BACKGROUND OF THE INVENTION

Field of The Invention:

The present invention is directed to the broad field of enzyme process technology. More specifically the present invention is directed to a method for removing contaminants from resins, particularly ion exchange and fractionation resins by use of enzyme treatment. The present invention also is directed to a method of producing purified corn sweeteners, particularly corn syrup and high fructose corn syrup by use of resins previously cleaned with enzymes.

Description of the Related Art:

Resins provide a powerful tool in analytical chemistry for separation of organic or inorganic ionic or nonionic species. The resins are typically contained within a column or vessel. Resins are manufactured from substances such as cross linked polystyrene, which have been chemically cross linked together, also known as DVB (di-vinyl benzene), in both macroporous (large holed) and gel (small holed) forms. Additional resins used in food processing are formed from acrylates. Resins can typically be reused by regenerating the resin. Deactivation or fouling of the resin by contaminants can destroy the resin. In addition to thermal instability (desulfonation or loss of functionalization) ionic, particulate (sludge) and polymeric materials cause fouling.

An ion exchange resin is a synthetic organic polymer, often based on cross- linked polystyrene, that has been derivatized by the addition of charged groups to produce materials that will exchange counterions when suspended in aqueous solutions. Cationic exchange resins have fixed acidic substituents based on, for example, sulfonic acid which is a strong acid exchanger or carboxylic acid which is a weak acid exchanger. Anionic exchange resins have fixed substituents based on for example, quaternary ammonium or

ethoxyamine groups or amines which are weak base exchangers. Other functional groups may be attached to the resin skeletin to provide more selective behavior. The degree of derivatization and the extent of cross-linking of the resin determines the overall capacity for ion exchange. A technique called chromatography is used to separate mixtures of substances based on their ability to partition between a liquid and solid phase. Ion-exchange chromatography is defined as a type of chromatography in which a solid support is an ion- exchange material used to separate mixtures of charged molecules or ions. This can be carried out on a preparative scale or as a modifcation of High Pressure Liquid Chromatography.

Chromatographic separations depend on differences in exchange potential, elution agent, column length and loading, flow rate particle size and temperature. Chromatographic separations can be used for a wide variety of uses including the purifcation of corn sweeteners such as corn syrup (CSU) and High fructose corn syrup (HFCS). Corn sweeteners are manufactured by hydrolyzing corn starch. A dextrose equivalent (D.E.) number is given to the finished ingredient depending on the degree of conversion allowed in the reaction. The lower the number, the lower the degree of conversion. Higher numbers indicate more complete conversion with 92 D.E. indicating complete conversion from corn starch to dextrose (Corn Sugar). High fructose corn syrup can be enzyme converted to 97 D.E. and fractionated to 97-99 D.E. Corn sweeteners have been used in ice-cream and yogurt fruit preparations for years. Two reasons dominate the list of reasons for corn sweetener incorporation. First, corn sweeteners can give desirable body or "chewiness" to finished products that sucrose alone can't provide. Second, certain corn sweeteners impart more sweetness per pound than sucrose so they are less expensive to use. Obviously, because of the great value of corn sweeteners an improved method to regenerate resins which can be used to prepare purified corn sweeteners would be of great benefit. Currently, Ion Exchange and fractionation chromatography are the methods of choice in purifying corn sweeteners, however, the columns or vessels which contain the resin frequently become contaminated and are difficult to clean. The current method of choice used for regeneration of resins involves the use of chemicals. The chemicals typically used include but not limited to: hydrochloric acid (HC1), sodium hydroxide (NaOH), ammonium hydroxide (NH ₄ OH) and sodium carbomate

(Na ₂ CO ₃ ). Preliminarily, the resin is rinsed with a rinsing agent, usually water and then taken through a series of chemical treatment steps. However, it is not unusual that even after chemical treament or regeneration that the resin is still contaminated with microorganisms, as for example bacteria.

It is an object of the present invention to provide a method for cleaning resins which utilize the action of enzymes, specifcally, proteases, lipases, carbohydrates and alpha amylases in a process area where enzyme use has never been utilized. The present invention is especially well-suited for cleaning ion exchange and fractionation resins used to purify corn sweeteners.

SUMMARY OF THE INVENTION

The present invention relates to an enzymatic method used to clean resins, particularly ion exchange and fractionation resins. The method can be used alone or in combination with chemical methods. The method allows for improved production of liquids that contain contaminants as for example proteins, carbohydrates, lipids, and residual unconverted starches that require fractionation or ion exchange treatment. The invention also relates to a the use of resins for the production of corn sweeteners, in particular, corn syrup and high fructose corn syrup.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to an enzymatic method of removing contaminants from resins, in particular ion-exchange and fractionation resins.The enzymes are exposed to the contaminated resin and optionally recirculated through the resin for a particular time designated. The amount of time the enzyme is recirculated is a production factor. The shorter the time allotted for treatment of the resin the larger the enzyme dose necessary to clean the resin.

The present invention can incorporate the novel use of enzymes in combination with traditional chemical treatment means to remove contaminants from resins. Enzymes have a characteristic optimum pH at which their activity is maximal. The pH-activity profiles of enzymes reflect the pH at which important proton-donating or proton-accepting groups in the

enzyme catalytic site are in their required state of ionization. The optimum pH of an enzyme is not necessarily identical with the pH of the normal surroundings, which may be just above or below the optimum pH.

The present invention can be served with exhisting proteases, lipases, carbohydrases and alpha amylases which operate at a p.H. of 1-9, more preferably between

6-8 and still more preferably between 6.5-7.5 and a temperature optimum of 90-180, more preferably 120-130 F.

While one skilled in the art would appreciate that a variety of enzymes could be used in the present invention depending on the contaminant in question. The following enzymes are routinely used and are offered as examples only and are not intended to limit the scope of the invention. The generic name is followed by the trade name in parenthesis.

Protease/peptidase (Alcalase, Neutrase, Flavourzyme)

Beta Glucanase (Finizym, Cereflo)

Lipase (Palatase, Lipozyme) Alpha Amylase (BAN, Termamyl Type L)

Carbohydrase (Viscozyme)

Multi-enzyme complexes include but are limited to: Alpha Amylase, Beta Glucanase, Protease (Ceremix) Carbohydrases (Viscozyme)

For purposes of this application, Protease/ Peptidase is defined as a class of enzymes which hydrolyze (breakdown) proteins into short chains, soluble peptides and amino acids. Beta Glucanase is defined as a class of enzymes which hydrolyze beta glucans

(1,4-beta-, 1,3-beta glucans) into oligosaccharides and disaccarides. The 1 ,4-beta and 1,3-beta designations refer to the chemical bonding between the carbon atoms in the molecule upon which the class of enzymes act upon.

Oligosaccharides are defined as short chains of dextrose molecules chemically bonded together. These chains are typically 3-5 glucose units in length.

Disaccharides are defined as two dextrose units linked together. These are only formed in minor amounts with beta glucanases.

Lipases are defined as a class of enzymes which hydrolyze both short and long chain fatty acids, from the 1,3-ester chemical bonding sites, into mono-or diglycerides and fatty acids. Short chain triglycerides refer to triglycerides with sidechains with twelve carbon molecules or less, i.e. esters of lauric acid and below. Long chain fatty acids refer to fatty acids with more than twelve carbon molecules i.e. esters above lauric acid.

Alpha amylases are defined as a class of enzymes which act at random to hydrolyze the alpha- l,4glucosidic lingages in amylose and amylopectin, resulting in the formation of soluble dextrins and oligosaccharides. Alpha- 1 ,4-glucosidic linkage refers to the chemical bonding between the glucose molecules which compose starch. The number 1 carbon in one glucose molecule is bonded to the number 4 carbon of the adjoining carbon.

It is this bond which is attacked by alpha amylase, resulting in the break down products of starch, dextrins and oligosaccharides.

There are two basic types of starch: Amylose and Amylopectin. Amylose is strictly composed of alpha- 1 ,4-glucosidic linkages and is decomposed by alpha amylase. Amylopectin is the second type of starch and is composed of both alpha- 1,4-glucosidic, and alpha- 1 ,6-glucosidic bonds. Alpha amylases cannot break down 1,6 bonding, so amylopectin is more difficult to hydrolyze by alpha amylase.

Carbohydrase is defined as a broad class of enzymes which hydrolyze non- starch polysaccharides (beta- l ,4-alpha-l,4-alpha- 1,5). This class of enzymes includes: arabanasc, cellulase, beta-glucanase, hemi-cellulase, and xylanase. Arabanase hydrolyzes arabans into oligoarabans and arabanose. Cellulase hydrolyzes cellulose into oligocelluloses and cellubiose. Hemi-cellulase hydrolyzes hemi-cellulose into oligopentosans and pentosans. Xylanase hydrolyzes xylans into oligoxylans and xylose.

If two or more contaminants are identified then the optimum pH and temperature of each enzyme to be used is checked and, if possible, the enzymes are added in combination as a multienzyme complex. When two enzymes are used in combination it is rare that both enzymes are used at their optimum pH and temperature. In this case a "common" overlap area where both enzymes have a relatively high activity is used. If the pH and temperature optimum of the two or more enzymes vary greatly then they are added successively. Typically, the two or more enzymes will be used in approximately the same ratio as the contaminant or contaminants. Examples of multienzyme complexes include but are not limited to:

Carbohydrases, including arabanase, cellulase, beta-glucanase, hemi- cellulase, and xylanase (Viscozyme) Alpha Amylases, Beta Glucanases, Proteases (Ceremix)

One skilled in the art would appreciate that as advances are made and enzymes with greater tolerances especially at lower pH ranges are identified one skilled in the art would adapt these enzymes for use in in the present invention.

In another preferred embodiment of the invention, the contaminant is identified prior to enzymatic treatment. Although this step is not an absolute requirement for cleaning to occur it will save money, time, and eliminate the use of ineffective enzymes and unsuccessful cleanings. Since enzymes typically have a high degree of specificity for their substrates, ideally, the selection of the enzyme or enzyme mixture that is used to treat the resin is conducted after the contaminant or contaminants are identified. However, one skilled in the art will appreciate that it is sometimes difficult or impossible to determine the source of contaminants of the resin. Therefore, the selection of enzymes must sometimes be conducted without knowledge of contaminants.

In another preferred embodiment of the invention, prior to the treatment of the one or more enzymes the resin is pH adjusted to reside within the optimum pH range of the chosen enzyme(s). If a cation resin is to be treated, then the resin capacity should be completely exhausted using either NaOH or Na-,CO ₃ until the pH reaches a level of 6-8, optimally 6.5-7.5. If is used to exhaust the resin care must be taken as CO ₂ will form in the unit, increasing the overall pressure in the vessel.

A weak or strong base anion resin is ready to be treated after sweetening off. If the pH in the vessel is greater than 7.5, HCl should be used to complete the exhaustion of the resin.

In another preferred embodiment of the invention, the resin is thoroughly rinsed with a washing agent, as for example water, prior to tratment with one or more enzymes to remove an initial concentration of contaminant still undergoing a reaction, as for example undergoing ion-exchange. In the sugar and corn wet milling industry this step is called

"sweetening off. For purposes of this application "sweetening off' is defined as the rinsing of syrups, sugars, liquors, or other carbohydrate based liquids from the resin and vessel prior

to chemical and/or enzymatic treatment, until the total amount of organic carbon in the vessel or unit is below 3500 parts per million (ppm), more preferably 2500 ppm. Residual syrup is defined as syrup which remains in a vessel after 'sweetening off" has occurred. For purposes of this application "Initial concentration of contaminant" is defined as a level of contaminant greater than 2500 ppm, more preferably 1000 ppm and still more preferably 100 ppm. For purposes of this application "Residual level of contaminant" is defined as a level of contaminant less than 2500 ppm, more preferably 1000 ppm, and still more preferably 100 ppm.

The washing agent i.e. water, is not required to be potable although the purest water possible is preferred. Optionally, salt can be added to the water to aid in the

"sweetening off process. Preferably the water should be softened or, if available, condensate water recovered from evaporators should be used. In suger and corn wet milling industries, condensate water is the water of choice.

The resin is exposed to the washing agent until such time that the remaining initial level of contaminant that is undergoing ion exchange is eliminated. In the sugar and corn wet milling industries this rinsing with a washing agent or "sweetening off process must continue until the levels of measured total organic carbon (TOC) remaining in the unit is at a maximum of 2000 ppm, with a more preferred level at less than 1000 ppm, and a still more preferred level at less than lOOppm. For purposes of this application "total organic carbon" (TOC) is defined as a measure of all the organic carbon in a sample. Since the ultimate oxidation of organic carbon is measured as carbon dioxide, the burning of a small sample in a combustion chamber, measuring the amount of carbon dioxide emitted and subtracting the contribution from inorganic carbon, the total organic carbon level can be determined. These levels of total organic carbon or residual sugar, remaining in the unit will not interfere with the cleaning process. Levels greater than 2000 ppm may interfere with the process. The use of the rinsing agent also allows one skilled in the art to adjust the temperature of the resin before the treatment of the contaminant with one or more enzymes. The temperature of the rinsing agent used should be within or slightly greater than the chosen enzyme(s) optimum range for temperature since the pH adjustment will reduce the temperature in the vessel.

In another preferred embodiment of the invention, after treatment is complete the enzyme solution is rinsed with a solution, typically water, until the enzyme concentration in the vessel is exhausted.

In another preferred embodiment of the invention, the resin is regenerated using well-known chemical procedures. Typical concentration and chemicals used for regeneration include: For a weak base anion type resin 4% NaOH solution at 96-1 12 kg/m ³ and 5% Na-,CO ₃ solution at 112-1 18 kg/m ³ is recirculated for 30-60 minutes at 40 C. For a strong base anion resin, 4% NaOH solution at 112-120 kg/m ³ and 7% Na ₂ CO ₃ solution at 80-96 kg/m3 is recirculated for 30-60 minutes at 40 C. The invention now being generally described, the same will be better understood by reference to certain examples which are included herein for purposes of illustration only and are not to be considered to be limiting thereof.

Examples:

EXAMPLE 1. PROCEDURE FOR DETERMINATION OF CONTAMINANTS IN RESIN:

Unnecessary time and money can be saved if the type of contaminant is identified in the resin prior to selection and treatment of the contaminant or contaminants with the enzyme or enzymes. The elucidation of the contaminant is conducted by conventional methods. Typically, one skilled in the art would be aware of resin contaminants as for example: proteins; lipids; as for example fats and oils; fibers, as for example cellulose, hemi- cellulose, beta-glucan, starch, as for example amylose, amylopectin or other carbohydrates as for example arabose or xylanose. The following procedure is routinely used by one skilled in the art to identify contaminants present in the resin. The analysis for all contaminants should be carried out after filtering the resin from the flasks. Only the solution should be subjected to testing. The tests are typically harsh and involve strong oxidizing agents which will react with resins. Two grams of the resin sample is placed in a beaker, which contains a magnetic stir bar, and is filled with water to a total volume of 200ml. The pH and temperature of the solution is adjusted to the optimum pH of the enzyme being evaluated. The enzyme is

weighed so that the total percentage of enzyme in solution is equal to 3-5% of the total weight of solution. The goal at this point is to determine the composition of the contaminant. Each sample is checked using only one enzyme. The solution is mixed for 30-60 minutes and then amalyzed appropriately for the source of contamination. Analysis can be done using a number of methods, most of which are "standard methods" for a particular industry. For resins in a corn wet mill or sugar refinery, the most likely contaminants will be: protein, starch, beta-glucans, fats and lipids, with protein being the largest.

Analysis for protein can be accomplished using the Kjeldahl method from Standard Analytical Methods of the members of the Corn Refiners Association. Inc. ; Corn

Starch (Unmodified), Method B-48. There are many variations of the Kjeldahl procedure but this method is adequate and sensitive.

Analysis for fats and lipids can be accomplished using the Crude Fat (Hexane Extractable) method from Standard Analytical Methods of the members of the Corn Refiners Association, Inc. ; Feedstuffs, Method G-1 1. There are other variations of this procedure but this method is the most applicable.

Analysis for beta-glucan (corn fiber) can be accomplished using the Crude Fiber method from Standard Analytical Methods of the Members of the Corn Refiners Association. Inc. ; Feedstuffs, Method G-12. There are other variations of this procedure but this method is the most applicable

Analysis for beta-glucan (corn fiber) can be accomplished using the Crude Fiber method from Standard Analytical Methods of the _. Members of the_ Corn Refiners Association, Inc. ; Feedstuffs, Method G-12. There are other variations of this procedure but this method is the most applicable Analysis for beta-glucan (corn fiber) can be accomplished using the Crude

Fiber method from Standard Analytical Methods of the Members of the_ Corn Refiners Association. Inc. ; Feedstuffs. Method G-12. There are other variations of this procedure but this method is the most applicable

Analysis for starch can be accomplished by adding a few drops of standard iodine solution to the resin solution. If a purple, green or blue color forms in the flask upon addition, then starch in its native form is present.

EXAMPLE 2. Conditions For Enzymatic Treatment

Optionally, the contaminant or contaminant is identified by the method of Example 1. Although this is not a requirement it allows for the appropriate choice of an enzyme or enzymes for treatment as well as proper selection of chemical treatment for the contaminant. The total amount of contamination is estimated by and is expressed as a percentage of the total weight of resin plus contaminant. The resin is rinsed (Sweetened off) in the ion exchange vessel with a rinsing agent, as for example water, until the residual contaminant or contaminant concentration (substance undergoing ion exchange), as for example corn syrup or high fructose corn syrup is brought down to an acceptable level, typically, 1000-2500ppm total organic carbon. Although lower levels are even more desirable.

The pH of the resin is adjusted to reside within the pH range of the chosen enzyme or enzymes. For example, if a bacterial protease is chosen, the pH range is 5-9 , more preferably pH 5.5-7.5 and and temperature range is 20-75 C, more preferably 40-60 C. If a botanical protease is chosen such as Papain a pH range is between 2.5-10 more preferably 3-9.5 and a temperature range is between 30-60 C, more preferably 38-55 C.

The chemicals used to adjust the pH include but are not limited to NaOH, HCl, NH ₃ and Na ₂ CO ₃ . The appropriate enzyme is mixed with water and added to the contaminant. The dose is dependent on the type and level of contamination. The enzyme is recirculated through the ion exchange vessel for an appropriate time. The recirculation time is dependent upon the enzyme used, adherence to optimum enzyme conditions and extent of contamination. The circulation time is typically in the range of 1-24 hours. After treatment is complete the enzyme solution is rinsed with a solution typically water until the enzyme concentration in the vessel is exhausted. The resin can now optionally be regenerated using standard chemcal procedures. Typical concentration and chemicals used for regeneration include: For a weak base anion type resin 4% NaOH solution at 96-112 kg/m ³ and 5%

N-t-CO _j solution at 1 12-118 kg/m3 is recirculated for 30-60 minutes at 40 C. For a strong base anion resin, 4% NaOH solution at 1 12-120 kg/m3 and 7% Na2CO3 solution at 80-96 kg/m3 is recirculated for 30-60 minutes at 40 C.

EXAMPLE 3. ENZYMATIC TREATMENT OF CORN GLUTEN PROTEIN

CONTAMINANT

The present example illustrates the method used to remove the contaminant corn gluten protein from cation and anion ion exchange resin that could not be removed with either chemical treatment or water.

Initially the resin is treated by traditional chemical means. The procedure for the use of the chemical trial cleaning is to circulate 7% HCl (IN) through the cation resin

(Dowex 88) and 4% (IN) NaOH through the anion resin (Dowex 66) for four (4) hours, at

120 F, which is similar to plant conditions. The chemicals are rinsed from the columns and then adjusted appropriately for the enzyme cleaning trial.

The resin is rinsed (Sweetened off) using water, adjusted to the optimum enzyme operating temperature. The pH of the vessel is adjusted to 6.8 using a 5% sodium carbonate solution, which is near the enzyme optimum pH.

The enzyme used is a 0.5L peptidase (Neutrase). Thirty kg of peptidase is added per 1500 ft ³ of resin. This amount of enzyme is estimated to be 1% of the total amount of contaminant. The enzyme is added via suction through the cation and anion vessels. The solution is recirculated through the vessels for a total time of four hours. The enzyme is then rinsed from the vessel. Samples of the enzyme rinse is analyzed for total protein content by standard methods. (Kjeldahl analysis (Standard Analytical Methods of the Members of the Corn Refiners Association, Method B-48). The analyzed solutions are obtained after each hour of circulation with the following results. The amount of enzyme present in the sample which would be measured as protein is subtracted.

Hour % Protein in Solution % Protein in Solution

(HCl and NaOH only) (Enzyme Cleaning)

1 0.2% 0.5%

2 0.3% 0.8%

3 0.7% 1.1%

4 0.7% 1.4%

The solution in the vessels become milky brown in appearance within five minutes of enzyme addition. The values are extremely significant as prior to enzyme treatment, the protein present in the vessels could be seen through sight glasses on the vessels.

After two treatments no protein pieces could be seen and the service times of the units,

reduced by as much as 70 % returned to normal. It is clearly apparent from this example that enzyme treatment is much more efficient than the chemical treatment used in the prior art. The enzyme treatment is as much as 100% more efficient.