Development of a novel, rapid, and quantitative method for the measurement of carry-over Α-amylase content in sugar samples

Abstract

The present disclosure is directed to systems and methods for measuring the amount of amount of carryover α-amylase in sugar and in food products comprising sugar.

Description

DEVELOPMENT OF A NOVEL, RAPID, AND QUANTITATIVE METHOD FOR THE MEASUREMENT OF CARRY-OVER A-AMYLASE CONTENT IN SUGAR SAMPLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit to U.S. Provisional Patent Application Serial No. 63 / 762306, filed February 24, 2025, which claims the benefit and priority, under 35 U.S.C. § 119(e) and any other applicable laws or statutes, to U.S. Provisional Patent Application Serial No. 63 / 655120, filed June 3, 2024, both of which is incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to devices and methods for measuring the amount of carryover a-amylase in sugar.BACKGROUND

[0003] The use of industrial enzyme preparations as processing aids is widespread in food and ingredients manufacturing. Enzymes must be inactivated in finished goods in order to be classified as processing aids and not active ingredients. Depending on the type of enzyme used and the manufacturing process, their inactivation may only be partial leading to residual enzyme activity in the finished product. In the sugar industry, enzymes such as dextranases and amylases arc used to break down plant polysaccharides such as dextrans and starch that impact the performance of manufacturing operations. Alpha- amylases used in sugar manufacturing and refining are known to be relatively thermoresistant. Therefore, a-amylase activity may carry over into raw and refined sugar even when heat treatment processing steps designed to inactivate these enzymes have been performed. The carry-over amylase can have significant consequences on the sugar manufacturing and refining industries.

[0004] Amylase, even in small quantities, can impact the quality of food when contaminated sugar is used in combination with starch-based products or texturizing agents (e.g. in yogurts, drinks, sauces, desserts, etc.). Indeed, the residual amylase can depolymerise starch, thereby impacting the quality of processed food products over their shelf lives through a gradual change in their viscosity. Methods for the analysis of carry-over amylase in raw and refined sugar samples which employ a commercially available dyed tablet product or a high sensitivity colorimetricMatter No. 91598-426237 detection system for the measurement of low levels of a-amylase are known. While the methods arc optimized to achieve the necessary sensitivity to detect low levels of amylase, they typically lacked reproducibility. Alternatively, some of these methods are targeted towards specific food products such as cereals. Therefore, there is a need for robust, widely applicable methods for amylase detection.

[0005] The present disclosure is directed to devices and methods for quantitative, rapid, highly sensitive, linear and repeatable measurement of carry-over amylase in sugar. The present disclosure is directed to devices and methods that do not require calibration curves or filtrations and are compatible with the laboratory instrumentation present in sugar analysis laboratories. The present disclosure is directed to devices and methods that allow the user to establishment pass I fail amylase thresholds relevant to their processes and differentiate accurately between samples containing industrially significant levels of amylase and samples considered to have insignificant levels.SUMMARY

[0006] Systems and methods for quantifying a-amylase in a food product are described.

[0007] In a first aspect, the present disclosure is directed to a method of quantifying carryover a-amylase in a food product comprising increasing sample concentration of the food product and employing an assay temperature more than about 40 °C.

[0008] In some embodiments, the method comprises increasing sample concentration by implementing a centrifugation step.

[0009] In some embodiments, the method comprises increasing sample concentration by implementing a filtration step.

[0010] In some embodiments, the method comprises the assay temperature is about 70 °C.

[0011] In some embodiments, the method comprises including incubated reagent blanks to account for auto-hydrolysis that occurs during the method.

[0012] In some embodiments, the method comprises using a buffer with a pH of about 5.4 to about 6.5.

[0013] In some embodiments, the food product is sugar.Matter No. 91598-426237

[0014] In some embodiments, the food product is a product comprising sugar.

[0015] In some embodiments, the concentration of the a-amylase in the food product is greater than about 0.1 mU / g.

[0016] In some embodiments, the method comprises using ancillary enzymes that are stable above 70 °C.

[0017] In a second aspect, the present disclosure is directed to a food product identified with a concentration of carryover a-amylase comprised in the food product. In some embodiments, the food product comprises sugar-. In some embodiments, the concentration of the a-amylase in the food product is greater than about 0.1 mU / g.BRIEF DESCRIPTION OF DRAWINGS

[0018] The following description accompanies the drawing(s), all given by way of nonlimiting examples that may be useful to understand how the described method and composition may be embodied.

[0019] Figure 1 is a schematic showing an overview of a sample preparation procedure for three different food sample types;

[0020] Figure 2 is a schematic showing an overview of the analytical procedure highlighting the steps that are altered in comparison to existing protocols / assay; (1) change in incubation conditions and, (2) addition of a sample specific centrifugation step;

[0021] Figure 3 is a schematic illustrating the principle underlying the amylase SD assay for the detection of amylase in sugar;

[0022] Figure 4 is a graph showing the incubation of white sugar (WS) and black sugar (BS) samples (0.0625 g / mL extracted) over 5 hours at 40 °C in amylase SD buffer, pH 5.4;

[0023] Figure 5 is a graph showing the incubation of WS and BS samples (0.1 g / mL extracted) over 5 hours at 70 °C in amylase SD buffer, pH 5.4;

[0024] Figure 6 is a graph showing the incubation of WS1 over 5 hours, with and without temperature and extraction modifications;

[0025] Figure 7 is a graph showing the incubation of WS2 over 5 hours, with and without temperature and extraction modifications;

[0026] Figure 8 is a graph showing the incubation of blanks at 70 °C in the modified amylase SD assay over 300 minutes;Matter No. 91598-426237

[0027] Figure 9 is a graph showing the incubation of WS1 and WS2 in the modified amylase SD assay over 60 minutes.DETAILED DESCRIPTION

[0001] One or more examples of these embodiments are described herein and illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. Accordingly, aspects and features of every embodiment may not be described with respect to each embodiment, but those aspects and features are applicable to the various embodiments unless statements or understandings are to the contrary.

[0002] The term “about” as used herein refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.”

[0003] The present disclosure is directed to an amylase SD assay configured to measure the amount of a- amylase that is carried over into sugar used for manufacturing food products, referred to as carryover a-amylase. The assay may include the step of sample preparation, as the a-amylase enzyme needs to be present at concentration levels within the limits of detection and the linear range. Additionally, the sample may need to be clarified for optical measurement. The procedure in Figure 1 outlines the steps in one embodiment of the amylase SD assay used for three specific sample types (powdered sugar, liquid sugar, and foodstuffs). The identified steps allow for accurate and precise measurement of a-amylase in sugar concentration samples. In some embodiments, such an amylase SD assay can be applied to similar sample types with no or minimal optimization.

[0004] Figure 2 highlights two changes to the standard Amylase SD assay (K-AMLSD 04 / 19; ICC Standard No. 303). First, incubation time under the standard Amylase SD assay (04 / 19) conditions is set at 10 minutes. This incubation time cannot allow for accurate and precise measurement of a-amylase in high concentration sugar samples as the enzyme concentration inMatter No. 91598-426237 such samples is too low. The amylase SD assay in the present disclosure includes an increased incubation time (up to 5 hours) to allow for sufficient substrate hydrolysis to afford a measurable absorbance increase for samples containing very low levels of a-amylase. In some embodiments, the increased incubation time can range from about 15 minutes to about 6 hours, including any time or range comprised therein. For example, in some embodiments, the incubation time may be increased by about 5 hours.

[0005] Second, the incubation temperature under the standard Amylase SD assay (04 / 19) conditions is set at about 40 °C. Industrially relevant thermostable a-amylases have optimal activity at higher temperatures. The amylase SD assay in the present disclosure includes an increased incubation time. By increasing the incubation temperature, the enzyme activity is increased and therefore lower levels of a-amylase can be detected and quantified over shorter incubation times than would be necessary at about 40 °C. In some embodiments, the increased incubation temperature can range from about 60 °C to about 80 °C, including any temperature or range comprised therein. For example, in some embodiments, the incubation temperature may be about 70 °C. Such increased temperature may allow for an increased reaction rate and / or an accelerated substrate hydrolysis, and therefore enhanced measurement of low levels of a -amylase.

[0006] In some embodiments of the amylase SD assay, incubated reagent blanks (no sample) may be to account for minor auto-hydrolysis of the substrate that takes place at the elevated temperature. While sample-specific blanks are created in the standard amylase SD assay to account for any color arising from the sample, incubated reagent blanks are typically not included when the assay is carried out at about 40 °C and auto-hydrolysis of the substrate does not occur to any significant extent at this temperature during the normal recommended timeframe of the assay.

[0007] In some embodiments of the amylase SD assay, centrifugation and / or filtration may be performed for samples, such as yogurt, that might have residual solid particles remaining post sample preparation. These solid particles may impact optical reading by obstructing the light path, which would generate inaccurate results. To remove these particles, a centrifugation and / or filtration step is introduced, and the supernatant post centrifugation is used for the optical reading step.Matter No. 91598-426237EXAMPLES AND METHODOLOGY

[0008] Example 1: Principle of Amylase SD assay

[0009] The amylase SD assay principle is shown in Figure 3. The amylase substrate (EtPNPG7) is a DP7 maltodextrin covalently attached to the dye molecule 4-nitrophenol and protected at the non-reducing end as an ethylidene acetal. The substrate is specific for a-amylases. The carry-over amylase in sugar hydrolyses the substrate to produce a shorter unblocked maltodextrin fragment which is subsequently broken down into glucose by the ancillary thermostable a-glucosidase to release the phenolic dye in solution. The amylase SD assay is stopped by adding an alkaline solution that inactivates a-amylase. Sample specific blanks are created to account for any color arising from the sample. Incubated reagent blanks are also created to account for minor auto-hydrolysis of the substrate that takes place at elevated temperatures. The absorbance is read at 400 nm and the absorbance difference (AAbs - sample absorbance, minus sample blank absorbance, minus contribution from incubated blanks) value obtained is used to calculate amylase activity in the sample from the extinction co-efficient of 4-nitrophenol. No calibration curves are required.

[0010] Example 2: Comparison of a- Amylase activity measured in SD mU / g, and AAbs for White Sugar (WS) and Brown Sugar (BS) samples over 5 hours with and without modifications.

[0011] Table 1 shows results achieved (Amylase SD mU / g) for two White Sugar (WS) and two Brown Sugar (BS) samples using the standard Amylase SD protocol / assay (K-AMYLSD 04 / 19) versus the results achieved for the assay described in this disclosure. In the K-AMYLSD assay one Unit of activity is defined as the amount of enzyme, in the presence of excess thermostable a-glucosidase, required to release one micromole of p-nitrophenol from EtPNPG7 in one minute at 40 °C and pH 5.4. In the amylase SD assay described in this disclosure, one Unit of activity is defined as the amount of enzyme, in the presence of excess thermostable a-glucosidase, required to release one micromole of p-nitrophenol from EtPNPG7 in one minute at 70 °C and pH 5.4.

[0012] Table 1 : a-Amylase activity measured in SD mU / g for WS and BS samples over 5 hours with and without additional modifications. ND: Not detected (absorbance below 0.02).Matter No. 91598-426237

[0013] The present disclosure is directed to an amylase SD assay that results in more rapid quantification of a-amylase in samples WS1 and WS2. Samples BS1 and BS2 contain levels of a- amylase that are not detectable (ND) in the standard protocol / assay, even at significantly extended incubation times, but a-amylase in these samples is quantifiable using this assay.

[0014] For every 10 °C increase in incubation temperature, a sensitivity increase of approximately 1.5-fold may occur. Following this trend, it is possible to extrapolate that an increase in incubation temperature from 40 °C up to 90 °C would increase the reaction rate approximately 12-fold, assuming the target a-amylase exhibits sufficient stability at this temperature (Table 2). While it may not be possible to run the Amylase SD assay at 90 °C, as it is limited by the stability of the ancillary a-glucosidase (from Bacillus stearothermophilus') employed, which exhibits poor stability at temperatures above 70 °C, it is possible to find examples of a-glucosidase enzymes in literature that exhibit good stability and activity above 90 °C. Replacing the ancillary enzyme in order to support a higher assay incubation temperature would generate further increased sensitivity.

[0015] Table 2: Change in enzyme activity measured (U / mL) at increasing temperaturesMatter No. 91598-426237* Theoretical value extrapolated from other data

[0016] Example 3: Impact of combined temperature and extraction modifications

[0017] The impact of the combined modifications were investigated. Figures 4-7 show data generated with all samples extracted using the modified extraction protocol (0.1 g of sugar in 1 mL of extract) as described in this disclosure and show the main effects of the increased temperature (about 40 °C to about 70 °C) over the time periods specified (up to 300 minutes). Generally, the increase in temperature results in an increase in rate of the enzyme-substrate reaction of approximately 5 -fold. Direct comparisons of the rates of reactions resulting from the changes can be seen in Figures 6 and 7 for WS1 and WS2.

[0018] The impact of the substrate auto-hydrolysis on the reaction was investigated and it was determined that substrate and buffer must be incubated alongside the samples and subtracted from the sample absorbance in order to obtain accurate test results for the samples. The auto-hydrolysis of the substrate is a reaction that increases over time in a linear fashion (Figure 8). The hydrolysis of substrate negatively impacts absorbance readings for samples, impacting the results more significantly on the low end of the absorbance scale.

[0019] The assay allows for quantification of a-amylase in samples containing very low levels of the analyte (>0.14 mU / g), approximately 48-times lower than the levels measurable in the previous protocol / assay (>6.63 mU / g) when taking into account the increase in incubation time and the increase in concentration of sample in the extract (Table 3). The added benefit of a 4.5- fold faster rate of reaction is not reflected in the LOQ values above, but combining all threeMatter No. 91598-426237 components, namely the increased assay temperature, the increased sample concentration and the increased reaction time, deliver an assay that is >200-timcs more sensitive than the standard Amylase SD (K-AMYLSD) method, provided that the substrate autohydrolysis is accounted for by running incubated reagent blanks alongside real analyte samples.

[0020] Table 3: Comparison of the Limit of Quantification (LOQ) for the assay described and previous protocols

[0021] For the application of differentiation between samples containing relatively low but industrially significant levels of amylase, such as samples WS1 and WS2, using the assay protocol, it is possible to accurately quantify the level of amylase in both samples within a 60 minute incubation period (r2 = 0.999 and r2 = 0.992) (Figure 9). This would not have been possible in the previous K-AMYLSD protocol / assay, as analysis of WS1 and WS2 does not result in a measurable increase in absorbance over this time period (ND).

[0022] The figures provided herein are not necessarily to scale, although a person skilled in the art will recognize instances where the figures are to scale and / or what a typical size is when the drawings are not to scale. While in some embodiments movement of one component is described with respect to another, a person skilled in the art will recognize that other movements are possible. Additionally, a number of terms may be used throughout the disclosure interchangeably but will be understood by a person skilled in the art. Further, to the extent features, sides, or steps are described as being “first” or “second,” such numerical ordering is generally arbitrary, and thus such numbering may be interchangeable. Still further, in the present disclosure, like-numbered components of various embodiments generally have similar features when those components are of a similar nature and / or serve a similar purpose. Lastly, the present disclosure includes some illustrations and descriptions that include prototypes, bench models, or experimental design. A person skilled in the art will recognize how to rely upon the present disclosure toMatter No. 91598-426237 integrate the techniques, systems, devices, and methods provided for into a product in view of the present disclosures.

[0023] The following numbered embodiments are contemplated and are non-limiting:1. A method of quantifying carryover a-amylase in a food product comprising: increasing sample concentration of the food product and employing an assay temperature more than about 40 °C.2. The method of clause 1, any other suitable clause, or any combination of suitable clauses comprising implementing a centrifugation step.3. The method of clause 1, any other suitable clause, or any combination of suitable clauses comprising implementing a filtration step.4. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising an assay temperature is about 70 °C.5. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising incubating reagent blanks.6. The method of clause 5, any other suitable clause, or any combination of suitable clauses, comprising incubating reagent blanks to account for autohydrolysis that occurs during the method. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising using a buffer with a pH of about 5.4 to about 6.5.7. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising sugar as the food product.8. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising a product comprising sugar as the food product.9. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising a concentration of a-amylase greater than about 0.1 mU / g in the food product.10. The method of clause 1, any other suitable clause, or any combination of suitable clauses, comprising using ancillary enzymes that are stable above 70 °C.11. A food product identified with a concentration of carryover a-amylase comprised in the food product.Matter No. 91598-42623712. The food product of clause 11 , any other suitable clause, or any combination of suitable clauses, comprising sugar as a constituent of the food product.13. The food product of clause 11, any other suitable clause, or any combination of suitable clauses, comprising a concentration of a- amylase greater than about 0.1 mU / g in the food product.

[0024] While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific exemplary embodiments of the disclosure have been shown by way of example. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular disclosed forms; the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims. Although this disclosure refers to specific embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the subject matter set forth in the accompanying claims.

Claims

Matter No. 91598-426237CLAIMS:

1. A method of quantifying carryover a-amylasc in a food product comprising: increasing sample concentration of the food product; and employing an assay temperature more than about 40 °C.

2. The method of claim 1, wherein increasing sample concentration comprises implementing a centrifugation step.

3. The method of claim 1, wherein increasing sample concentration comprises implementing a filtration step.

4. The method of claim 1, wherein the assay temperature is about 70 °C.

5. The method of claim 1, wherein the method further comprises including incubated reagent blanks to account for auto-hydrolysis that occurs during the method.

6. The method of claim 1, wherein the method comprises using a buffer with a pH of about 5.4 to about 6.5.

7. The method of claim 1, wherein the food product is sugar.

8. The method of claim 1, wherein the food product is a product comprising sugar.

9. The method of claim 1, wherein the concentration of the a-amylase in the food product is greater than about 0.1 mU / g.

10. The method of claim 1, wherein the method comprises using ancillary enzymes that are stable above 70 °C.

11. A food product identified with a concentration of carryover a-amylase comprised in the food product.

12. The food product of claim 11, wherein the food product comprises sugar.

13. The food product of claim 11, wherein the concentration of the a-amylase in the food product is greater than about 0.1 mU / g.

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