6 times the filtration rate, respectively. They also removed components from wheat starch hydrolysates and added one-by one to potato starch hydrolysates and evaluated their effects on the filtration rate. The filtration rate of potato starch hydrolysates decreased after adding gluten, pentosanes, soluble, or the propanol extract of defatted wheat starch.
The influence of amylose-lipids and amylose protein complexes, pentosanes and nongelatinized starch on filtration rate of wheat starch hydrolysates were observed by others authors [1, 2, 3, 5].
Matser and Steeneken [6] made the comparison of filtration characteristics of maize and wheat starch hydrolysates. Maize starch hydrolysates showed good filtration characteristics. The filtration of wheat starch hydrolysates was a highly inefficient operation that removes only a minor part of the impurities. Even after a second filtration, the filtration rate remained much lower than that of maize starch hydrolysates.
Nebesny and Rosicka [9] claimed that in the process of enzymatic hydrolysis of starch, it is necessary to use lysophospholipase and xylanase-cellulase, which decompose amylose-fat complexes and arabinoxylanes, which bring about an increase of filtration rate, in addition to limiting the risk of clouding that takes place as a result of precipitation of xylanes at low temperature.
CONCLUSIONS
The use of additional enzymes during enzymatic hydrolysis of corn and wheat starches profitable influence on production process and quality of hydrolysates.
Simultaneous application of alpha amylase and lysophospholipase in production process of corn hydrolysates increases the filtration rate and decreases viscosity, colour indicator and iodine absorbance of hydrolysates. The carried research indicates that: optimal lysophospholipase dosage is 0.025%, sufficient reaction time - 1h, rise of starch concentration to 40% DS create by 12% increase of filtration rate.
Additional application of b-glucanase and pentosanase enzyme complex in production of wheat hydrolysates, independently on the method of their introduction to production process, makes 2.5-times rise of filtration speed of hydrolysates.
REFERENCES
Bowler P., Towersey P.J., Gilliard T., 1985. Some effects of the minor components of wheat starch on glucose syrup production. Starch/Stärke 37, 351-356. Derez F.G.H. Carbohydrate refining process and enzyme compositions suitable for use therein. European patent application: EP0219269. 1987. Ducroo P., 1987. Improvements relating to the production of glucose syrups and purified starches from wheat and other cereal starches containing pentosans. European patent application: EP0228732. Hebeda R.E., Leach H.W, 1974. The nature of insoluble starch particles in liquefied corn-starch hydrolysates. Cereal Chemistry 51, 272-28. Konieczny-Janda G., Richter G., 1991. Progress in the enzymatic saccharification of wheat starch. Starch/Stärke 43, 308-315. Master A.M., Steeneker P.A.M., 1998. Filtration characteristics of maize and wheat starch hydrolysates. Cereal Chemistry 75, 3, 241-246. Master A.M., Steeneker P.A.M., 1983. Origins of poor filtration characteristics of wheat starch hydrolysates. Cereal Chemistry 75, 289-293. McCleary B.V., Gibson T.S., Allen H., Gams T.C., 1986. Enzymic hydrolysis and industrial importance of barley b-glucans and wheat flour pentosans. Starch/Stärke 38, 433-437. Nebesny E., Rosicka J., 1998.: Enzymatic hydrolysis of wheat starch into glucose. Starch/Stärke 50, 337-341. Niemann C., Unbehaum S., Meuser F., 1995. Preparation of porous starches and their properties. Starch Convention, Detmold. PN-78/A-74701. Polish standard: Starch Hydrolysates. Methods of Testing. Raucher , K., 1956. Untersuchung von Lebensmitteln, Fachbuchverlag, Leipzig, 2, 125. Schenk F.W., Hebeda R.E., 1992. Starch hydrolysis products. VCH Publishers: New York.
Lucyna Słomińska
Starch and Potato Products Laboratory
Armii Poznań 49, 62-030 Luboń, Poland
ph. (+4861) 893 46 05 ext. 106
fax. (+4861) 893 46 08
e-mail: ls@man.poznan.pl
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