GB2536304A

GB2536304A - Allulose syrups

Info

Publication number: GB2536304A
Application number: GB1507032.9A
Authority: GB
Inventors: David Woodyer Ryan; Lloyd-Jones Peter
Original assignee: Tate and Lyle Ingredients Americas LLC
Current assignee: Primary Products Ingredients Americas LLC
Priority date: 2015-02-24
Filing date: 2015-04-24
Publication date: 2016-09-14
Anticipated expiration: 2035-04-24
Also published as: GB201507032D0; AR103756A1; GB2536304B

Abstract

An allulose syrup has a total dry solids content of from 70% to 80% by weight, and comprises allulose in an amount of at least 90% by weight on a dry solids basis. The pH of the syrup is from 3.0 to 5.0. The syrup can be used as a low-calorie replacement for convention sucrose, high fructose corn syrup, etc.. Microbial stability of the syrup is achieved by the relatively high solids content of the syrup. The pH of the syrup has been found to be optimal in minimising allulose degradation and hydroxymethylfurfural formation, whilst minimising undesirable colour formation over time.

Description

Allulose Syrups

Field of the Invention

The present invention relates to allulose syrups, use of allulose syrups in the manufacture of food or beverage products, and food and beverage products made using the allulose syrups.

Background of the Invention

Many food and beverage products contain nutritive sweeteners such as sucrose (generally referred to as 'sugar' or 'table sugar'), glucose, fructose, corn syrup, high fructose corn syrup and the like. Although desirable in terms of taste and functional properties, excess intake of nutritive sweeteners, such as sucrose, has long been associated with an increase in diet-related health issues, such as obesity, heart disease, metabolic disorders and dental problems. This worrying trend has caused consumers to become increasingly aware of the importance of adopting a healthier lifestyle and reducing the level of nutritive sweeteners in their diet.

In recent years, there has been a movement towards the development of replacements for nutritive sweeteners, with a particular focus on the development of low or zero-calorie sweeteners. One proposed alternative to nutritive sweeteners is allulose (also known as D-psicose). Allulose is known as a "rare sugar", since it occurs in nature in only very small amounts. It provides around 70% of the sweetness of sucrose, but only around 5% of the calories (approximately 0.2 kcal/g). It may therefore essentially be considered to be a 'zero calorie' sweetener.

In view of its scarcity in nature, production of allulose relies on the epimerization of readily available fructose. Ketose-3-epimerases can interconvert fructose and allulose, and various ketose-3-epimerases are known for carrying out this conversion.

US patent no. 8,030,035 and PCT publication no. W02011/040708 disclose that Dpsicose can be produced by reacting D-fructose with a protein derived from Agrobacterium tumefaciens, and having psicose 3-epimerase activity.

US patent publication no. 2011/0275138 discloses a ketose 3-epimerase derived from a microorganism of the Rhizobium genus. This protein shows a high specificity to D-or L-ketopentose and D-or L-ketohexose, and especially to D-fructose and D-psicose. This document also discloses a process for producing ketoses by using the protein.

Korean patent no. 100832339 discloses a Sinorhizobium YB-58 strain which is capable of converting fructose into psicose (i.e. allulose), and a method of producing psicose using a fungus body of the Sinorhizobium YB-58 strain.

Korean patent application no. 1020090098938 discloses a method of producing psicose using E. co/i wherein the E. coli expresses a polynucleotide encoding a psicose 3-epimerase.

Allulose is present in processed cane and beet molasses, steam treated coffee, wheat plant products and high fructose corn syrup. D-allulose is the C-3 epimer of D-fructose and the structural differences between allulose and fructose result in allulose not being metabolized by the human body to any significant extent, and thus having "zero" calories. Thus, allulose is thought to be a promising candidate as a replacement for nutritive sweeteners and as a sweet bulking agent, as it has no calories and is reported to be sweet while maintaining similar properties to sucrose.

A convenient product form for allulose is an allulose syrup, i.e. a syrup comprising allulose and water. It has been found that allulose syrups may be susceptible to degradation over time (i.e. gradual reduction in allulose content), to color formation, to the formation of impurities (such as hydroxymethylfurfural -HMF), and to inadequate microbial stability.

The object of the present invention is to provide an allulose syrup that addresses the above problems.

Summary of the Invention

According to a first aspect, the present invention provides an allulose syrup having a total dry solids content of from 70% to 80% by weight, and comprising allulose in an amount of at least 90% by weight on a dry solids basis, wherein the pH of the syrup is from 3.0 to 5.0.

In an embodiment, the total dry solids content of the allulose syrup is from 71% to 78% by weight. In another embodiment, the total dry solids content of the allulose syrup is from 71% to 73% by weight. In another embodiment, the total dry solids content of the allulose syrup is from 76% to 78% by weight.

In an embodiment, the pH of the allulose syrup is from 3.5 to 4.5. In an embodiment, the pH of the allulose syrup is from 3.8 to 4.2.

In an embodiment, the allulose syrup comprises allulose in an amount of at least 95% by weight on a dry solids basis.

In an embodiment, the allulose syrup comprises less than 1000 ppm of HMF.

In an embodiment, the allulose syrup comprises sulfur dioxide in an amount of from 1 to 20 ppm.

In an embodiment, the allulose syrup comprises less than 10 parts per billion of isovaleraldehyde.

In an embodiment, the allulose syrup comprises less than 2 parts per billion of 2-aminoacetophenone.

In an embodiment, the allulose syrup further comprises one or more additives. In an embodiment, the one or more additives may include a stability-enhancing additive. In an embodiment, the one or more additives may include a buffer. In an embodiment, the one or more additive may be selected from the group consisting of ascorbic acid or salts thereof; isoascobic acid (erythorbate) or salts thereof; citric acid or salts thereof; acetic acid or salts thereof; salts of bisulfite or metabisulfite; and tocopherol acetate.

According to a further aspect, the present invention provides a process for preparing an allulose syrup according to the first aspect.

According to a further aspect, the present invention provides the use of the allulose syrup according to the first aspect in the preparation of a food or beverage product.

Detailed Description

The present invention is based on the finding that allulose syrups with improved storage stability can be prepared by careful control of certain parameters.

The term "allulose" as used herein refers to a monosaccharide sugar of the structure shown as a Fischer projection in below Formula I. It is also known as "D-psicose": CH2OH C=0 H-C -OH H-C -OH H-C -OH Formula (I) CH2OH According to a first aspect, the present invention provides an allulose syrup having a total dry solids content of from 70% to 80% by weight, and comprising allulose in an amount of at least 90% by weight on a dry solids basis, wherein the pH of the syrup is from 3.0 to 5.0.

The total dry solids content of the allulose is from 70% to 80% by weight. For example, the total dry solids content may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% by weight, as well as all intermediate values. In an embodiment, the total dry solids content of the allulose syrup is from 71% to 78% by weight. In another embodiment, the total dry solids content of the allulose syrup is from 71% to 73% by weight. In another embodiment, the total dry solids content of the allulose syrup is from 76% to 78% by weight.

It has been found that, although the stability of the allulose syrup is generally highest towards the lower end of the total dry solids content range of the invention, microbial stability is generally highest towards the higher end of the total dry solids content range of the invention. Accordingly, the selection of a suitable total dry solids content within the range of the invention can be made depending on the key attribute for the particular application.

The pH of the allulose syrup is from 3.5 to 4.5. For example, the pH of the syrup may be 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5, as well as all intermediate values.

In an embodiment, the pH of the allulose syrup is from 3.8 to 4.2. In an embodiment, the pH of the allulose syrup is about 4.0.

It has been found that allulose degradation and HMF formation can be minimized by increasing the pH, but that undesirable color formation is also promoted by increasing the pH. It has been found that the pH according to the present invention is optimal both in terms of minimizing allulose degradation and HMF formation, and minimizing undesirable color formation.

It is surprising that allulose syrups have been found to be most stable in the above range of pH, since monosaccharide syrups have previously been found to be most stable at lower pH, e.g. between 2.2 and 3.0 (Smirnov V, Geispits K; Stability of Monosaccharides in Solutions of Different pH; BioChem. Moscow, 1957, 22:849-854).

The allulose syrup comprises allulose in an amount of at least 90% by weight on a dry solids basis (i.e., of the total dry solids present in the allulose syrup, at least 90% by weight is allulose). For example, the allulose syrup may comprise allulose in an amount of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% by weight on a dry solids basis, as well as all intermediate values. In an embodiment, the allulose syrup comprises allulose in an amount of at least 95% by weight on a dry solids basis.

In an embodiment, the allulose syrup comprises less than 1000 ppm of HMF. For example, the allulose syrup may comprise less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm or less than 100 ppm of HMF.

In an embodiment, the allulose syrup further comprises one or more additives. In an embodiment, the one or more additives may include a stability-enhancing additive. In an embodiment, the one or more additives may include a buffer. The incorporation of a buffer in the allulose syrup maintains the pH of the allulose within the desired range for a longer period of time, such that storage stability is further enhanced.

In an embodiment, the buffer may be selected from the group consisting of ascorbic acid or salts thereof; isoascobic acid (erythorbate) or salts thereof; citric acid or salts thereof; acetic acid or salts thereof; salts of bisulfite or metabisulfite; and tocopherol acetate. In the case of salts, suitable salts include alkali metal salts, particularly sodium and potassium salts, and especially sodium salts. Specific examples of buffers useful in the present invention include ascorbate, isoascorbate, sodium citrate, sodium acetate, totopherol acetate and metabisulfite.

The concentration of buffer included in the allulose syrup may be around 0.2% in the case of ascorbic acid or salts thereof; isoascobic acid (erythorbate) or salts thereof; citric acid or salts thereof; acetic acid or salts thereof; and tocopherol acetate. The concentration of buffer included in the allulose syrup may be around 0.02% in the case of salts of bisulfite or metabisulfite.

The allulose syrup of the present invention preferably has a shelf-life of at least 6 months. In particular, the allulose syrup of the present invention preferably maintains an allulose content of at least 95% on a dry solids basis for at least 6 months, preferably at least 9 months, at least 12 months or more than 12 months.

According to a further aspect, the present invention provides a process for preparing an allulose syrup. The process comprises: Providing an allulose syrup; adjusting the dry solids content of the allulose syrup such that it is from 70% to 80% by weight; adjusting the allulose content of the allulose syrup such that allulose is present in an amount of at least 90% by weight on a dry solids basis; and adjusting the pH of the allulose syrup so that it is from 3.0 to 5.0. The process optionally comprises adding one or more additives to the syrup.

The description of the embodiments of the allulose syrup herein applies mutatis mutandis to the process for preparing an allulose syrup.

According to a further aspect, the present invention provides the use of the allulose syrup according to the first aspect in the preparation of a food or beverage product, as well as food or beverage products made using the sweetener syrup.

Food or beverage products which may be contemplated in the context of the present invention include baked goods; sweet bakery products (including, but not limited to, rolls, cakes, pies, pastries, and cookies); pre-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (including, but not limited to, fruit pie fillings and nut pie fillings such as pecan pie filling, as well as fillings for cookies, cakes, pastries, confectionary products and the like, such as fat-based cream fillings); desserts, gelatins and puddings; frozen desserts (including, but not limited to, frozen dairy desserts such as ice cream -including regular ice cream, soft serve ice cream and all other types of ice cream -and frozen non-dairy desserts such as non-dairy ice cream, sorbet and the like); carbonated beverages (including, but not limited to, soft carbonated beverages); non-carbonated beverages (including, but not limited to, soft non-carbonated beverages such as flavored waters and sweet tea or coffee based beverages); beverage concentrates (including, but not limited to, liquid concentrates and syrups as well as non-liquid 'concentrates', such as freeze-dried and/or powder preparations); yogurts (including, but not limited to, full fat, reduced fat and fat-free dairy yogurts, as well non-dairy and lactose-free yogurts and frozen equivalents of all of these); snack bars (including, but not limited to, cereal, nut, seed and/or fruit bars); bread products (including, but not limited to, leavened and unleavened breads, yeasted and unyeasted breads such as soda breads, breads comprising any type of wheat flour, breads comprising any type of non-wheat flour (such as potato, rice and rye flours), gluten-free breads); pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads (including, but not limited to, jellies, jams, butters, nut spreads and other spreadable preserves, conserves and the like); confectionary products (including, but not limited to, jelly candies, soft candies, hard candies, chocolates and gums); sweetened breakfast cereals (including, but not limited to, extruded (kix type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals); and cereal coating compositions for use in preparing sweetened breakfast cereals. Other types of food and beverage product not mentioned here but which conventionally include one or more nutritive sweetener may also be contemplated in the context of the present invention. In particular, animal foods (such as pet foods) are explicitly contemplated.

Examples:

The invention will now be further described and illustrated by means of the following examples, it being understood that these are intended to explain the invention, and in no way to limit its scope.

Summary

It was determined from stability experiments that allulose syrup produced in Loudon, TN has a more rapid purity degradation that allulose syrup produced in Decatur, IL (Example 1). The main difference between these syrups was starting pH. A pH -time -temperature factorial designed experiment was carried out to determine the effect and interplay of pH, time and temperature on purity, HMF, color formation in allulose syrup (Example 2). It was determined that low pH had an effect on purity and high pH had an effect on color and there was a very narrow range where both color and compositional purity were stable. An accelerated stability study (Example 3) was carried out with pH values around the narrow range of predicted stability and also with additives and at different % dry solids. It was determined that % dry solids and optimal pH of around 3.8 to 4.0 were critical to storage stability. Microbial stability was also investigated (Example 3) and was very stable at 77% and less stable at 72%, thus putting a lower bracket on dry solids for syrup shelf life stability. Finally, a more detailed study of additives with respect to stability at two different dry solids levels and optimal pH was carried out (Example 4). Some of the additives reduced the change in color composition and HMF.

Example 1.

Allulose Syrup lot number YP14J01502 was used for this study. Each sample consisted of 3500 mL of allulose syrup in a 4 quart square plastic container. The sampling was carried out at 0 and 2 months.

Analytical Samples were analyzed using the following test methods: For pH and color the samples were analyzed at a standard DS.

Table 1 Analytical methods DS TN 27501 pH TN 60710 Carbohydrate composition TN 67395-67409 HMF TN 39945-46 Color TN 22725 Figure 1. Composition 0.5 1 1.5 2 2.5 Time (months) Swcomposition 25C tom position 30C 35C 94 92 90 88 86 84 82 80 Purity (% a Ilu lose) Allulose composition dropped significantly in the course of 2 months as seen in Figure 1. The material at all three temperatures investigated was no longer in specification at 2 months. There was a clear trend that higher temperature resulted in greater change in composition.

Changes in color were minor (Figure 2). At 35 °C (higher than recommended storage temp) the syrup did go beyond 2, which is a recommended upper limit. However at 25 and 30 °C (recommended storage temp), the color change was minimal and did not exceed 2.

HMF increased in each sample over 2 months (Figure 3). The sample at 35°C was out of specification on HMF after 2 months and the 30 °C sample had just passed the limits of 100 ppm. The starting value for HMF was higher than previously prepared material and it is likely that this starting number will be substantially reduced during normal process improvement.

Figure 2. Color 9 8 7 4-1 3 2 1 0 a\\\ti c's 0.5 1 1.5 2 2.5 Time (months) Figure 3. HMF Figure 4. pH The pH value decreased similarly over 2 months for each sample. It is noteworthy that the pH started lower in the Loudon, TN prepared material than previously studied material which started at a pH of 4.0 rather than 3.4.

The main difference in composition between the two products is the material produced in Loudon, TN had a lower initial pH by approximately 0.6 pH unit. Additionally, pH in the previous study remained above pH3.5 for the first 5 months (Figure 5). Color development (Figure 2) was lower in this study at 25 °C for two months (0.67) 0.5 1 1.5 2 2.5 Time (months) E 120 0. 0.

40 20 0 3.45 3.4 3.35 3.3 0. 3.25 3.2 3.15

0 0.5 1 1.5 Time (months) 2 2.5 -4-25C -0-30C compared to the previous study Figure 6 at the same time and temp (1.32). HMF increased by 20 ppm at 25 °C and 120 ppm at 35 °C in this study (Figure 3) in comparison to 40 ppm and 140 ppm at respective times and temperature in the previous study (Figure 7).

Overall it appears that color develops faster at higher pH, while composition degrades more quickly at lower pH and that the optimal pH window may be surprisingly narrow.

Figure 5 -Decatur study pH * 4C 0250 a. 35C X 50C T" 3.5 2.5 4.5 *,- 1 2 3 4 5 6 Time (Months) Figure 1 -Decatur study Color before heat, detailed Figure 7 -Decatur study HMF content, detailed 180 160 140 X 4C c 120 a 100 M 25C 35C s a X 50C * 4 * 4 0 1 2 3 4 5 6 Time (Months) 0 1 2 3 4 5 6 Time (Months) * 4C 25C 35C X 50C 9 8 7 6 5 Color via TN 22725 Figure 8 -Decatur study Allulose content Table 4 Carbohydrate profiles for lot # L014J03155 (Stored in Railcar STSX4000) (Normalized % wt of saccharide components) The compositional change seen in this stability storage study was additionally confirmed in the bulk product in railcars (Table 4). The compositional changes were slightly less at 3 months in railcars, than two months at 25 °C in the storage study (i.e. 2% in 3 months vs. 3% in 2 months). This effect due to volume of container was demonstrated in the previous study as well, when 300 gallon totes were less susceptible to compositional changes than 1 quart containers. A likely explanation may be that pH is more stable in larger containers as the surface area to volume ratio is smaller. An alternative explanation could be that the average temperature was lower in the larger containers, however, this was not directly measured over the course of storage.

Allulose Purity %DSB Time (months) HPLC Components Allvlose *:.peKtit, Ott.6: 33.6 10/26/14 95.42 0.11 1.08 0.6 1.38 0 1.41 1/21/15 93.42 0.24 1.91 0.69 2.00 0.49 1.25 -2.00 0.13 0.83 0.09 0.62 0.49 -0.16 :After.vaitcar iflerence The first commercial batch of allulose syrup prepared in Loudon, TN rapidly changed composition and was no longer in specification after 2 months. Other attributes of the syrup remained within specifications. This result is in contrast with slow compositional changes over 6 months with previously prepared material in a Decatur plant trial in 2013 (Figures 5-8). The primary physical different in those two syrups appears to be pH, therefore additional studies are underway to determine the robustness of stability with respect to pH as well as other physical properties and additionally how to reprocess the out of specification material efficiently.

Example 2: pH -time -temperature investigation Samples of two key process streams with different carbohydrate composition were subjected to different temperatures and pH for periods of time up to 8 hours. Sub samples were taken at intervals and their carbohydrate composition and pH analysed.

Substrates Two substrates; were utilized in this experimental plan. To understand the impact of DS on the rate of degradation these same streams after evaporation conditions will be tested. Their approximate compositions are shown below: Table 2-1 Sample carbohydrate profiles These two streams were entered into the DOE software Design ExpertTM with the following ranges for the variables of interest: * tl* lui ose Substrate 1 0 1 97 2 Substrate 2 22 70 5 3 Table 2-2 Time, temperature and pH ranges The Box-Behnken design produced the following experiments to conduct for one of the feed streams.

Table 2-3 Box-Behnken experimental runs \*IW 3.0 120 4.25 3.0 167.5 0.5 3.0 167.5 8 3.0 215 4.25 3.75 120 0.5 3.75 120 3.75 167.5 4.25 3.75 167.5 4.25 3.75 167.5 4.25 3.75 167.5 4.25 3.75 167.5 4.25 3.75 215 0.5 3/5 215 8 4.5 120 4.25 4.5 167.5 0.5 4.5 167.5 4.5 4.25 Three samples of each starting material were taken. The pH measured and recorded.

One sub sample of each was adjust to pH 3.0, the next adjust to pH 3.75 and the final one 4.5 using dilute HCI or sodium carbonate. After completing this for each of the four substrates there were 12 stock solutions. The pH method is described below in the Tim Low 0.5 3.0 120 High 8 4.5 215 analytical section. Each stock solution was only made on the morning of its testing as there is a noticeable drift in pH over time even at room temperature.

For each of the stock solutions 50m1 was poured into labelled glass jars. To determine the heating curve a syrup sample was run and its temperature tracked.

The lids were closed on the sample containers then placed into the different temperature ovens and the time started.

Samples were removed from each oven for each stock material at the time intervals shown below. Samples were chilled quickly in an ice bath and submitted for the carbohydrate and HMF analysis. Sub samples diluted to the standard DS were submitted for colour and pH.

Table 2-4 Time, temperature and pH ranges Analytical Samples were analyzed using the following test methods: For pH and color the samples were analyzed at a standard DS, which will be determined by that of the lowest DS stock sample.

Table 2-5 Analytical methods DS TN 27501 pH TN 60710 Carbohydrate composition TN 67395-67409 HMF TN 39945-46 Color TN 22725 3.0 0.5 167.5 3.75 4.25 4.5

P ip 8.0

The heat up rate of samples in the 215°F oven was measured and is shown in the chart below.

The sample data was entered into the experimental design software for each of the feed streams. Initial models, using starting pH and absolute values for allulose, were weak and contained significant lack of fit. Substituting final pH for initial pH, and allulose loss, defined as (allulose loss / allulose purity initial), for absolute allulose content, greatly improved the models.

Table2-6 Substrate 1A and 1B analyses.

The perturbation plots shown below shows the responses from the three parameters, pH (A), temp, (B) and time (C). It shows: * Increase time there is a linear increase in % allulose loss.

* Increase pH there is reduction in % allulose loss.

* Increase temperature and there is an increase in % allulose loss 00 lime /minutes SOml Syrup Glass Jar Heat Up Profile (215°F Oven) Fructuse Substrate 1A 3.39 0.69 92.72 3.43 4.2 14.0 Substrate 1B 3.25 0.0 92.49 4.26 4.5 25.5 Perturbation

B CA

Deviation from Reference Point (Coded Units) Perturbation

A

Deviation from Reference Point (Coded Units) Figure 2-7 Perturbation plots of degradation factors on Substrate IA (top) and 'I B (bottom) (A = pH, B = temperature and C = time) The model predicts that at high pH, allulose loss reaches a minimum and then begins to increase. This is likely an artifact of fitting the available data and could be confirmed with additional runs.

The following charts are plotted at 4 hours.

% all lose loss % allulose loss A: ph % allutose loss A. ph Figure 2-8 Contour plots of degradation factors on Substrate 'IA (top) and Substrate 'I B (bottom) at experimental time of 4 hours Another factor is the DS of the stream to be evaporated. The two charts below can be used to compare the rate of degradation between the lower DS (14%) Substrate 1A and the higher DS (25%) Substrate 1B. They show that at the higher pH values (>3.6) there is not much difference predicted in their respective rates of degradation. However at lower pH (<3.6) there are noticeably steeper curves of allulose loss with increasing temperature in the higher DS Substrate 1B. This suggests that higher DS results in more rapid allulose degradation.

Figure 2-9 Allulose degradation effect of temperature at different pH values in 4 hours Temp °F 4.4 3.3 % allulose loss 1.00 0.80 0.60 x 0.40 Substrate 1A 0.20 -3.0 0.00 Substrate 1B 7" 0.60 8 0.40

R

1.00 -0.80 0.20 -0.00tt.1/4 160 180 Temp °F

N 4.4

--k',,pH 3.0 Increasing pH reduces the rate of allulose loss but increases the rate of color formation, see contour plot for Substrate 1B below.

OCI color

A\I-Th \,c* \ : \*,****,\\.

* \' **,.\\\*',:\C-\"'\\* *\\A *\'Nw\'t\: 1/2q' \\$\'* \*\\' A: pH Figure 2-10 Color formation effect of temperature and pH on Substrate 1B in 4 hours Another observation from the testing was the final pH of the tested samples was typically lower than the starting one. The plot below shows the impact of temperature and starting pH on the change in pH.

pH change A: pH Figure 2-11 pH change observed on Substrate 1B in 4 hours The information presented suggests a dynamic and complicated set of equilibrium reactions is likely occurring.

Substrate 2A and 2B Table 2-11Substrate 2A and 2B analysis.

In both substrate 2A and 2B almost no change was observed in the final allulose purity across the range of pH, time and temperature. Changes in fructose were small and did not generate a substantiated model. The dextrose was modelled to vary according to the plot below.

ffruolso UlOse::.

Substrate 2A 13.76 78.91 2.33 2.0 3.00 3.75 35.1 Substrate 2B 15.56 74.47 2.95 4.1 2.92 3.56 58.1

E A: pH

Figure 2-12 Dextrose change observed on Substrate 2A in 8 hours The increase in DP2 content was shown to be only dependent on time, see chart below.

One Factor C: time Figure 2-13 DP2 formation in Substrate 2A as a function of time Despite only minor changes in carbohydrate profile being observed in the experiments, color and HMF do increase at high temperature and for HMF specifically, at low pH, see charts below.

cies:cp space. g 6 -

H F ati A; pH

Figure 2-13 HMF formation on Substrate 2A in 8 hours A similar plot was made for the effect of pH and temperature on color after 8 hours, shown below: color \\:Ta:6\,h,:%:...* *:;::i.,n:c \\' *V\:: N'1/4.1/4 \ ,\ k.'1/4'\",k%,\\ 4.\\&N.*:, :v\:*\e'"\\ '\'\\ \\ ::,.,::::.:::._:***:,\:::::::::::::::::g:: , :5::::'5::'' \\ \ 'K \\\\ k \\N' \\\\\ \\* b:,.:*,*:;:w.':*,::':;N\--' - \''W\ ' ''\' \ '' :::::::::::::::::::::::* kw-,.,u,:-*\ .'. \\\ \* * *,.:\':.' ,.,. \\ \\ \<\ W:',::::N*0.....,.',--\ :*:' Y'z:::...:::: .'::.....-......' ,\.. \\ ,w, \\, ' '. ll A: ph

Figure 2-14 Color formation on Substrate 2A in 8 hours a (3) E m The pH has less effect on the rate of color formation in contrast to the high allulose content streams (substrate 1A and 1B). The temperature trend is similar with increasing temperature increasing color and the model is sensitive, particularly at the high temperatures, the red zone shown above starts at colors of 15 units and goes into the thousands.

Conclusions

For the high purity allulose streams * Increase time there is a linear increase in % allulose loss.

* Increase pH there is reduction in % allulose loss * Increase temperature and there is an increase in % allulose loss * Increase DS and the rate of allulose loss increases * Manufacturing and storing a high purity allulose syrup with minimized color, HMF and maximized purity requires very narrow and specific operating parameters and final product parameters.

Example 3 -Storage stability Based on Example 2, it was determined that pH, temperature and DS were critical to maintaining product purity. Final allulose syrup product samples were subjected to a range of pH and different DS and temperatures. Another series of samples will have sodium metabisulfite and sodium citrate added. Separate sub samples will be taken at pre-determined intervals and their carbohydrate composition, color, HMF, DS and pH analyzed.

Table 2-15 Sample carbohydrate profile Table 3-2 Nominal screening experiments to be run.

Campaign 1 3.4 Product 77.0 0.5 2.2 93.0 4.3 pH Temp °C DS % Additive 3.4 40 77 -3.4 50 77 3.4 40 71 3.4 50 71 3.5 40 77 3.5 50 77 - 4.0 40 77 - 4.0 50 77 - 4.0 40 77 Sodium citrate 4.0 50 77 Sodium citrate 4.0 40 77 Sodium metabisulfite 4.0 50 77 Sodium metabisulfite 4.5 40 77 4.5 50 77 Methods Samples of starting material were taken. The pH and DS measured and recorded. One sub sample of each was taken as is, the next adjusted to pH 3.6, another 4.0 and the final one 4.7 using dilute HCI or sodium carbonate. One subset of starting material was diluted to 71% DS. Another subset of the pH 4.0 batch had sodium citrate or sodium metabisulfite added. Sealed sample containers were placed into different temperature ovens at 40 and 50 °C. Extracts from each of the samples were removed from each oven periodically. Samples were chilled quickly in an ice bath and analyzed for carbohydrate composition, HMF, color and pH.

Table 3-3 Analytical methods DS TN 27501 pH TN 60710 Carbohydrate composition TN 67395-67409 HMF TN 39945-46 Color TN 22725 In general pH dropped over the course of the experiments, see Figure 3-2 and Figure 3-3. The decrease in pH is more pronounced in samples starting at higher pH, and the pH drops faster at higher temperature. It appears that the pH of each sample becomes more stable around a value near 3.0 to 3.3.

Two of the samples that were adjusted to start at pH 4.0 had buffer systems added. The first was with 75 ppm sodium metabisulfite (MBS) and the second was 60 ppm sodium citrate (NaCit). Based on this data there are only small effects seen from the additives.

Figure 3-2 Samples pH change at 40°C pH (40°C) a b Days Figure 3-3 Samples pH change at 50°C The pH drift data above at pH 4.0 matches a previous stability study in which the product pH started at 3.9 and samples were stored at 50°C, demonstrating the same trend in a separate experiment.

Figure 3-4 Samples pH change at 50°C compared to initial study Allulose purity dropped in all samples following the trend of higher temperature, lower pH and longer time resulting in faster allulose losses. The pH 4.0 samples with additives show a similar rate of allulose loss as the pH 4.0 sample with no additive. This may be explained by the similar pH changes observed above.

pH (50°C) 4.6 4.6 4.4 -63-3.3.1 68 4.2 6446 3 6 pH z. W'.3...

46,6,4,0 21I MRS /6'1.6 6h1 6i3:4 "34'6. 'z. Days 20 Day

Surprisingly, the sample with only a slightly lower DS, (71% vs. 77%) starting at pH 3.37 showed much less allulose loss than its equivalent pH sample at 77% DS. The rate of allulose loss at 71% DS was approximately half that at 77% DS, demonstrating that a narrow range of DS has a dramatic and unexpected effect on alldose syrup stability. Similar effects are not observed for similar monosaccharide syrups such as glucose or high fructose corn syrups within such a narrow range of pH.

Figure 3-5 Samples Allulose change at 40°C Figure 3-6 Samples Allulose change at 50°C AIWiese Purity (4OT) 737 0171% Os mas oft 8,-3 6 00. Za 97 -," 90.5 12

Axis Title Aliulose Purity (50°C) 71%0S NaCit Color was measured and plotted against time, see below. High pH, longer time and high temperature increase the color formation. By increasing the pH it is possible to mitigate the carbohydrate degradation, however there is a limit as increasing the p1-1 too much will lead to unacceptable color in the final product. This results in a surprisingly narrow pH range being acceptable for long term storage of allulose syrup. This range appears to be between pH 3.5 and 4.5 when both color and composition stability are considered.

Color (40°C) 2.0 8-8.87 pH 3 37 psi 13% 08 asas-s..0 2.5 to Figure 3-7Samples color change at 40°C Color (50°C) as0--3.31 p! 833..Y.31,0 pH MOS pH NaCit _..... _.... .... ... Days... .... ..... ..... _.... .... ..... .... ....

Figure 3-8 Samples color change at 50°C The change in HMF over time for these samples is shown below. Low pH, high temperature and longer time contributing to increased HMF formation.

Figure 3-9 HMF at 40°C HMF (40aC) -4-4,411p11 44-4 0 p13 M1311 4214.1 Nu< 4 pH 2(3

U oV 12 Days

Figure 3-10 HMF at 50°C Microbial stability T80 T=1 T=2w T=4 Control samples: One larg container 1250 mL sterile glass Jar RCM MI Cara' DITIZMIZE Mr= E:I=INNIIIIS CIE= CIO Experimental samples: <10 <10 <10 72602 372603 372604 72605 thpatik * 'NIIIIIMINC 0723 Replicates:3 CMS=2S EfEEI SM Plating: 12/point 25 NEDIElati

CNC ENEMSZO

IMINUERNE CCM

WC^1051 k":',\ <lo no no 770 770 770 770 <10 <10 <10 <10 <10 <10 <10 3357:00 kr&"1/43: 309"OCC Livah 311,10)...."s.'''., 290800ils: 260,500,Y'.,: 317,507 402:500 i?..\ 7"(KV 298,xmZO,hxh27,000 (> ,OZ 287"01:12,5: \ NS;51. 425:':',-.\&' 265 ills: NISVS 440 list5*Va 55."K' <10 <10....',,..4, 40 ats.-SS, <10 CO &;. -<10h\1 <10 55 &\a <10 <10 E <11).k'' '7,"0 <10 <10 'S'.,1 jr <1 0 kK.WNN <10 <10 <10<10 <10 CO, , <10 Cn <10 <10 (2&62k;', CD Sk.

i:t V: N cu '::\, , <10.:>X <10 g:X<>: l l(8) Temperature ("C) LIMS Container: 12 Min total cal: 1,800 grams S5 OS level LIMS 422405 585.2* CAA,.

2,400 4/35:AV*2&-. <10:ks:. \2,2N <10 4:050..,CYC\\I 5544.' \ CYO <10 2,250 \''.al S.5\0'.^\) 420:b.Ck'.5'' "On& N\.5' 2,300:x <10 "a: <10Lgyp\ 770 2,71.105,"MC; it\lin i'N'SN.

3,25.N.",;;; 155 co& <10 2:21-10&>1 \?. 150 S. \&..' 2,850 L Ts',56,1*N, 175 5, \ <10<10 z.,500;;;;":,,, <10 &;..;;.z <Th.& ,z <10 3,150 <10 \SS <10N <10\ 2,8% co <10;\ h; 55. ones S8.6 \ISA Replicates: 3 Effilar5 Container: 12 °66.::°:°:°: OM Mal Er3CMI CM4

CNC :ECM

1:05:888:185:5:5:5:5853$ Plating, 12/paint Min total gal: 1,800 grams 2$ riZEI riZEI Microbial stability was assessed 72% and 77% dry solids by a challenge study with osmophilic yeasts and molds.

Put 250 grams of each DS level control sample into two sterile glass jars (250 grams x 2 for each moisture level, total 4 containers). Put 1,000 grams of each DS level sample into two sterile Nalgene containers (1,000 grams x 2 for each DS level, total 4 containers). Inoculate osmophilic mold and yeast separately (less than 1% of total volume) for each 1,000 gram sample (8 containers total). Mix and leave containers at RT for 2-3 hours to equilibrate the inoculum. Take 250 grams to put into a 250 mL sterile glass jar to make triplicates for each testing condition (24 x 250 mL glass jars).Take initial sample for plating (T=0). Start incubation at 25 and 35 °C. Take sample at intervals as planned for plating.

At 77% DS osmophilic yeasts and molds were rapidly made non-viable. However at 72% DS, allulose syrup took 4 weeks to completely kill all viable yeasts and molds.

Based on the findings above, final product stability is optimized in a narrow range of pH, from 3.5 to 4.5 and more preferably in a pH range from 3.8 to 4.2 in order to optimize the trade-off between carbohydrate stability and color/HMF formation. Lower pH was shown to increase the rate of compositional degradation and HMF formation, while higher pH was shown to result in more rapid formation of color. Lower DS reduces the rate of degradation in all parameters, however a final product DS of less than 70% will likely have a water activity that is too high to maintain good microbial stability. Higher DS results in more rapid degradation. Therefore an optimal DS of 7178% is required for long term stability of allulose syrup and more preferably a DS of 71- 73% should have the highest stability. In cases where microbial stability is the key attribute necessary, 76-78% DS would have the best microbial stability.

Example 4 -Stability improvement with syrup additives Additives have an effect on stability. These additives may stabilize the syrup by buffering the pH to help control at pH 4.0 and also to minimize oxidation.

One temperature 30°C (86°F) has been used to assess effect on stability.

Approximate composition of campaign 1 material: AtiLifiStd,:% pH Q Starting Material 94 3.3 77.5 Method Allulose Syrup lot number YP14J01502 was used for this study. Each sample consisted of 1000 mL of syrup in a plastic container. Two gallons of this material were pH adjusted to 4.0 using 1M sodium carbonate (NaCO3), by slow and careful addition and regular pH measurement at 1:1 dilution. This material was then split into two separate containers and one was diluted to 71% DS (11.5 lbs 77% DS syrup, plus 0.97 lbs water).

After dilution, the samples were subsampled into 500 mL plastic containers. Fresh 10% solutions (25 mL) of Ascorbate, Isoascorbate, Sodium Citrate, Sodium Acetate, and 1% tocopherol Acetate, and metabisulfite were prepared and pH adjusted with Sodium carbonate to -4.0 pH. 10mL of these solutions were added and mixed in with the corresponding samples as in Table 1 The following samples will be prepared as above and then placed in the 30°C oven and sampled as table 2 below.

Table 1: Samples

DS pli Ascorbate lsoascorbate Sodium Sodium Tocopherol Metabisulfite Citrate Acetate Acetate 71 4.0 0.2% 71 4.0 0.2% 4.0 0.2% 71 4.0 0.2% 71 4.0 0.2% 71 4.0 0.02% 77 4.0 0.2% 77 4.0 0.2% 4.0 0.2% 77 4.0 0.2% 77 4.0 0.2% 77 4.0 0.02% The sampling and testing schedule is detailed in Table 2.

Table 2 -Stability Robustness Samples Sample x Color Before Heat DS RI (% as is) Carbohydrates (% as is) LL 0. 5

I

0 Months x x x x x 1 Months x x x X 2 Months x X x x 4 Months x X x x 6 Months x x x x x Results Some of the additives reduced the change in color composition and HMF.

Advantages of the Invention: A syrup form that is more stable has benefits in that it can be stored for longer time periods and still be saleable, it has broader customer appeal, it can be shipped to geographic locations that require lengthy shipping and holding times. Additionally, improved product stability means that the product as used will retain a higher quality of composition and taste. This is beneficial from a calorie labelling position and final consumer product quality position.

Preferred features of the invention include: The allulose syrup is envisioned to comprise 70-80% dry solids by weight, and >90% allulose on a dry solids basis, a measured pH between 3.0 and 5.0 and a shelf life of at least 3 months.

* Preferred ranges for the dry solids include 71-78%, 71-73% or 76-78% * Preferred pH ranges are between 3.5 and 4.5 or between 3.8 and 4.2 * Preferred allulose content is >95% allulose on a dry solids basis * Preferrably, the syrup has a limited amount of the following compounds a less than 1000 ppm hydroxymethylfurfural HMF o sulphur dioxide concentration less than 20 parts per million o Isovaleraldehyde concentration measured less than 10 parts per billion o 2-aminoacetophenone concentration of less than 2 parts per billion * Optionally the syrup can have any of the following compounds alone or in combination thereof o stability enhancing ingredient including one or more of: 1) ascorbic acid or salts thereof, 2) isoascobic acid (erythorbate) or salts thereof, 3) citric acid or salts thereof, 4) acetic acid or salts thereof, 5) salts of bisulfite or metabisulfite, and/or 6) tocopherol acetate * allulose syrup with a concentration >95% with a shelf-life of at least 6, 9, 12 months, or more than 12 months

Claims

CLAIMS: 1. An allulose syrup having a total dry solids content of from 70% to 80% by weight, and comprising allulose in an amount of at least 90% by weight on a dry solids basis, wherein the pH of the syrup is from 3.0 to 5.0.
2. An allulose syrup according to Claim 1, wherein the total dry solids content of the allulose syrup is from 71% to 78% by weight.
3. An allulose syrup according to Claim 1 or 2, wherein the total dry solids content of the allulose syrup is from 71% to 73% by weight.
4. An allulose syrup according to Claim 1 or 2, wherein the total dry solids content of the allulose syrup is from 76% to 78% by weight.
5. An allulose syrup according to any preceding claim, wherein the pH of the allulose syrup is from 3.5 to 4.5.
6. An allulose syrup according to Claim 5, wherein the pH of the allulose syrup is from 3.8 to 4.2.
7. An allulose syrup according to any preceding claim, wherein the allulose syrup comprises allulose in an amount of at least 95% by weight on a dry solids basis.
8. An allulose syrup according to any preceding claim, wherein the allulose syrup comprises less than 1000 ppm of HMF.
9. An allulose syrup according to any preceding claim, wherein the allulose syrup comprises sulfur dioxide in an amount of from 1 to 20 ppm.
10. An allulose syrup according to any preceding claim, wherein the allulose syrup comprises less than 10 parts per billion of isovaleraldehyde.
11. An allulose syrup according to any preceding claim, wherein the allulose syrup comprises less than 2 parts per billion of 2-aminoacetophenone.
12. An allulose syrup according to any preceding claim, wherein the allulose syrup further comprises one or more additives.
13. An allulose syrup according to Claim 12, wherein the one or more additives include a stability-enhancing additive.
14. An allulose syrup according to Claim 12, wherein the one or more additives include a buffer.
15. An allulose syrup according to Claim 12, wherein the one or more additive is selected from the group consisting of ascorbic acid or salts thereof; isoascobic acid (erythorbate) or salts thereof; citric acid or salts thereof; acetic acid or salts thereof; salts of bisulfite or metabisulfite; and tocopherol acetate.
16. A process for preparing the allulose syrup according to any of Claims 1 to 15, wherein the process comprises: providing an allulose syrup; adjusting the dry solids content of the allulose syrup such that it is from 70% to 80% by weight; adjusting the allulose content of the allulose syrup such that allulose is present in an amount of at least 90% by weight on a dry solids basis; and adjusting the pH of the allulose syrup so that it is from 3.0 to 5.0.
17. A process according to Claim 16, wherein the process further comprises adding one or more additives to the syrup.
18. Use of the allulose syrup according to any of Claims 1 to 15 in the preparation of a food or beverage product.