THE INFLUENCE OF ADDITIONAL ENZYMES TREATMENT OF CORN AND POTATO MIX STARCHES ON FILTRATION PROPERTIES OF HYDROLYSATES

Lucyna Słomińska, Danuta Wiśniewska, Joanna Niedbach

ABSTRACT

Starch hydrolysates were prepared by hydrolysis of potato and corn starch mixture with a heat-stable bacterial alpha amylase (Termamyl 120 LS) or with a heat-stable bacterial alpha amylase (Termamyl 120LS) and next with a fungal alpha amylase (Finizym 1600L). Additionally so-called filtration enzyme action was applied (Finizym, Neutrase, Lecitase, SP 348, Gammazym LPL). Introduction of these enzymatic preparations influenced on the increase of filtration rate of hydrolysates and decrease of their viscosity and transmittance value. “Filtration enzyme” did not influence on the carbohydrate composition.

Key words: starch, alpha-amylase, hydrolysates, carbohydrate, filtration rate, viscosity..

INTRODUCTION

Starch hydrolysates are prepared by conversion of starch with acid or enzymes. Hydrolysates are filtered to remove suspended solid and insoluble impurities. They influence unfavorably on the filtration rate of hydrolysates. Their amount depends on botanical source of starch. The hydrolysates after enzymatic conversion of corn starch contain the insoluble particles in the range of 0.2-1.0 ľm. These particles consist of nongelatinized starch [1, 3] or amylose-lipids complexes [5]. The impurities which exist in corn starch hydrolysates are connected with chemical composition of corn starch. Corn starch contains several times more protein (corn starch 0.35%, potato starch 0.06%) and lipids (corn starch 0.87%, potato starch 0.05%) than potato starch [17]. Protein present in hydrolysates is disadvantages considering its negative effect on the color and aroma of the hydrolysates and its contributes to the formation of foam during the process of its production [10, 11]. Similarly lipids content in starch is also unprofitable during starch hydrolysis. In the process of heating starch suspension lipids form amylose-lipids complexes that have negative effect on ability of water assimilation as well as swelling and solubility of starch [2, 4]. They also cause opalescent and cloudiness of starch slurry and give worse filtration characteristic of product. Application of potato and corn starch mixture in hydrolysates production reduces the amount of lipids and protein in product which is undesirable for technological process. Application of so-called filtration enzymes additionally increases of filtration rate of hydrolysates [8, 12].

The attempts of hydrolysates filtration improvement was also made by application of membrane filtration [6, 15, 16].

In this study the effect of “filtration enzymes” on the hydrolysates filtration obtained from potato and corn starch mixture was studied.

MATERIALS AND METHODS

Starch

Potato starch (Factory of Potato Industry - Poland), corn starch (Avebe - Holland) were used.

Enzyme

Termamyl 120 type LS (Novo Nordisk, Denmark) - a mixture of outstanding heat-stable alpha-amylases produced by selected strains of Bacillus licheniformis. The enzyme activity was 120 KNU/g (KNU = Kilo Novo Units alpha-amylase – the amount of enzyme which breaks down 5.26 g starch per hour at Novo Nordisk’s standard method for determination of alpha amylase).

Finizym (Novo Nordisk, Denmark) – a fungal beta glucanase produced by a selected strain of Aspergillus niger. The enzyme activity was 200 FBG/g (FBG = Fungal Beta-Glucanase unit – the amount of enzyme which, under Novozymes’ standard conditions degrades barley beta-glucan to reducing carbohydrates with a reduction power corresponding to 1 µmol glucose per minute).

Neutrase (Novo Nordisk, Denmark) – a protease produced by selected strain of Bacillus subtilis. The enzyme activity was 0.5 AU/g (AU Anson Units).

Lecitase (Novo Nordisk, Denmark) - a purified phosfolipase A₂ manufactured from porcine pancreas. The enzyme activity is was 10.000 IU/ml (IU – the amount of enzyme producing 1 µmol of free fatty acid per minute under international standard conditions. SP 348 (Novo Nordisk, Denmark) – a complex carbohydrate mixture with beta glucanase and pentosanase activities.

Gammazym LPL (Gammazym, Germany) – a lysophospolipase derived from Aspergillus niger.

Fungamyl 1600L - a fungal alpha-amylase produced by selected strain of Aspergillus oryzae. The enzyme activity was 1600 FAU/g (FAU = Fungal Alpha-Amylase unit – the amount of enzyme which breaks down 5.26 g starch per hour at Novozymes’ standard method for determination of alpha-amylase).

Analysis

• The following determinations were carried out:

• the content of reducing sugars according to the modified Schoorl-Rogenbogen method [14],

• the carbohydrate composition by HPLC using Ostion LG/KS 0803 column (samples -20µL- were eluted at 1.5 cm³ · min^-1 using water at 85°C),

• the viscosity measured in Brabender Typ E and at 50°C using Rheotest RV 2,

• the filtration rate (the volume of hydrolysate – ml - filtered through Büchner filter of a diameter 7 cm covered filter paper under pressure 60 kPa within 1 min),

• the colour factor using Minolta CR-300 series Chroma Meters,

• the ash content by Kieldhal’s method [13],

• the total and soluble protein content by the Lowry’s method [9],

• the lipid content [7],

• the average diameter of starch performed in Analysette 20 f-ma Fritsch.

• the transmittance value (transmittance at 720 nm)

Procedure

Potato and corn starches mixed in 1:1 ratio were slurried in water to obtain a starch concentration of 35% DS. Hydrolysis was performed with the application of Termamyl LS used in amount of 0.15% at 97C and pH 6.0-6.5 within 1 h. Ca⁺⁺ in amount of 40 ppm was used as an activator.

Action of additional enzymes (Finizym, Neutrase, Lecitase, Sp 348, Gammazym LPL) was carried out before or after alpha-amylase treatment using following enzymes dosages: 0.05: 0.1 and 0.15% at 50°C within 1,2 3, 7 h.

Liquefied by Termamyl action starch was further hydrolysed by the use of simultaneous action of Fungamyl and Finizym at 55°C and pH 5.0 by 20h.

RESULTS AND DISCUSSION

Starch properties

The properties of the tested starches used as substrates for alpha- amylase action are shown in Table 1.The measurement of absolute starch colour by the L*a*b* colour system indicate that potato starch has better colour parameters. Negative value of chromaticity coordinate “a” which indicates affinity to green colour and positive value of chromaticity coordinate “b” which indicate affinity to yellow colour are lower for potato starch.

The size of tested starches granules is different. The potato starch granules diameter is almost three times higher than corn starch granules.

Action of enzymatic preparations in production of maltodextrine from potato and corn starches mixture

Dextrose equivalent and carbohydrate composition

Table 2 illustrates the influence of additional enzyme action, called futher so-called filtration enzymes (Finizym, Neutrase, Lecitase, SP 348, Gammazym LPL) on liquefaction process carried out by alpha-amylase (Termamyl ). It can be observed that DE of samples is similar but differences exist in their carbohydrate composition. The application of so-called filtration enzymes influences on the decrease of G2-G7 by 20-44% but G8-G10 only by 3-13% or increase by 8-16% in comparison to samples without “filtration enzymes”. The level of higher sugar (Gn) increases by 13-20%.

Filtration rate

The application of each enzyme from so-called filtration enzymes gives by 28-86% higher filtration rate (Fig. 1) The highest influence can be observed in the case of Finizym application.

Transmittance value

Figure 2 shows the positive influence of “filtration enzymes” on transmittance value – the decrease of transmittance value by 13 – 35%. The best results are also obtained for Finizym application similarly as in the case of filtration rate.

Finizym, which obtains a beta glucanase, was chosen for further research. It indicates that beta glucans are the substances mainly responsible for the high viscosity of hydrolysates.

Influence of Finizym action on filtration rate of liquefied starch

Reaction time and enzyme dosage

The research work of application of enzyme dosages: 0.05, 0.10,and 0.15% indicate that filtration rate increases when enzyme dosage was enhancement from 0.05 to 0.1% but further increase has no influence on filtration rate (Fig. 3a). Sufficient enzyme dosage was 0.1%.

Prolongation of reaction time of Finizym from 1-to 3 h influences positively on filtration rate ; increases by 30% filtration rate (Fig. 3b). In the case of using of Finizym after Termamyl LS action the filtration rate was by 15% lower.

Sequence of enzyme action

Finizym action was used before and after alpha-amylase action (Fig. 4). Both ways of Finizym application cause the increase of filtration rate however better results are obtained when in the first place Finizym was used and then liquefaction by Termamyl LS was carried out.

Viscosity

Viscosity measurements of hydrolysates after 1, 2 and 3 h action of Finizym show that during prolongation of enzyme action time the hydrolysate viscosity decreases (fig. 5). After 1h of enzyme action the viscosity is 29.6 MPa·s, but after 2 h of reaction is by 22% lower and after 3 h – by 41% lower.

Finizym and Fungamyl action

The simultaneous action of Fungamyl and Finizym influences on following factors: filtration rate (increase by 69%), viscosity (decrease by 13%) and transmittance value (decrease by 25%) (table 3). Carbohydrate composition stayed without any influence on additional action of “filtration enzyme”

• “Filtration enzymes” used in production of maltodextrine from corn and potato starch combination do not have any influence on DE hydrolysates but increase the amount of higher sugars.

• Among selected “filtration enzymes” Finizym action has the best influence on filtration rate of hydrolysates.

• Finizym action increases filtration rate of liquefied and saccharified starch, decreases of viscosity and transmittance value of obtained hydrolysates.

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