PL239536B1 - Method for one-stage liquefaction of native starch - Google Patents

Abstract

The subject of the application is a method for one-stage liquefaction of native starch using α-amylase. This process is characterized in that α-amylase derived from the Bacillus amyloliquefaciens KU12 strain is used as the enzyme, which is added in an amount of 0.012 to 0.1 U/mg of native starch to a 1 - 40% solution of native starch, and the liquefaction is carried out at pH of the solution from 4.5 to 6.5, mixing with a shear rate from 100 to 200 s-1 and in the temperature range from 70°C to 90°C, with the addition of calcium ions Ca2+ above the temperature of 82°C .

Description

The subject of the invention is a method of one-stage liquefaction of native starch of various origins, omitting the preliminary conventional method of starch germination at 105-140 ° C, α-amylase (endo-a-1,4-glucanase or a-1,4-glucan glucanohydrolase; EC-3.2 .11) is an active polypeptide that catalyzes the hydrolysis of starch, including its two fractions (amylose and amylopectin). It cleaves the internal? -1,4-glycosidic bond of these substrates from the reducing end randomly with the slow release of oligosaccharides of varying degrees of polymerization (DP), dextrins and small amounts of glucose. The concentration of the hydrolysis products and their degrees of depolymerization depend on the type of enzyme, substrate mass, conditions and the time of hydrolysis (Ezeji and Bahl, J. Biotechnol, 125, 27-38, 2006). Until now, the conventional starch liquefaction process in biotechnological industries has mainly been carried out in two stages: sprouting and liquefaction. In these stages, a suspension of native starch, flour or starch biomass (15-30%) is first subjected to a jet-cooking process at high temperature above 105 ° C (5-10 min) with the use of thermophilic α-amylase, ( Atrim, Patent US 5180669; 1991; Thisted et al., US 7713723 B1,2000; Udeh and Targoński, Patent PL 199207 B1, 2008; Udeh et al., Patent PL 211354 B1, 2012; Anderson and Poulsen, Patent US 9139822 B1 , 2015). At this stage, the starch is generally partially liquefied by the pre-added thermophilic α-amylase from B. licheniformis to the 10-15 degree of polymerization (DP) (Crab and Mitchinson, TIBTECH, 15, 349-352, 1997; Anderson and Poulsen, US Patent 9139822 B1.2015). Dupont, a company producing commercial a-amylase (Spezyme Alpha - 13775 AAU / g), recommends using it for this process in the amount of 0.2-0.3 kg / ton of starch, which corresponds to approximately 3.7 - 5.5 U / g. mg of starch (www.dupont.com). However, at the stage of proper liquefaction, the gruel is subjected to further hydrolysis, during which secondary liquefaction takes place during 1.5 to 2 h at 90 ° C with the addition of a second portion of a specific concentration of α-amylase (Veit et al., WO 2002038787 A2, 2001 ; Anderson and Poulsen, Patent US 9139822 B1,2015). The above-described technological activities increase the costs of processing the substrate intended for the fermentation process, because the flow of gruel is impeded in the germination stage due to its high viscosity (Veit et al, WO 2002038787 A2, 2001; Udeh and Targoński, Patent PL 199207 B1,2008; Anderson and Poulsen, Patent US 9139822 B1, 2015) which requires additional activities and energy expenditure.

Solutions were developed for the production of highly thermostable α-amylases by strains (Pyrococcus woesei., P, furiosus, Thermococcus hydrothermalis) isolated from extreme environments (Koch et al., Arch. Microbiol., 155, 572-578, 1991; Dong et al., Appl. Environ. Microbiol., 63, 3669-3576, 1997; Bertoldo and Aniranikan, Curr, Opinion Chem. Biol., 6, 151-160, 2002). These a-amylases are not widely used in the starch industry due to the high production costs (growth and synthesis from 70-90 ° C). Their synthesis is unprofitable on an industrial scale due to mat efficiency (Crab and Mitchinson, TIBTECH, 15, 349-352, 1997). Moreover, these types of α-amylases have a limited ability to hydrolyze starch even at lower temperatures after germination (Bertoldo and Antranikan, Curr. Opinion Chem. Biol., 6, 151-160, 2002; Anderson and Poulsen, Patent US 9139822 B1, 2015) . Also known are many potential mesophilic and thermophilic microorganisms which produce by submerged or surface cultivation; native starch hydrolyzing α-amylases (Ozdermir et at., Ann. Microbiol., 62, 1367-1375, 2012; Udeh and Targoński, Patent PL 199207 B1,2008; Anderson and Poulsen, Patent US 9139822 B1, 2015; Tang et al. ., patent application US 2016/0208230 A1). Most of them have not found industrial application because they are not active at high temperatures.

The term "starch germination" means the dissolution of starch particles suspended in water (starch suspension) by a temperature that exceeds its germination temperature in order to destroy its amorphous and crystalline structures and to form a hydrogel.

By the term "germination temperature" is meant the lowest temperature value at which germination of the starch begins. The exact value of the starch germination temperature is determined by known methods and depends on the type of starch, its origin, variety and growing conditions. These values vary depending on the origin of the starch.

The term "conventional starch sprouting" means heating the starch suspension to above 105 ° C (105-140 ° C) for 5 to 15 minutes with the addition of a specified concentration of thermostable α-amylase.

By the term "starch biomass" is meant both whole grains derived from cereal and similar plants, as well as tubers from tuberous and root plants, containing starch as

Spare material. They can be pre-prepared by grinding fresh material or / and dried and ground. Then subjected to the action of various enzymes, e.g. cell wall decomposition (cellulase, xylanase), branching (cellubiosis, β-glucosidase, etc.) and proteolytic (protease) in the stage of substrate pre-treatment for one-stage hydrolysis or starch liquefaction.

The publication of the patent PL 217670B1 discloses a method of single-step enzymatic hydrolysis of instantized starch with a starch content in the dry substance of not less than 95%, characterized by a value of glucose equivalent (DE) not higher than 2, in order to obtain a maltose or glucose hydrolyzate. In this method, the enzyme which breaks the bonds is added, in common or separate streams, to the instantized starch solution having a pH of 5.0 to 5.5 to obtain a maltose hydrolyzate or of a pH of 4.8 to 5.2 to obtain a glucose hydrolyzate. glycosidic type α-1,4 from the center of the starch chain (liquefying enzyme) and breaking down α-1,4 and α-1,6 bonds of starch macromolecule (saccharification enzyme) or an enzyme that breaks down only the α-1,4 bond sequentially from the end of the chain macromolecules (saccharification enzyme), followed by a one-step process of instantized starch hydrolysis by simultaneous liquefaction and saccharification for an appropriate time, depending on the expected final degree of saccharification, for maltose hydrolyzate at a temperature above 70 ° C, and for glucose hydrolyzate at a temperature below 70 ° C.

There is a known strain of Bacillus amyloliuefaciens, which was isolated from a soil sample from Abuja, Nigeria. The nucleotide sequence of the gene encoding the 16S rRNA of this strain has been deposited in the GenBank bioinformatics database, NCBI, USA under no. KX870880 and marked KU12.

Unexpectedly, it turned out that this strain produces α-amylase, which enables the liquefaction of native starch in a dynamic process with simultaneous sprouting and its liquefaction in one step.

The essence of the one-stage liquefaction of native starch using α-amylase is that the enzyme is α-amylase derived from the Bacillus amyloliquefaciens KU12 strain, which is added in an amount from 0.012 to 0.1 U / mg of native starch to 1 - 40 % of native starch solution. The liquefaction is carried out at a solution pH of 4.5 to 6.5, with mixing at a shear rate of 100 to 200 sec ^-1 and in a temperature range of 70 ° C to 90 ° C depending on the origin of the starch. Above 82 ° C, the addition of calcium ions Ca ^{2+ is used} .

Preferably, the process temperature is achieved in no more than 2 minutes.

The process according to the invention using the α-amylase derived from the strain Bacillus amyloqiucfackns KU12 allows the process to be carried out in the temperature range from 70 ° C to 90 ° C at a low enzyme concentration, quickly reducing the viscosity of the native starch undergoing liquefy. This solution allows to shorten the liquefaction time, reduce energy expenditure and enzyme concentration. This has the effect of reducing the total cost compared to the conventional liquefaction method. In this way, a hydrolyzate is obtained with a viscosity that allows it to be easily transported and further processed in the sugar process. This product contains from 1 to 9 DP (degree of polymerization) and other malto-oligosaccharides. This hydrolyzate can be directly used for the production of sugar syrup for various technological processes and in the method of simultaneous saccharification and fermentation under high gravity (SSF-VHG) ((Nguyen et al., Ind. Crops Products, 56, 160- 165; 2014) of starch with a high soluble mass.

The method of one-stage liquefaction of native starch according to the invention is detailed below in the Examples.

P r z k ł a d 1

The process of producing α-amylase hydrolyzing native starch by the strain Bacillus amyloliquefaciens KU12

The strain was stored in 20% glycerol at minus 80 ° C, then cultivated in nutrient agar broth prepared according to the manufacturer's (BTL-Polska) recommendations for 48 h at 37 ° C. Agar slants with the following composition were prepared in 100 cm ³ (w / v): 1 g of soluble starch (POCH), 1.5 g of plain broth (BTL-Poland), 2 g of agar. The previously amplified cells were transplanted onto agar and left in a thermostat at 37 ° C for 24 h.

The grown cells were inoculated onto a sterilized and cooled seed medium in a volume of 50 ml at pH 6.5 in a conical flask with a capacity of 300 ml. The seed medium had the following composition per 100 cm ³ (w / v): 1 g soluble starch. (POCH), 1.5 g of regular broth (BTL-Poland). The seed culture was carried out on a rotary shaker at 37 ° C for 24 h. The obtained cells were used to inoculate the production medium.

PL 239 536 B1

The production medium had the following composition at 100 cm ³ (w / v); 1 g of starch; 0.2 g, KH4PO4; 0.05 g (NH4) SO4; 0.05 g of NaCl; 0.02 g CaCl2.4H2O, 0.01 g MgSO4.7H2O; 0.5 g yeast extract (BIOCORP). Before sterilization, the pH of the medium was adjusted to 6.5. After sterilization, the medium was cooled down and sterile inoculated with cells from the seed culture in an amount of 1% in relation to the volume of the production medium. The production culture was carried out on a rotary shaker at 37 ° C for 72 h.

Starches for the production of this enzyme may be native, modified, or starch biomass, ground, dried, hydrolysed or non-hydrolyzed. The concentration of dried starch biomass in the medium may be from 5% to 10% and of other sources of purified starch from 1 to 2%. The production medium contains a total of organic nitrogen compounds in a concentration of 0.2% to 2% and inorganic nitrogen in a concentration of 0.05% to 2%, while the production culture can be carried out in the temperature range from 28 ° C to 45 ° C and at pH 5.5 to 7.5 (preferably at pH 6.5) and for a period of 24 to 96 hours.

After completion of the culture, the obtained fluid, grown at 6000 x g, was centrifuged for 15 minutes at 4 ° C. The filtrate, devoid of microbial biomass, was used for the determination of total enzyme activity using a 1% buffered starch solution, pH 5.5. The amount of released reducing sugars was analyzed according to the DNS method (Miller, Anal. Chem., 31, 426-428, 1959). Based on the analysis of the amount of released reducing sugars after the enzymatic reaction, the activity of α-amylase was calculated and determined. This activity is expressed in IU / ml. One unit determines the amount of reducing sugars secreted by the enzyme, expressed in μmol of glucose per unit of time (1 minute) depending on the conditions of the enzymatic reaction. Total protein content analysis was determined by the Lowry method (Lowry et al., J. Biol. Chem., 193, 1, 265-275; 1951).

The general and specific activities after purification according to the known methods of the invention were respectively 68.0 U / mL and 1746 U / mg of protein (after gel filtration) at the temperature of 60 ° C. This enzyme is active in the range of pH 3.0 to 9.0, optimally at pH 5.5. Operating temperature range; it ranges from 30 ° C to 100 ° C, with the optimal activity being between 60 ° C and 80 ° C. In the temperature range of 60 ° C to 80 ° C, it shows activity without the addition and / or with the addition of Ca ²⁺ ions, while at temperatures above 82 ° C, Ca ²⁺ ions are indicated, and above 90 ° C it is necessary.

The term-α-amylase is understood to mean both the enzyme preparation obtained by submerged culture, containing α-amylase together with other impurities derived from the components of the culture media, and pre-purified α-amylase obtained in the processes of preliminary purification using the known precipitation method (acetone, ethanol, (NH4) 2SO4) and α-amylase purified to homogeneity by known methods (ion exchange chromatography, hydrophobic chromatography, molecular filtration).

P r z k ł a d 2

One-stage liquefaction of native corn starch

A 30% aqueous solution of native corn starch with a pH of 5.5 was prepared. The α-amylase obtained according to the method described in Example 1 was used for the tests in a dose of 0.06 U / mg of native starch. The enzyme was added to the starch suspension in two combinations: without the addition of Ca ²⁺ ions and with the addition of Ca ²⁺ in the amount of 5 mM. Then, each of the samples in two combinations was mixed with tangential stress, i.e. a shear rate of 150 s- ¹ , and simultaneously heated to two assumed measurement temperatures of 80 ° C and 90 ° C, respectively. Therefore, the study was carried out for four variants of samples. For each of the samples, the temperature for the given measurement was reached within 2 minutes. The programmed temperature was constant throughout the liquefaction process, which lasted 30 minutes. Additionally, control tests were performed in the same way as for the actual measurements, in which no enzyme was added. A rheometer (Kinexus pro + Malvern Instruments Ltd rheometer, Worcestershire, UK) integrated with a microprocessor capable of analysis per unit time was used to measure the viscosity change occurring during the one-stage liquefaction of 30% native corn starch at two different temperatures (80 ° C and 90 ° C). (realtime measurement). A graph showing the measurement results of viscosity changes during the one-stage liquefaction of native starch is shown in Fig. 1.

The descriptions used in the chart have the following meanings:

- (D) 30% 80 ° C and (A) 30% 90 ° C - means the controls with 30% aqueous solution of native corn starch without the addition of the α-amylase enzyme, carried out at 80 ° C and 90 ° C, respectively;

PL 239 536 BI

- (B) 30% E 90 ° C and (C) 30% E + Ca 90 ° C - means samples of 30% aqueous solution of native maize starch with the addition of the α-amylase enzyme, carried out at 90 ° C respectively without addition and with the addition of Ca ²⁺ ions;

- (E) 30% E 80 ° C and (F) 30% E + Ca 80 ° C - means samples of 30% aqueous solution of native maize starch with the addition of the α-amylase enzyme, carried out at the temperature of 80 ° C without addition and with the addition of Ca ²⁺ ions.

The results obtained show that the viscosities decreased in all measurements from an initial value of more than 1000 Pa s (control) to a final value of less than 0.01 Pas. The exception was the measurement at 90 ° C without the addition of Ca ²⁺ ions, in which the viscosity was about 0.01 Pa s. it is not limited to the use of only that proven starch.

Example 3

Percentage of reducing sugars produced by α-amylase hydrolyzing native starch during liquefaction at various temperatures

Table 1 shows the percent hydrolysis of 30% native corn starch at 80 ° C and 90 ° C with the addition of 5 mM Ca ²⁺ in 30 minutes using the enzyme at a dose of 0.06 U / mg starch. At 90 ° C about 42.8% of the final degree of hydrolysis in the presence of Ca ^{2+ was found} , while at 80 ° C this value was 43.2%. The test conditions were analogous to example no. 3. These values were calculated according to the known methodology (Sujka et al., Ital. Food Sc 4, 4, 433-439, 2006).

Time (min) Temperature ( ° C). 80 90 5 27.4 ± 0.2 ^j 27.3 ± 0.37 10 30.6 ± 0.1 ' 33.0 ± 0.6 ^h 15 34.6 ± 0.3 ^s 37.5 ± 0.2 ^f twenty 40.0 ± 0.4 ^d 38.5 ± 0.4 ^e 25 41.1 ± 0.1 ^c 41.1 ± 0.0 ^c thirty 43.2 ± 0.5 ^ab 42.8 ± 0.3 ^a ___

Tab. 1. Percentage of hydrolysis after one-stage liquefaction of 30% maize starch during 30 minutes at 80 ° C and 90 ° C in the presence of 5 mM Ca ²⁺ at pH 5.5. Values are the mean ± standard deviation. Means with different letters differ significantly, p <0.05.

Example 4

Chromatogram of the hydrolyzate produced by α-amylase hydrolyzing native starch

The chromatogram of the hydrolyzate produced by the native starch hydrolyzing α-amylase is shown in Fig. 2. High performance liquid chromatography analysis of the hydrolyzate after one-stage liquefaction of native maize starch under the conditions described in example 2 showed that the hydrolysis product consisted of different degrees of polymerization from DPI to DP 9 and so, for example, the DPI in the shown chromatogram is glucose (rightmost peak) etc. and furthermore contains other unidentifiable malto-oligosaccharides. For these analyzes, a column (Benson Carbohydrate column BP-200 Ag + - Benson Polymeric Inc., Nevada, USA) designed for carbohydrate separation was used according to the manufacturer's recommendation.

Literature

1. BERTOLDO ANTRANIKAN.G., (2002) Starch hydrolysing enzymes from thermophilicarchaea and bacteria. CURR. OPINION CHEM. BIOL, 6, 151-160.

2. GRABB D. W., MITCHINSON (1997) Enzymes involved in the Processing of starch to sugars. TIBTECH, 15, 349-352:

3. DONG G, VIEILLE C., SAVCHENKO A., ZEIKUS GREGORY J "(1997) Cloning, sequencing and expression of the gene encoding extracellular α-amylase from Pyrococcus

Furiosus and biochemical characterization of the recombinant enzyme. APPL. ENVIRON. MICROBIOL., 63, 3569-3576.

4. EZEJI C.T., BAHL H., (200) 6 Purification, characterization and synergistic action of phytate-resistani α-amylase and α-glucosidase from Geobacillus ihermacienitrificans HRO10. J. BIOTECHNOL., 125,27-38.

5. KOCH R., SPREINAT A., LEMKE K., ANTRAIK1AN G., (1991) Purification and properties of a hyperthermoactive α-amylase from the archeabacterium Pyrococcus woesei. ARCH. MICROBIOL., 155, 572-578.

6. KY-CHU 5., PHAM T, BUI T.K, NGUYEN T., PHAM K, NGUYEN T., HO P., LE T. (2016)

Simultaneous liquefaction, saccharification and fermentation at a very high gravity of rice at pilot scale for potable ethanol production and distillers dried grains composition., FOOD AND BIOPRODUCTS PROCESSING, 98, 79-85.

7. LOWRY O.H., ROSEBROUGH N.J., FARR A.L., RANDAL RJ., (1951) Protein measurement with the Folin-Phenol reagents. J. BIOL. CHEM., 193, 265-267.

8. MILLER G.L., (1959) Use of dintrosalicylic acid reagent for determination of reducing sugars. ANAL. CHEM., 31, 426-428.

9. NGUYEN C., LE T., KY-CHU. S., (2014) Pilot scale simultaneous saccharification and fermentation at very high gravity of cassava flour for ethanol production. INDIUM. CROPS PRODUCTS., 56, 160-165.

10. OZDERMIR S., MATPAN F., OKUMUS V., DUNDAR A., ULUTUSM.S., KUMURU M., 11 (2012) Isolation of a thermophilic Anoxybacillus flavithermus sp.nov. and production of thermostable α-amylase under solid state fermentation (SSF). ANN. MICROBIOL., 62, 1367-1375.

11. SCRICHUWONG S., ORIKASA T "MATSUKIJ., SHIINA T. KOBAYASHI T., TOKUYASU K., (2012) Sweet potato having a low temperature-gelatinizing starch as a promising feedstock for bioelhanol production. BIOMASS AND BIOENERGY, 39, 120-127.

12. SONI K.S., GOYAL N "GUPTA K.J., SONI R., (2012) Enhanced production of α-amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in hydrolysis of raw potato starch. STARCH / STARKE, 64, 64-77.

13. SUJK.A M. UDEH K.O., JAMROZ J., (2006) α-Amylolysis of native com, potato, wheat and rice granules. ITAL. j. FOOD SC., 4, 18, 433-439.

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Patent claims

Claims (2)

Patent claims 1. Method of one-stage liquefaction of native starch using α-amylase, characterized in that the enzyme is α-amylase derived from the strain Bacillus amyloliquefaciens KU12, which is added in an amount from 0.012 to 0.1 U / mg of native starch to 1-40 % native starch solution and liquefaction is carried out at a solution pH of 4.5 to 6.5, with mixing at a shear rate of 100 to 200 s ^-1 and in the temperature range from 70 ° C to 90 ° C, with a temperature above 82 ° C, the addition of calcium ions Ca ^{2+ is used} . 2. The method according to p. The process of claim 1, wherein the process temperature is achieved in no more than 2 minutes.