# Patent

## Title
Method for increasing asparaginase activity in a solution

## Abstract

A stable asparaginase solution is disclosed. In one aspect, drinking water is treated to reduce the level of chlorine to enhance the residual enzyme activity of asparaginase. The treatment can occur by removing chlorine constituents or by supplying additives that reduces the level of chlorine. Additives can include reducing agents and chlorine scavengers. Removal technologies can include use of activated carbon, ion exchange, and air stripping.

## Background
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a method for reducing the amount of asparagine, a precursor of acrylamide, in a food product. More specifically, this invention relates to increasing the stability of the enzyme asparaginase in a solution.
2. Description of Related Art
As discussed in U.S. Pat. No. 7,037,540, acrylamide has been found in thermally-processed foods containing asparagine. The level of acrylamide formed in some food products can be reduced by adding the enzyme asparaginase to the food product prior to cooking the food product.
The addition of acrylamide reducing enzymes such as asparaginase to food products on a commercial scale, as opposed to a batch scale, presents several challenges. For example, the enzyme asparaginase must contact free asparagine to facilitate the hydrolysis of asparagine. As the enzyme is typically supplied in a relatively concentrated form, the enzyme is ideally mixed and diluted in a water-based solution prior to contacting the food product with the enzyme solution. For example, contacting the food product with the enzyme solution can comprise forming a dough and admixing an enzyme solution with the dough.
A known way to quantify the activity of an enzyme is by referring to the enzyme in terms of units. One unit of enzyme activity is defined as the amount of enzyme required as a catalyst to convert one micromole of substrate in one minute. Thus, knowing the relative concentration of a substrate or compound such as asparagine in a food product, and the amount of food product, one can calculate the units of enzyme, such as asparaginase, required to convert the desired chemical compound, in this case, asparagine, into a different chemical compound.
For previously unknown reasons, even when excess doses (meaning more than the mathematically expected amount required to convert all the asparagine in the food product) of the asparaginase enzyme are used in a food product, such as potato mash or corn masa, there oftentimes are still measurable levels of asparagine in the dough. Because it is desired to reduce the level of acrylamide formed when certain foods are thermally processed, it would be desirable to have a system and method of maximizing the effectiveness of an enzyme used to reduce acrylamide pre-cursors in food products made on a commercial scale.
SUMMARY OF THE INVENTION
The present invention, in one aspect, is directed towards a method of making a stable asparaginase solution from drinking water by removing chlorine from the water. In one aspect, chlorine is removed by ion exchange, reverse osmosis, activated carbon, and/or by air stripping. In one aspect, additives such as reducing agents and chlorine scavengers are used to treat the drinking water. The treated water, in one aspect, is then admixed with asparaginase to make an asparaginase solution. The above as well as additional features and advantages of the present invention will become apparent in the following written detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives and advantages thereof, will be best understood by reference to the following detailed description of illustrative embodiments when read in conjunction with the accompanying drawings, wherein:
FIG. 1a is a graphical representation of the residual enzyme activity after various treatments of drinking water; and
FIG. 1b is a graphical representation of the residual enzyme activity of various salt water mixtures.



DETAILED DESCRIPTION
In one embodiment, the present invention is directed towards providing a water-based solution that enhances asparaginase stability and preserves asparaginase activity. Enhanced asparaginase activity can translate into more effective acrylamide reduction in food products because asparagine is a pre-cursor of acrylamide. As used herein, the term “enzyme activity” is expressed in units. Each unit of asparaginase can hydrolyze one micromole of asparagine in one minute.
In one embodiment, the food product in which it is desired to reduce the level of acrylamide formed upon thermal processing is formed from a dough. The term “fabricated snack” means a snack food that uses as its starting ingredient something other than the original and unaltered starchy starting material. For example, fabricated snacks include fabricated potato chips that use a dehydrated potato product as a starting material and corn chips that use masa flour as its starting material. It is noted here that the dehydrated potato product can be potato flour, potato flakes, potato granules, or other forms in which dehydrated potatoes exist. When any of these terms are used in this application, it is understood that all of these variations are included. By way of example only, and without limitation, examples of “fabricated foods” to which an asparaginase solution can be added include tortilla chips, corn chips, potato chips made from potato flakes and/or fresh potato mash, multigrain chips, corn puffs, wheat puffs, rice puffs, crackers, breads (such as rye, wheat, oat, potato, white, whole grain, and mixed flours), soft and hard pretzels, pastries, cookies, toast, corn tortillas, flour tortillas, pita bread, croissants, pie crusts, muffins, brownies, cakes, bagels, doughnuts, cereals, extruded snacks, granola products, flours, corn meal, masa, potato flakes, polenta, batter mixes and dough products, refrigerated and frozen doughs, reconstituted foods, processed and frozen foods, breading on meats and vegetables, hash browns, mashed potatoes, crepes, pancakes, waffles, pizza crust, peanut butter, foods containing chopped and processed nuts, jellies, fillings, mashed fruits, mashed vegetables, alcoholic beverages such as beers and ales, cocoa, cocoa powder, chocolate, hot chocolate, cheese, animal foods such as dog and cat kibble, and any other human or animal food products that are subject to sheeting or extruding or that are made from a dough or mixture of ingredients.
The use of the term “fabricated foods” herein includes fabricated snacks as previously defined. The use of the term “food products” herein includes all fabricated snacks and fabricated foods as previously defined.
As referred to herein, the thermally-processed foods include foods that can be treated with an asparaginase solution, by way of example and without limitation, all of the foods previously listed as examples of fabricated snacks and fabricated foods, as well as French fries, sliced potatoes, yam fries, other tuber or root materials, cooked vegetables including cooked asparagus, onions, and tomatoes, coffee beans, cocoa beans, cooked meats, dehydrated fruits and vegetables, heat-processed animal feed, tobacco, tea, roasted or cooked nuts, soybeans, molasses, sauces such as barbecue sauce, plantain chips, apple chips, fried bananas, and other cooked fruits.
According to some such embodiments, the desired ingredients for making the dough are mixed together with water, and the desired amount of asparaginase is also mixed with treated water to make an asparaginase solution. The asparaginase solution can then be added to the dough. In one embodiment, an asparaginase solution is mixed directly with desired ingredients to make a dough. The dough can then be made into a thermally processed food product.
In a commercial facility, the water used to form the dough and the asparaginase solution is that water that is readily available to the facility, which is typically the drinking water supplied to an end-user from the local municipal water supply. As used herein, “drinking water” shall mean the water supplied from a potable water supply, and includes, but is not limited to, water from a municipal water supply. Almost all U.S. municipal water supplies add enough chlorine to drinking water so the drinking water has residual chlorine at the customer's tap. Many municipal water districts add chloramine to drinking water because chloramine is more stable than chlorine. As used herein, chlorine is defined as oxidizing forms of chlorine and includes, but is not limited to chloramine and hypochlorites. Similarly, non-oxidizing forms of the chloride ion, such as provided by hydrochloric acid (HCl) and sodium chloride (NaCl), are excluded from the definition.
The present inventors have discovered that certain characteristics of drinking water, for example, the presence of chlorine, reduces the activity of the asparaginase enzyme to a point where it is not useful in a commercial setting for the production of food. As used herein, “residual enzyme activity” (expressed as a %) refers to the enzyme activity of a control divided by the enzyme activity of a sample, and provides a relative measurement of enzyme activity under various test conditions. The present inventors have also identified methods and systems for mitigating the effect of drinking water on enzyme activity and preserving the residual enzyme activity of asparaginase such that it may be useful in a commercial setting. The following examples are illustrative of the foregoing.
EXAMPLE 1
Four Solutions were formed from aliquots, each aliquot having an initial equal Asparaginase (Novozymes A/S) activity added, and each aliquot, diluted with distilled water or drinking water such that each Solution had a total volume of about 50 ml. The drinking water for Solution Nos. 3 and 4 was drinking water supplied from the North Texas Municipal Water District to Plano, Tex. USA. The water types used in each solution are described in the following Table 1a.







TABLE 1a







Type of water used to make an asparaginase solution.








Solution



No.
Type of Water





1
distilled water


2
distilled water


3
drinking water from the North Texas Municipal Water



District to Plano, TX USA


4
drinking water from the North Texas Municipal Water



District to Plano, TX USA mixed with 0.1 N hydrochloric acid



added to achieve a pH of 6









Each of Solution Nos. 2-4 were heated at about 35° C. for about 40 minutes before measuring enzyme activity, and pH using Solution No. 1 as the control for residual enzyme activity comparison. Solution No. 1 was refrigerated for about 40 minutes at a temperature of about 10° C.
The values measured are shown in Table 1b below:







TABLE 1b







Residual enzyme activity of distilled water and drinking water.









Solution No.
pH of Solution
Relative Activity





1
6.93
100%


2
7.00
103%


3
8.22
38%


4
7.55
48%









It should be noted that the test results for the enzyme activity and residual enzyme activity were conducted using the Test Method described at the end of this disclosure. As compared to Solution No. 1 (control), Solution No. 2 did not lose any enzyme activity. Solution No. 3 was slightly alkaline, having a pH of about 8.22, and the asparaginase enzyme lost about 62% of its activity after about 40 minutes at about 35° C. The addition of dilute hydrochloric acid to drinking water (Solution No. 4) lowered the pH to about 7.55, and the asparaginase lost about 52% of its activity after about 40 minutes of being heated at about 35° C. Consequently, it appears that the alkalinity of Solution No. 3 is responsible for some loss of enzyme activity. It is generally recognized that pH has an impact on asparaginase activity and the asparaginase activity is higher when the pH is between about 4 and about 7.
EXAMPLE 2
Four Solutions were formed from aliquots, each aliquot having an initial equal
Asparaginase (Novozymes A/S) activity, and each aliquot, diluted with de-ionized water or drinking water such that each Solution had a total volume of about 50 ml. The water types used in each Solution are described in the following Table 2a:







TABLE 2a







Asparaginase Solutions made from different water supplies.








Sol. No.
Type of Water





1
De-ionized water (control)


2
Drinking water from the North Texas Municipal Water



District to Plano, TX USA


3
Drinking water from supplied to residents of Duncanville,



TX USA


4
Water used in a food manufacturing process in Mexicali, Mexico









Each of Solution Nos. 2-4 were heated at about 35° C. for about 40 minutes before measuring chlorine levels, water hardness, pH, and enzyme activity. The control was not heated. The measured values are shown in Table 2b below.







TABLE 2b







Residual enzyme activity and water chemistry of three different


drinking water solutions.













Free
Total
Total





Chlorine
Chlorine
Hardness


Solution No.
(mg/L)
(mg/L)
(mg/L)
pH
Activity















1
0
0
0
6.89
100%


2
1.0
1.0
232
7.47
9%


3
0.02
0.02
90
7.87
85%


4
0.02
0.06
28
8.00
89%









This data clearly demonstrates the negative impact chlorine has on residual enzyme activity. For example, Solution No. 1 (control) had no chlorine and had the highest residual enzyme activity. Solution No. 2 had the lowest level of residual enzyme activity, and the highest level of free chlorine and total hardness.
Solution No. 3 had a relatively low concentration of free chlorine, and a moderate hardness level, with a residual activity of over 80%. Solution No. 4 had a free-chlorine concentration similar to that of Solution No. 3, and a lower hardness level, resulting in a slightly higher residual activity. Table 2b demonstrates that the residual enzyme activity of asparaginase is inversely proportional to the level of chlorine.
EXAMPLE 3
Four Solutions were formed from aliquots, each aliquot having an initial equal Asparaginase (Novozymes A/S) activity, and each aliquot, diluted with de-ionized water or drinking water such that each Solution had a total volume of about 50 ml. The water types for each sample are listed in Table 3a below.







TABLE 3a







Type of chlorinated water used to make an asparaginase solution








Sol. No.
Type of Water





1
De-ionized water (control)


2
De-ionized water + sufficient hypochlorite to yield 12 ppm



chlorine


3
Drinking water from the North Texas Municipal Water



District to Plano, TX USA


4
Drinking water from the North Texas Municipal Water



District to Plano, TX USA, filtered through a BRITA filter



three times









Each of Solutions Nos. 2-4 were heated at about 35° C. for 40 minutes before measuring free chlorine, total hardness, pH and residual enzyme activity.
The values measured are shown in Table 3b below:







TABLE 3b







Residual enzyme activity of solutions having various


levels of chlorine.












Free Chlorine
Total Hardness




Solution No.
(mg/L)
(mg/L)
pH
Activity





1
0
—
4.81
100%


2
Not Measured
—
5.87
4%


3
Not Measured
228
7.10
21%


4
0
20
4.99
102%









As Table 3b above indicates, the addition of chlorine to deionized water as shown by Solution 2 or chlorine's presence in drinking water as shown by Solution 3 clearly lowers the residual activity of the asparaginase enzyme. Further, the removal, or absence of chlorine, clearly results in an increased activity of enzyme, as demonstrated by the residual activity level of enzyme in deionized water in Solution 1 and as demonstrated by the residual enzyme activity in BRITA filtered water in Solution 4. The chlorine level for Solution 2 was not measured because Chlorine in the form of sodium hypochlorite was added to the solution. Also, because drinking water was being, the relative level of chlorine in Solution 3 was known to mimic drinking water levels.
EXAMPLE 4
The objective of this test was to analyze the effect of chlorine on enzyme activity by adding an amount of chlorine found in drinking water to deionized water having no chlorine to ascertain the effects of chlorine on asparaginase activity.
Four Solutions were formed from aliquots, each aliquot having an initial equal Asparaginase (Novozymes A/S) activity, and each aliquot, diluted with de-ionized water or drinking water such that each Solution had a total volume of about 50 ml. The water types for each sample are listed in Table 4a below.







TABLE 4a







Type of chlorinated water used to make an asparaginase solution








Solution No.
Type of Water





1 (control)
deionized water


2
acidified deionized water with sufficient sodium



hypochlorite to result in water having 1.2 ppm chlorine



and sufficient hydrochloric acid to result in an acidic pH


3
acidified deionized water with sufficient sodium



hypochlorite to result in water having 0.2 ppm chlorine



and sufficient hydrochloric acid to result in an acidic pH


4
Drinking water from the North Texas Municipal Water



District to Plano, TX USA









Each of Solutions Nos. 2-4 were heated at about 35 C for 40 minutes before measuring chlorine, pH and residual enzyme activity. Solution 1 was not heated. The values measured are shown in Table 4b below:







TABLE 4b







Residual enzyme activity of solutions having various


levels of chlorine.












Free Chlorine
Total Chlorine




Solution
(mg/L)
(mg/L)
pH
Activity














1
—
—
4.85
100


2
1.2
1.2
4.69
3


3
0.1
0.2
4.62
108


4
0.8
1.1
6.84
14









The data in Table 4b above demonstrates that when chlorine alone is added to water, the residual asparaginase activity is substantially lowered. However, at relatively low levels, chlorine has less impact on the residual enzyme activity.
EXAMPLE 5
Five solutions were prepared to ascertain the potential effects of drinking water modification on the residual activity of asparaginase. Each solution was formed from aliquots, each aliquot having an initial equal Asparaginase (Novozymes A/S) activity, and each aliquot, diluted with de-ionized water or drinking water such that each Solution had a total volume of about 50 ml. The citric acid was added to make the solutions slightly acidic. The water types used in each Solution are described in the following Table 5a:







TABLE 5a







Modifications to drinking water








Solution No.
Type of Water





1
De-ionized water


2
Drinking water from the North Texas Municipal Water



District to Plano, TX USA


3
Acidified Drinking water from the North Texas



Municipal Water District to Plano, TX USA with



sufficient citric acid to have 100 ppm citric acid


4
Acidified Drinking water from the North Texas



Municipal Water District to Plano, TX USA with



sufficient citric acid to have 100 ppm citric acid and



950 ppm of EDTA


5
Acidified Drinking water from the North Texas



Municipal Water District to Plano, TX USA with



sufficient citric acid to have 100 ppm citric acid and



with sufficient thiosulfate to have 10 ppm of sodium



thiosulfate









Each of Solutions Nos. 2-5 were heated at about 35° C. for 40 minutes before measuring free chlorine, total chlorine, pH and residual enzyme activity. Solution 1 was not heated. The values measured are shown in Table 5b below:







TABLE 5b







Residual enzyme activity of various treated drinking water solutions.












Free
Total Chlorine

Residual


Solution
Chlorine (ppm)
(ppm)
pH
Activity














1
0
0
—
100%


2
0.2
1.2
7.53
12%


3
0.4
0.8
5.81
32%


4
1.0
1.0
6.45
100%


5
0.1
0.4
5.65
86%









FIG. 1a is a graphical representation of the residual enzyme activity after various treatments of drinking water. The enzyme activity is represented by the bars in the bar chart and the total chlorine concentration is represented by the line (150). As evidenced by the data, thiosulfate (added at a level about 5 times greater than the chlorine concentration in drinking water) decreased the chlorine concentration and increased enzyme activity to 86% (140). The drinking water, having a total chlorine of 1.2 ppm had a relatively low residual activity of only 12% (110). Citric acid decreased the level of chlorine in drinking water and increased the enzyme activity to 32% (120).
Enzyme activity (130) in drinking water with EDTA was equivalent to the enzyme activity of de-ionized water (100), but EDTA only slightly decreased the total chlorine. Without being bound by theory, Applicants believe that the EDTA may either jacket and thereby protect the enzyme from chlorine or believe that EDTA may tie up the chlorine. For example, the chlorine still shows up when tested, but the reaction a reversible reaction between EDTA and chlorine may prevent chlorine from oxidizing or otherwise reacting with asparaginase. Thus, the EDTA appears to inactivate the chlorine. Consequently, in one embodiment, additives can be added that inhibit the chlorine from reducing the activity of asparaginase and/or that inactivate the chlorine.
EXAMPLE 6
Five solutions were prepared to ascertain the potential effects of hard water constituents commonly found in drinking water. Each solution was formed from aliquots, each aliquot having an initial equal Asparaginase (Novozymes A/S) activity, and each aliquot, diluted with de-ionized water or drinking water such that each Solution had a total volume of about 50 ml. Each salt solution had salt added to achieve a salt concentration of 5 mM (5 millimolar), which is roughly double the calcium carbonate concentration found in drinking water from Plano, Tex. For example, referring to Table 3b above, the Total Hardness for Solution No. 3 (Plano Drinking water) is 228 mg/L which corresponds to about 2.28 mM. The various types of salts used in each Solution are described in the following Table 6a:







TABLE 6a







Salts added to de-ionized water.








Solution No.
Type of Water





1
De-ionized water


2
De-ionized Water + Sodium Chloride


3
De-ionized Water + Calcium Chloride


4
De-ionized Water + Magnesium Nitrate


5
De-ionized Water + Sodium Bicarbonate









Each of Solutions Nos. 2-5 were heated at about 35° C. for 40 minutes before measuring residual enzyme activity. The values measured are shown in Table 6b below:







TABLE 6b







Residual enzyme activity of various salt water mixtures.










Solution
Activity






1
100%



2
99%



3
101%



4
96%



5
102%









FIG. 1b is a graphical representation of the residual enzyme activity of various salt water mixtures and graphically shows the results from Table 6b above. The added salt had no apparent effect on the enzyme stability. Consequently, it is believed that chlorine is responsible for most of loss of asparaginase activity.
EXAMPLE 7
Two aliquots having an initial equal Asparaginase Activity were diluted equally with deionized water (Cell 1) and tap water (Cell 2) to make a first asparaginase solution and a second asparaginase solution. Each solution was held for 30 minutes at room temperature and then each asparaginase solution was then added to corn masa. Asparagine in the masa was measured 5 minutes and 10 minutes after the enzyme was added to the masa and the values measured are shown in Table 7 below.







TABLE 7







Asparagine level in corn masa using enzyme mixed with drinking


water and deionized water.









Type of Water Used for

Asparagine


Enzyme Dilution
Masa Sample
(ppm)












Deionized water
5 minutes after first
3.6



asparaginase solution added



Deionized water
10 minutes after first
2.9



asparaginase solution added



Drinking water from the
5 minutes after second
37.2


North Texas Municipal
asparaginase solution added



Water District to Plano, TX




USA




Drinking water from the
10 minutes after second
24.2


North Texas Municipal
asparaginase solution added



Water District to Plano, TX




USA









The level of asparagine in the corn masa shown in Table 7 above, demonstrates that the resultant level of asparagine is highly dependent on the underlying diluted asparaginase solution. In the embodiment shown above, the level difference was on the order of about one magnitude in the level of asparagine in corn masa following treatment by de-ionized water versus drinking water.
The data shown above clearly indicates that the active chlorine level must be lowered to maximize the residual activity of asparaginase. Because the de-ionized water and distilled water are expensive, the present invention provides a way to maximize residual enzyme activity by selectively removing and/or inactivating chlorine from drinking water or other water source.
Any method known in the art that can reduce the concentration of enzyme activity reducing components in drinking water can be used, including but not limited to, treating drinking water to reduce the concentration of the activity reducing component by filtration of drinking water through activated carbon, an air stripper (to volatilize the chlorine), reverse osmosis systems, and/or ion-exchange resins. Drinking water can also be treated by mixing drinking water with deionized water or distilled water in sufficient amounts to lower the concentration of activity reducing components to make a stable enzyme solution.
As used herein, a “scavenger” is any additive that preserves enzyme activity by reacting with chlorine. Consequently, scavengers for enzyme reducing components can be added to the drinking water. For example, in one embodiment, thiosulfate, a scavenger for chlorine is added to the drinking water. Further, other additives can be used to inactivate the chlorine. For example, because chlorine is a strong oxidization agent, reducing agents can also be added to the drinking water to react with the chlorine. Reducing agents are known in oxidation-reduction chemistry to be compounds that are electron donors and oxidizing agents are known to be electron acceptors. Consequently, in one embodiment, one or more reducing agents (e.g., electron donors) can be added to a source of drinking water to inactivate or neutralize the chlorine. Examples of reducing agents include, but are not limited to stannous chloride dihydrate, sodium sulfite, sodium meta-bisulfite, ascorbic acid, ascorbic acid derivatives, isoascorbic acid (erythorbic acid), salts of ascorbic acid derivatives, iron, zinc, ferrous ions, and combinations thereof.
In one embodiment, the present invention reduces the total chlorine concentration to a level that is between about 0 and less than about 0.5 ppm and preferably between 0 and about 0.1 ppm.
In one embodiment, asparaginase can then be mixed with the treated water to make a stable asparaginase solution and the asparaginase solution can then be mixed with food product. In one embodiment, drinking water is sufficiently treated and a stable enzyme or asparaginase solution occurs when the residual enzyme activity is at least about 80% and more preferably at least about 90% for at least 30 minutes and more preferably for at least about 4 hours after the enzyme has been added to treated drinking water. In one embodiment, the residual enzyme activity is at least about 90% for the time required to get an asparaginase solution admixed into a dough.
Armed this disclosure, one skilled in the art, will be able to ascertain and provide the necessary water compositions to result in the desired residual enzyme activity.
Food products the asparaginase solution can be added to include, but are not limited to, doughs, slurries, and any other consumable products where it is desired to lower the level of acrylamide. For example, in one embodiment, the asparaginase solution is added to a potato slurry made from potato flakes. In one embodiment, the potato slurry is made by adding the asparaginase solution to potato flakes. In one embodiment, the asparaginase solution is used for added water and is added to a flour composition to make a dough. In one embodiment, the asparaginase solution is added to corn masa.
In one embodiment, the present invention comprises a system for providing a stable solution of asparaginase that can be added to a food ingredient having asparagine. In one embodiment, the system comprises a treatment system to treat water. The treatment system can remove components such as chlorine through an activated carbon or with other removal methods listed above and/or the treatment system can provide additives including, but not limited to, reducing agents, chlorine scavengers, or EDTA that enhances the activity of asparaginase to a level that is higher than if the additive had not been added. The treated water can then be routed to a mix tank where asparaginase can be diluted therein to make a stable asparaginase solution. The asparaginase solution can then be metered in or otherwise added to a dough used to make a fabricated food, or thermally processed as described above. The dough can then be further processed (e.g., formed by extrusion and sheeting and thermally processed) as well known in the art. Those skilled in the art, armed with this disclosure will understand that the present invention can be used anywhere an asparaginase solution is desired to reduce the level of acrylamide in a food product.
In one embodiment, the invention comprises a system comprising a source of drinking water and a source of asparaginase, a treatment system operable to enhance the activity of asparaginase in the treated water to a level that is higher than if the treatment had not been occurred, and a delivery system operable to mix the treated drinking water and asparaginase. In one embodiment, the delivery system comprises a mix tank that receives treated water form the treatment system and asparaginase.
The Test Method for used to determine asparaginase activity for the Examples in this application is shown below:

    
    
        I. Background The SIGMA procedure for asparaginase activity used a Tris buffer at pH 8.6 (Sigma catalogue A 4887). Because food grade asparaginase has low activity at pH-8.6, the assay was changed to pH 7.0 with MOPS (3-morpholinopropanesulfonic acid).
        II. Principle:

L-Asparagine+H2OAsparagine>L-Aspartate+NH3

        III. Conditions: T=37 C, pH=7.0, A436, Light path=1 cm
        IV. Method: Spectrophotometric Stop Rate Determination
        V. Reagents
        
            a. 100 mM MOPS sodium salt (3-morpholinopropanesulfonic acid). Weigh out 2.09 g of MOPS (Sigma M5162). Dissolve in about 60 ml of DI water at room temperature. Add sodium hydroxide to adjust pH to 7.0. Make up to 100 ml with DI water. Store in refrigerator when not in use.
            b. 189 mM L-Asparagine Solution Weigh out 0.25 g of L-asparagine anhydrous, and dissolve in 10 ml DI water. Store in refrigerator when not in use. After refrigeration, sonicate to dissolve asparagine crystals before using.
            c. 6 mM Ammonium Sulfate Standard Solution ((NH4)2SO4 Standard) Weigh out 0.079 of ammonium sulfate on an analytical balance, and record weigh to 0.0001 g. Dissolve and make up to volume with 100 ml with DI water. Store in refrigerator when not in use.
            a. 1.5 M Trichloroacetic acid (TCA) Weigh out 2.45 g of trichloroacetic acid. Dissolve and make up to 10 ml with DI water.
            b. Ammonia Color Reagent: Test kit for Ammonia Nitrogen High, Nesslerization, LaMotte Code 3642-SC, VWR Cat. No. 34186-914. The reagent # 2 contains mercury.
            c. Asparaginase Enzyme Solution: Immediately before use, prepare a solution containing 2.0-4.0 units/ml of asparaginase in room temperature deionized water. If enzyme is frozen, thaw completely in lukewarm water before taking an aliquot for dilution. For typical enzyme concentrations, 0.1 ml of enzyme solution can be diluted to 50 ml.
        
        
        VI. Procedure:
        
            a. Set heating block for vials to 37 C.
            b. Use an adjustable micropipette to transfer the following reagents into vials (ml):
        
        
    
    




















Enzyme



Reagent


Reagent
Test
Blank
Std. 1
Std. 2
Std. 3
Blank







A (Buffer)
1.00
1.00
1.00
1.00
1.00
1.00


B (L-ASN)
0.10
0.10
—
—
—
—


C (Ammonium Std.)
—
—
0.25
0.50
1.00
—


DI Water
0.90
0.90
0.85
0.60
0.10
1.10


F (Enzyme Solution)
0.10
—
—
—
—
—









    
    
        
        
            c. Cap vials, and place in heating block at 37 C. Start agitation of heating block.
            d. Remove vials from heating block after 30 minutes. Decap vials, immediately add TCA reagent, and mix. Then add Reagent F (Enzyme Solution) to Enzyme Blank. For enzyme test solutions, the time between removal of the vials from the heating block and addition of TCA should be as short as possible. After TCA is added, time before ammonia measurement is not critical. For blanks and standards, time between removal from the heating block and addition of TCA is not critical.
        
        
    
    




















Enzyme



Reagent


Reagent
Test
Blank
Std. 1
Std. 2
Std. 3
Blank







D (TCA)
0.10
0.10
0.10
0.10
0.10
0.10


F (Enzyme Solution)
—
0.10
—
—
—
—









    
    
        
        
            e. Pipet 0.20 ml of each solution into test tubes or vials. Add 4.30 ml of deionized water, 4 drops of LaMotte reagent #1, and 0.50 ml of LaMotte #2. Mix solutions and leave at room temperature for 10-20 minutes before reading absorbance at 436 nm in 1 cm cell. Zero the spectrophotometer with DI water.
        
        
        VII. Calculation of Results
        
            a. The enzyme activity is calculated from a calibration curve for ammonia (umole/0.2 mL).
            b. Description of Calculation Steps.
            
                i. Calculation of ammonium sulfate standard solution concentration:
            
            
        
        
    
    






  
    
      
        
          m
          ⁢
          
            
          
          ⁢
          M
        
        =
        
        ⁢
        
          
            (
            
              0.0809
              ⁢
              
                
              
              ⁢
              g
            
            )
          
          *
          
            (
            
              1000
              ⁢
              
                
              
              ⁢
              m
              ⁢
              
                
              
              ⁢
              
                M
                /
                M
              
            
            )
          
          *
        
      
    
  
  
    
      
        
        ⁢
        
          
            (
            
              2
              ⁢
              
                
              
              ⁢
              NH
              ⁢
              
                
              
              ⁢
              
                3
                /
                NH
              
              ⁢
              
                
              
              ⁢
              4
              ⁢
              SO
              ⁢
              
                
              
              ⁢
              4
            
            )
          
          /
          
            (
            
              
                (
                
                  132.14
                  ⁢
                  
                    
                  
                  ⁢
                  g
                  ⁢
                  
                    /
                  
                  ⁢
                  mole
                
                )
              
              *
              
                (
                
                  0.1
                  ⁢
                  
                    
                  
                  ⁢
                  L
                
                )
              
            
            )
          
        
      
    
  
  
    
      
        =
        
        ⁢
        
          
            12.24
            ⁢
            
              
            
            ⁢
            m
            ⁢
            
              
            
            ⁢
            M
          
          =
          
          ⁢
          
            
              mmole/L
            
            =
            
            ⁢
            
              
                umole/ml
              
            
          
        
      
    
  




    
    
        
        
            
            
                 Where 0.0809 g is weight of ammonium sulfate for standard
                ii. Calculate umole of NH3 in 2.2 ml standards:

umole of NH3 in 2.2 mL=(NH3 umole/mL of standard solution)*(mL of standard)

                iii. Calculate umole of NH3/0.2 mL:

umole of NH3/0.2 mL=(umole of NH3 in 2.2 ml)*(0.2 mL)/(2.2 mL)

                iv. Calculate regression curve with
                
                    x=A436
                    y=NH3 umole/0.2 mL
                
                
                v. From calibration curve, umole of NH3/0.2 ml is calculated:

umole of NH3/0.2 mL=(slope)*(A436)+Intercept

                vi. The activity of the diluted enzyme solution is calculated with the following formula:

Units/ml enzyme=(umole of NH3 liberated)*(2.20)/(0.2*30*0.1) where

            
            
            2.20 ml=Volume from Step 1 (Step 1 is enzyme assay solution.)
            0.2 ml=Volume of Step 1 used in Step 2 (Step 2 is color development.)
            30 minutes=Time of assay in minutes
            0.1 ml=Volume of enzyme used
            
                vii. The dilution factor is 50 mL divided by volume of concentrated enzyme diluted to 50 mL,
                viii. Concentration of enzyme solution before dilution==(units/ml of diluted solution)*(dilution factor)
            
            
        
        
    
    


While the invention has been particularly shown and described with reference to several embodiments, it will be understood by those skilled in the art that various other approaches to the preservation of the residual asparaginase activity in solution may be made without departing from the spirit and scope of this invention.

## Claims

1. A method for increasing asparaginase activity in a food product, said method comprising the steps of:
a)treating a drinking water from a municipal water supply, wherein said treating reduces the combined chlorine concentration to less than about 0.5 ppm wherein the treatment is selected from the group consisting of adding a reducing agent, adding a chlorine scavenger, air stripping and combinations thereof;
b)admixing asparaginase with said treated water to make an asparaginase solution; and
c) adding said asparaginase solution to a food product.

2. The method of claim 1 wherein said treating step a) further comprises filtration of the drinking water through activated carbon.

3. The method of claim 1 wherein said treating step a) further comprises using an ion-exchange resin.

4. The method of claim 1 wherein said treating step a) further comprises using reverse osmosis.

5. The method of claim 1 wherein said treating step a) comprises air stripping.

6. The method of claim 1 wherein said asparaginase solution comprises a residual activity of at least about 80%.

7. The method of claim 1 wherein said treating step a) comprises adding a reducing agent.

8. The method of claim 7 wherein said reducing agent comprises one or more agents selected from stannous chloride dihydrate, sodium sulfite, sodium meta-bisulfite, ascorbic acid, ascorbic acid derivatives, isoascorbic acid (erythorbic acid), salts of ascorbic acid derivatives, iron, zinc, ferrous ions, and combinations thereof.

9. The method of claim 1 wherein said treated water comprises a pH of less than about 8.0.

10. The method of claim 1 wherein said drinking water is treated with an additive sufficient to reduce the final total level of chlorine to a level that is lower than if the additive had not been added.

11. The method of claim 10 wherein said additive comprises thiosulfate.

12. The method of claim 1 wherein said treating step a) comprises adding a chlorine scavenger.

13. The method of claim 1, further comprising adding EDTA to the treated water.