1. Field of the Invention
The present invention relates to a method of enhancing the extraction of a proteinase inhibitor, and more specifically, to a method of improving the yield and purity of Proteinase Inhibitor-II (PI2) extracted from whole potatoes.
2. Background of the Prior Art
The extraction, isolation and purification of plant-derived proteins is well known in the field of biochemistry. In 1972, Melville and Ryan reported a large-scale preparation for isolating Chymotrypsin Inhibitor I from potato tubers (Melville, J. C. and Ryan, C. A. Chymotrypsin inhibitor I from potatoes. J. Biological Chem., 247: 3445-3453, 1972). According to the method of Melville and Ryan, potatoes were sliced with peels intact and soaked in a sodium dithionite solution, homogenized, and expressed through nylon cloth. The resulting juice was adjusted to pH 3, centrifuged at 1000xc3x97 g for 15 minutes at 5xc2x0 F., and the supernatant collected and fractionated with ammonium sulfate.
Purification was achieved through water washing and heat treatment whereby clear filtrates of heated fractions were pooled and lyophilized. Suspending the lyophilized powder in water, dialyzing it against water for 48 hours, and lyophilizing the resulting clear filtrate obtained a crude extract. Resuspended extract was then centrifuged and applied to a column of Sephadex G-75. Collected fractions containing the Inhibitor I were pooled, evaporated, and desalted on a column of Sephadex G-25. The resulting gel-filtered inhibitor product was determined to be approximately 90% Inhibitor I protein purified by dissociation on a Sephadex G-75 column and desalted on a column of Sephadex G-25.
The Ryan lab followed-up by reporting the isolation and characterization of Proteinase Inhibitor II in much the same manner as described for Inhibitor I (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976). Bryant et al. differentiated potato-derived proteinase inhibitors into two groups based on their respective stabilities to a temperature of 80xc2x0 C. for 10 minutes. Proteinase Inhibitor I (PI1) is characterized as a tetrameric protein composed of four hybridized isoinhibitor protomer species having a molecular weight of 39,000, whereas PI2 is characterized as a dimeric inhibitor comprising four isoinhibitor promoter species having a molecular weight of 21,000.
The extraction and isolation of proteinase inhibitor proteins from potatoes is described in WO 99/01474. Proteins from potato tubers are extracted in soluble form in an aqueous/alcohol extraction medium, such as dilute formic acid and 20% ethanol. The alcohol extract is heated to a first temperature to denature most of the unwanted proteins and cooled to a second temperature to form a precipitate phase constituting the debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. The heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium, such as dilute formic acid.
Recently, PI2 has been implicated in playing a role in extending satiety in subjects fed a nutritional drink composition containing PI2. U.S. patent application Ser. No. 09/624,922 describes that subjects reported a significant reduction in hunger for up to 3xc2xd hours post meal when fed a meal comprising a nutritional drink composition containing PI2. Likewise, fullness ratings were enhanced, and each study subject lost an average of 2 kg over a 30-day period without experiencing the adverse side effects typically associated with appetite suppressing compounds. Mechanistically, it is thought that as a trypsin and chymotrypsin inhibitor, when consumed by a subject, PI2 stimulates the release of endogenous cholecystokinin, a known putative feedback agent effective in reducing the desire to intake food.
Existing methods for the extraction of proteinase inhibitors involve several laborious and time consuming steps and result in losses of yield and reduced purity of the recovered proteinase inhibitor. In addition, the most promising prior art methods rely on the use of ethanol in the extractant solution which, at the concentrations used, makes the solution flammable. None of the prior art processes have been demonstrated on a commercial scale. Accordingly, a need exists for a large-scale extraction process to extract PI2 in a cost-effective and efficient manner meeting industrial qualitative and quantitative standards.
Plant material containing a desired proteinase inhibitor is combined with a solution of an organic acid and a salt. The plant material is comminuted, forming a slurry in the acid and salt solution, to reduce the particle size and increase the surface area of the particles to improve the efficiency of the extraction. The process of comminution is selected to reduce the particle size without denaturing the desired proteinase inhibitor through local heating effects. The acid and salt solution enhances the extraction of the proteinase inhibitor from the comminuted plant material and protects it against degradation by other compounds that may be released from the ruptured plant cells. Once extracted into solution, the proteinase inhibitor is isolated and purified by centrifugation, clarification, filtration and drying of the extract solution. The acid and salt are removed during the filtration stage so as not to adulterate the purified proteinase inhibitor product.
In a preferred embodiment, proteinase inhibitor II (PI2) is extracted from whole potato tubers. Organic acids known to be effective in the process include acetic, ascorbic, citric and formic acid. Formic acid was found to result in the highest purity and highest yield of the final PI2 product. The formic acid content of the solution is adjusted in the range of 0.5% to 2.5% w/w, with a preferred content of approximately 1.5%. Sodium chloride is added to the extractant solution to increase the solubility of the potato proteins. Sodium chloride concentrations of between 1 N and 3 N are used. with a preferred concentration of approximately 1.5 N. The solution is added to the potatoes in a weight ratio of between 1:1 and 1:10, with a preferred ratio of 1:2.5 extraction solution:potato, w/w, respectively.
Comminution is accomplished by grinding. A target particle size is in the range of 100 to 1500 xcexcm. In this range, product yields were increased and flow characteristics of the slurry were acceptable. Decreasing the particle size below 100 xcexcm resulted in a lower recovery of PI2 and did not improve the flow characteristics. Grinding for an extended period of time also resulted in a reduced PI2 yield, most likely due to an increase in temperature and the release of undesired proteases that reduce the PI2 yield. The formic acid and sodium chloride are efficiently removed during the filtration stage.
An object of the present invention is to provide an improved method for the extraction of proteinase inhibitors from plant materials.
Another object of the invention is to provide an improved method for the extraction of proteinase inhibitor II from potato tubers which does not rely on the use of ethanol in the extraction solution.
A further object of the invention is to provide a method of extracting proteinase inhibitor II from potato tubers that is efficient and cost-effective on a commercial scale.
The extraction and isolation of PI2 from potatoes begins with the addition of an organic acid, preferably formic acid, and a salt, preferably sodium chloride, to raw potatoes. The mixture is subjected to comminution to reduce the particle size of the potato particles and extract soluble proteins. Centrifugation is used to remove solids and the liquid fraction is heated at a temperature sufficient to denature many undesired proteins but not PI2. The solution is again centrifuged to remove the insoluble denatured proteins and the liquid fraction is microfiltered to remove relatively large particles. Ultrafiltration is used to remove the organic acid and salt and further purify the PI2 in the retentate.
A process for the extraction of PI2 from whole potatoes was developed in an attempt to maximize yield, minimize impurities, minimize cost, and achieve commercial feasibility. The extraction solution was evaluated based on the ability of the process to solubilize the PI2, protect the PI2 from degradation, and maximize total PI2 removed from the insoluble potato components, while minimizing the amount of co-solubilized proteins. The extraction solution incorporated the solubility and functional stability of PI2 in acidic media and at elevated temperatures. An extraction solution containing sodium chloride and formic acid has been found effective for this purpose. The ratio of extraction solution utilized to raw material extracted was minimized for cost purposes, while producing the maximum yield of PI2 per kilogram of raw potato tubers.
Reverse Phase HPLC Method
The amount of PI2, Kunitz and carboxypeptidase inhibitors was measured using reverse phase HPLC. A Microsorb C-18 column (4.6 mmxc3x97250 mm, 5 xcexcm particles with 300 Angstrom pore size; Varian Analytical Instruments) was used. Two mobile phase solvents were prepared, solvent A was 800 g deionized H2O, 150 g acetonitrile, and 0.95 grams trifluoroacetic acid, and solvent B was 850 g acetonitrile and 0.85 g trifluoroacetic acid. Approximately 50 mg of the sample was added to 100 ml of solvent A. The sample was vortexed for 30 seconds, and then centrifuged at 10,000 rpm for 10 minutes. The supematant was collected for RP-HPLC analysis. One hundred xcexcl of the sample was injected into the column, with the pump set at 800-2500 psig, and a temperature of 30.0xc2x0 C. The other flow rate, time, and solvent compositions are as set out in Table 1. The diode array of the detector was set at 220 nm.
An external standard was prepared to construct a standard curve for calibration of the column. Five mg of BSA were dissolved in 10 ml of solvent A. Four volumes, i.e., 25, 50, 75, and 100 xcexcl, were injected into the column. A calibration curve was generated from the results.
USPTO Backgrounds
USPTO Backgrounds