Patent Application
20080145900
This application is a Continuation application of U.S. patent application Ser. No. 10/026,753, filed Dec. 19, 2001, the contents of which are incorporated by reference in their entirety.
The invention relates to the processing of polysaccharides, such as starch, to produce starch hydrolysates. Specifically, the invention relates to starch hydrolysis processes that do not require a secondary liquefaction step or pH adjustments before and after liquefaction.
Grains such as corn have long been used as a source of starch. One of the well-known methods of separating and purifying starch for use in industrial processes is the wet-milling process. This method has developed into a highly specific and integrated system designed to separate the major components of a grain kernel as completely as possible (see Stanley A. Watson, Starch: Chemistry & Technology, Vol. II, Industrial Aspects, Academic Press, New York, 1967, pp. 30-51).
In general, starch conversion processing consists of liquefaction of a granular starch slurry to produce dextrins and saccharification of the liquefied starch into dextrose. Additional processing may include purification and isomerization of dextrose to produce glucose or other sugars, such as fructose.
To liquefy granular starch, heat is applied to the granular starch slurry to disrupt the insoluble starch granules to produce a water-soluble starch solution which is then liquefied by adding an enzyme, such as an α-amylase. Typically, the enzymatic liquefaction process involves treatment of the about pH 3.5 to 5.0 granular starch slurry with a base (such as calcium hydroxide, sodium hydroxide or sodium carbonate) to adjust the pH to between about 6.0 and 6.5, the optimum pH range of commonly used α-amylases, such as an α-amylase derived from Bacillus licheniformis. The adjusted suspension may be pumped through a direct steam injection cooker (jet cooker) to raise the temperature to between about 105°-110° C. for low temperature liquefaction. In high temperature liquefaction the pH adjustment occurs just after a jet cooker phase at a temperature of about 140-155° C. Following jet cooking, the cooked slurry is cooled to the secondary liquefaction temperature, the pH is adjusted to a range favorable to the selected α-amylase, and then the α-amylase is added. Alternatively, the starch suspension and added α-amylase may be held at a temperature of about 80-100° C. to partially hydrolyze the starch granules, and this partially hydrolyzed starch suspension then is pumped through a jet cooker at temperatures in excess of about 105° C. to thoroughly gelatinize any remaining granular structure.
Secondary liquefaction at a pH of about 5.5 to 6.0 is carried out to allow the α-amylase to continue hydrolysis and reduce the dextrins produced during primary liquefaction. The secondary liquefaction step generally is carried out for about 90 to 120 minutes after cooling the primary liquefact to approximately 95°±5° C. Processing time for liquefaction is selected to produce a DE of approximately 10-12. Dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as glucose on a dry weight basis. Unhydrolyzed granular starch has a DE of virtually zero, whereas the DE of glucose is defined as 100.
Liquefaction temperatures depend upon the source of the α-amylase. Alpha-amylases produced by wild-type strains of B. licheniformis are preferred by the industry because they are thermostable under typical jet cooking temperatures. The α-amylases are thermostabilized with, for example, starch and calcium ions, and generally the starch slurry is adjusted to pH values above about 6 to protect against rapid inactivation at typical liquefaction temperatures. The high pH requirement results in undesirable by-products, e.g., maltulose which ultimately lowers glucose yields.
The pH of the starch slurry suspension from the wet milling stage is about 3.8 to 4.8, and is adjusted upward by the addition of acid neutralizing chemicals, which are removed later by ion-exchange refining of the final starch conversion product. If the liquefact undergoes further processing, such as saccharification utilizing glucoamylase, a pH of 4.0-4.5 is required; therefore, the pH is adjusted again down from about pH 5.5-6.0.
Even though the liquefaction processes employing alpha-amylase currently function to meet the starch processors' operational and performance demands, areas for improvement still remain.
The present invention provides a novel single stage liquefaction process for starch using an acid-stable, thermostable α-amylase derived from a selected strain of Bacillus acidocaldarius that enables liquefaction without an adjustment of the pH.
A novel, low temperature starch conversion process comprises a single liquefaction step using a novel acid-stable, thermostable-α-amylase from a selected strain of Bacillus acidocaldarius. The process:
In another embodiment of the present invention, a low temperature starch conversion process comprises primary and secondary liquefaction steps using low dosages of an acid-stable, thermostable α-amylase from a selected strain of Bacillus acidocaldarius. In addition to requiring low enzyme dosages, the process;
In yet another embodiment of the present invention, a high temperature starch conversion process comprises a single liquefaction step using a novel acid-stable, thermostable α-amylase from a selected strain of Bacillus acidocaldarius. The process:
The novel starch conversion processes utilize fewer chemicals, enable fewer processing steps, operate at a single pH, and can take less time to produce a liquefact with a commercially acceptable DE value and suitable for further optional saccharification. Additionally, the processes produce fewer undesirable by-products during liquefaction, due at least in part to eliminating or reducing typical chemical additions and conducting liquefaction at a reduced pH.
Without wishing to be bound by theory, it is believed that the novel α-amylase is inactivated, consumed, or in some way altered by the end of one of the low temperature liquefaction process thereby enabling saccharification to proceed without the thermo-inactivation utilized in the other embodiments.
The invention comprises the discovery that an acid-stable, thermostable α-amylase from a selected strain of Bacillus acidocaldarius enables the conversion of starch to glucose in a single liquefaction step without pH adjustment of the starch slurry, or in two liquefaction steps utilizing low dosages of the enzyme, and further enables optional saccharification steps to proceed without a pH adjustment to acceptable saccharification enzyme pH ranges. Additionally, in at least one embodiment of the invention, there is no need to inactivate the Bacillus acidocaldarius α-amylase prior to saccharification.
“Alpha-amylase” (α-amylase) means an enzyme which cleaves or hydrolyzes the internal α (1-4) glycosidic bonds in starch largely at random to produce α 1-2 bonds resulting in smaller molecular weight maltodextrin, e.g., in starch, high molecular weight amylopectin or amylose polymers are hydrolyzed to oligosaccharides. Suitable α-amylases are the naturally occurring α-amylases as well as recombinant or mutant amylases derived from Bacillus acidocaldarius.
“Granular starch” or “starch granules” means a water-insoluble component of edible grains which remains after removal of the hull, fiber, protein, germ, and other soluble substances through the steeping, mechanical cracking, separations, screening, countercurrent rinsing and centrifugation steps typically used in a grain wet-milling process. Granular starch comprises intact starch granules containing almost exclusively packed starch molecules (i.e., amylopectin and amylose). When corn is processed, the granular starch component comprises about 99% starch; the remaining 1% being comprised of protein, ash, fiber and trace components tightly associated with the granules. The packing structure of granular starch retards the ability of α-amylase to hydrolyze starch. Gelatinization of the starch is utilized to disrupt the granules to form a soluble starch solution and facilitate enzymatic hydrolysis.
“Liquefaction” or “liquefy” means a process by which starch is converted to shorter chain and less viscous dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of α-amylase. In commercial processes, it is preferred that the granular starch is derived from a source comprising corn, wheat, milo, sorghum, rye potato etc. However, the present invention applies to any grain starch source which is useful in liquefaction, e.g., any other grain or vegetable source known to produce starch suitable for liquefaction. The temperature range of the liquefaction is generally any liquefaction temperature which is known to be effective in liquefying starch. The temperature of the starch is between about 80° C. to about 115° C., or from about 100 to about 110° C., or from about 105 to about 108° C. for low temperature liquefaction. High temperature liquefaction temperatures are between approximately 140-145° C.
“Saccharification” means the conversion of the liquid product (liquefact), in this case, the hydrolysis of the soluble dextrins to dextrose monomers using enzymes such as glucoamylase. Saccharification products are, for example, glucose and other saccharides such as disaccharides and trisaccharides.
“Starch solution,” means the water-soluble gelatinized starch which results from heating granular starch. Upon heating of the granules to above about 72° C., granular starch dissociates to form an aqueous mixture of loose starch molecules. This mixture comprises, for example, about 75% amylopectin and 25% amylose in yellow dent corn, and forms a viscous solution in water. In commercial processes to form glucose or fructose, it is the starch solution, or starch slurry, which is liquefied to form a soluble dextrin solution.
“Steep liquor” means a liquid which is drawn from steeped grain kernels during the steeping process. The steep liquor contains a significant portion of the soluble components of the grain.
The α-amylases suitable for practicing the present invention exhibit the characteristics of the KSTM #2037 α-amylase described in Japanese Patent Application No. JP10136979, filed on May 26, 1998, titled “Novel Acidic Alpha-amylase and Its Production, hereby incorporated by reference in its entirety. After the fermentation, the microbial cells were removed by conventional means, leaving the extracellular enzyme in solution. The enzyme containing solution was then concentrated using ultrafiltration and freeze-dried. The activity of the resulting enzyme preparation was found to be 2170 ASAA units/g in Lot Number 1, and 2520 ASAA units/g in Lot Number 2.
The α-amylase activity of the KSTM #2037 α-amylase was measured by determining the hydrolysis of soluble potato starch as described in JP10136979. In a typical run, 10 ml of 1% potato starch solution (pH 4.5 acetate buffer) in an 8×180 mm test tube is placed in a constant temperature bath maintained at 40° C. for more than 5 minutes. One ml of properly diluted sample is added with agitation and the mixture is incubated for 10 minutes in the constant temperature bath. After 10 minutes, the enzyme reaction is interrupted by rapidly placing 1 ml of the reaction mixture into 10 ml of N/10 HCl. Then, 0.5 ml of the resulting solution is mixed with 10 ml of an iodine solution, and the mixture is shaken well. After two minutes the optical density is determined by a photoelectric calorimeter at 660 millimicrons, using 10 mm cuvettes. The activity unit is calculated using the following equation:
where S: optical density obtained without enzyme
One Acid Stable Alpha Amylase Unit (ASAA Unit) is defined as that activity of enzyme which causes 1% blue value reduction of 1% potato starch solution at 40° C., for one minute. A unit of the diluted sample for purposes of this application generally is between approximately 2 and 5 ASAA. The novel α-amylase described above acts to hydrolyze starch substrate to form, mainly, maltopentaose and maltohexaose.
In low temperature liquefaction, as shown in
In either high or low temperature conventional, commercial liquefaction procedures, the commercial α-amylases continue hydrolysis during a secondary liquefaction step to reduce the size of the dextrins, although a second addition of α-amylase may be added to further hydrolyze the starch. The resulting liquefact from such commercial procedures is expected to include, in the absence of further treatment, by-products at least as a result of the chemical additions. Maltulose is a by-product which is produced at liquefaction pH values greater than 6.0. Maltulose is known to lower glucose yields in subsequent saccharification procedures.
As seen in
In the single jet cooker low temperature liquefaction process shown in
In the double jet cooker high temperature liquefaction process shown in
In the double jet cooker low temperature liquefaction process shown in
The following examples are representative, and are not intended to limit the advantages conferred through the use of the invention. However, one of ordinary skill in the art would be able to substitute conditions, reactors, grains, temperature, enzymes and the like according to the above disclosure.
The following examples performed starch liquefaction using a reactor composed of 50 feet of 0.24 inch diameter (0.21 inch i.d.) stainless steel tubing bent into an approximately 10 inch diameter coil that was 5.5 inches high. The coil was equipped with an 11.5 inch, in-line static mixer (Cole-Parmer #G-04669-60) mounted 4 feet from the anterior end of the coil. The posterior end of the coil was equipped with a Swagelok in-line adjustable pressure relief valve (#SS-4CA-5) set at a cracking pressure of about 20 psi. Starch slurry was fed to the coil at a rate of app. 70 ml/min with a piston metering pump. The coil was heated by immersion in a glycerol-water bath heated to a temperature of approximately 103-120° C. for low temperature liquefaction and approximately 140-155° C. for high temperature liquefaction. The temperature of the water bath was maintained using a circulating heater/temperature controller (Fisher Scientific model 7305).
Four commercial, thermostable α-amylases, listed below, were compared with KSTM #2037 α-amylase, as described below.
1) SPEZYME™ FRED L. [Genencor International] at 100 LU/g ds.
2) Termamyl™ LC. [Novo Nordisk] at 115 LU/g ds.
3) Termamyl™ SC. [Novo Nordisk] at 75 LU/g ds.
4) G-995 [Enzyme Bio-Systems] at 75 LU/g ds.
Each α-amylase was tested using the same starch slurry, but under conditions designed to optimize enzymatic performance. Specifically, for all four of the commercial α-amylases, a 35% dsb starch slurry having a pH of 4.0 was treated to contain 20 ppm calcium and 50 ppm SO2 to thermostabilize the enzymes and adjust the pH to 5.6. The KSTM #2037 α-amylase (2170 ASAA units/g) was tested at the slurry pH of 4.0 without any addition of the calcium or SO2 thermostabilizers. All five resulting slurry/enzyme mixtures were subjected to a low temperature jet liquefaction process, such as shown in
The DE of each of the five hydrolysates continuing in a secondary liquefaction step was then determined at 95° C. at selected intervals of time. The DE progression for all of the enzymes is shown in
Two of the three commercial enzymes discussed above, SPEZYME™ FRED L and Termamyl™ SC, also were compared to the KSTM #2037 α-amylase using high temperature liquefaction procedures, such as shown in
The KSTM #2037 α-amylase (2.5 ASAA units/g. ds and 5.0 ASAA units/g ds) also was tested at pH 3.5 using the above liquefaction procedure as described in U.S. Pat. No. 3,654,081, hereby incorporated by reference. The pH 3.5 starch slurry was prepared by suspending 9380 grams of corn starch in 14.2 liters of distilled water containing 50 ppm SO2 to produce a starch concentration of 35% dsb. The results were as follows: 0.5 hours, 8.4-9.0 DE; and 1.0 hours, 11.2-11.6 DE.
These comparison studies surprisingly suggest that the KSTM #2037 α-amylase enzyme, after primary low temperature liquefaction, for examples as shown in
Without wishing to be bound by any theory, it is believed that KSTM #2037 α-amylase is inactivated, or at the least, altered in some fashion at the end of the low temperature liquefaction process of
Concentration of KSTM #2037 α-Amylase
The concentration of the KSTM #2037 α-amylase enzyme required to produce at least a 9-10 DE liquefact in the primary jet liquefaction step was studied. In a typical jet-cooking experiment, KSTM #2037 α-amylase was added to 650 g of distilled water containing 50 ppm SO2 at concentrations of 100 ASAA and 150 ASAA units/g ds of starch. Starch (350 g from Cerestar, USA) was then added and mixed continuously to produce a 35% dsb slurry. The pH of the slurry was then adjusted to pH 4.0. The slurry was passed through a bench cooker maintained at 107° C. for 5 minutes then flashed to atmospheric pressure at 95° C. for secondary liquefaction. The results are summarized in Table 1, which shows that the 100 ASAA unit/g dose did not produce the targeted DE, and both dosages did not continue to hydrolyze starch under secondary liquefaction conditions.
The effect of the pH of the 35% tsb starch slurry on the liquefaction of starch by KSTM #2037 α-amylase was studied under the conditions of Example 1 using 150 ASAA units/g ds. The liquefact recovered after primary liquefaction was held at 95° C. with DE determinations made after primary and at 30 minute and 60 minute intervals. The results are shown below in Table 2.
The Table 2 results show that the KSTM #2037 α-amylase produced a DE of 9-10 under jet cooking conditions at starch slurry pHs between at least about 3.5 to 5.0 thereby showing that the pH of a conventional starch slurry need not be adjusted for liquefaction. It will be clear to those skilled in the art that eliminating this initial pH adjustment also eliminates the need for a second pH adjustment prior to addition of the saccharification enzyme, and particularly in view of the results shown in
KSTM #2037 α-amylase was tested to determine the effect of calcium on the KSTM #2037 α-amylase during liquefaction at pH 4.0. Calcium generally enhances the stability of thermostable α-amylases derived from Bacillus licheniformis and Bacillus stearothermophilus used in the industrial starch process. The effect of calcium on the thermostability of KSTM #2037 α-amylase under 107° C. low temperature jet cooking conditions of starch at pH 4.0 and 150 ASAA units/g is shown in Table 3.
Unlike other thermostable conventional α-amylases, the results in Table 3 show that calcium does not affect the hydrolytic activity of KSTM #2037 α-amylase under jet cooking conditions of the starch at pH 4.0. On the other hand, the results show that, if it is desirable to add calcium for another purpose, the calcium will not interfere with the activity of the KSTM #2037 α-amylase. Starches, such as ground corn, contain phytic acid which binds calcium thereby decreasing the performance of conventional α-amylases which require calcium addition for stability.
The high temperature jet-cooking process described in U.S. Pat. No. 3,654,081, hereby incorporated by reference, was used in this example to liquefy starch using thermostable alpha-amylase. A pH 3.5 starch slurry was prepared by suspending 9380 grams of corn starch in 14.2 liters of distilled water containing 50 ppm SO2 to produce a starch concentration of 35% DSB. The starch slurry was then gelatinized in a pilot plant steam jet-cooker at 140° C. with a 5 minute holding loop. The gelatinized starch was then flashed directly into a temperature-regulated vessel maintained at between 95° C.-98° C., and KSTM #2037 alpha-amylase was added at a concentration of 2.5 ASAA units/g. dsb. Samples were taken at 30-minute intervals to determine DE production. The enzyme reaction was terminated by heating the liquefied starch at 110° C. for 2 minutes. The results are shown below in Table 4.
Table 4 demonstrates that thermo-inactivation of the KSTM #2037 α-amylase should be performed prior to saccharification for processes which utilize liquefaction temperatures of less than and around 95° C. The increase in DE shown in the 110° C. inactivation step represents the type of increase that was seen during the low temperature liquefaction process described above,
KSTM #2037 α-amylase was tested to determine the effects of high liquefaction temperatures. The results shown in
The low temperature jet-cooking process shown in
Comparison of the results in Table 1 and Table 5 shows that, if low enzyme dosages are desired in a low temperature liquefaction process, acceptable DE values may be obtained within one hour of an added secondary liquefaction step (see
The data present in Examples 1 through 7 demonstrate the liquefaction of a starch slurry at a pH between 3.5-5.0 with no added calcium using varying concentrations of KSTM #2037 α-amylase and conventional high and low starch processing temperatures. The data further demonstrate that the KSTM #2037 α-amylase produces acceptable DE amounts in a single low temperature primary liquefaction step at concentrations of about at least 140 ASAA units/g dsb, and in a high temperature single liquefaction step, at concentrations of about 1-10 ASAA units/g. The data also shows that the KSTM #2037 α-amylase may be added in one or more dosages in low temperature liquefaction, and that a first lower dosage of approximately 10 to 35 ASAA units/g followed by a second addition of about 1-10 ASAA units/g may be used to produce acceptable DE amounts in a secondary liquefaction step. In the low temperature liquefaction process described above and shown in
The following examples discuss optional saccharification processes and compare the KSTM #2037 α-amylase liquefacts to conventional α-amylase liquefacts.
The KSTM #2037 α-amylase hydrolyzed, liquefied starch sample collected and described in the low temperature liquefaction process described in Example 1 was saccharified using glucoamylase (Optidex™ L-400) and glucoamylase containing different concentrations of pullulanase (Optimax™). Referring now to Table 7, KSTM #2037 liquefacts were saccharified with the following enzymes: “A” Set using 0.22 units/g of glucoamylase; “B” Set using a mixture of 0.22 units/g of glucoamylase and 0.069 acid stable pullulanase unit/g; “C” Set using a mixture of 0.22 units/g of glucoamylase and 0.147 acid stable pullulanase units/g; “D” Set using a mixture of 0.22 units/g of glucoamylase and 0.330 acid stable pullulanase units/g; and “E” Set using a mixture of 0.22 units of glucoamylase and 0.880 acid stable pullulanase units/g. Saccharification was carried out at 32% ds at 60° without any pH adjustment of the pH 4.0 liquefact. Samples were taken at different intervals of time and the composition of the reaction products was determined by using a standard high pressure liquid chromatographic method under the following conditions:
The results are shown below in Table 7 which demonstrates that using KTSM alpha-amylase liquefacts for saccharification results in typical dextrose levels. Additional saccharification studies were conducted at 103° C., 105° C., and 107° C. using Sets “B” and “D”. These additional studies demonstrated attainment of about 95% glucose in 24-48 hours.
The liquefact from KSTM #2037 α-amylase produced glucose syrup having greater than 95% glucose which is an acceptable target level for commercial saccharification processes. A control study was conducted using the SPEZYME™ FRED L enzyme described in Example 1. The liquefact for the control had a DE of 9.07 and was inactivated by lowering the pH to 4.0 to 4.2 at 95° C. The KSTM #2037 and SPEZYME™ FRED L liquefacts were saccharified with OPTIMAX™ 2080 under the same conditions shown in Table 7. The results of the comparison are in
Evaluation of the novel KSTM #2037 α-amylase on the liquefaction of starch in the pH range of 3.5 to 5.0 with no added calcium provided at least three different options for operating conditions. Those skilled in the art will recognize that the processes described below are subject to many variations that are included in the scope of the invention.
As shown in
In this process shown in
In this process shown in
It will be understood by those skilled in the art that a wide range of changes and modifications can be made to the preferred embodiment described above, depending upon the desired end product. It is therefore intended that the foregoing detailed description not be limiting of the invention which scope, including all equivalents, is defined by the following claims.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10026753 | Dec 2001 | US |
| Child | 12070679 | US |
