NON-CRYSTALLIZABLE D-ALLULOSE SYRUPS

Description

Field of invention

[0001] The invention relates to a D-allulose syrup, one of the advantageous properties of which is that it is less crystallizable than the syrups of the prior art. Another subject of the invention relates to a method for manufacturing this D-allulose syrup.

[0002] The invention is defined by the appended claims. Prior art

[0003] D-allulose (or D-psicose) is a rare sugar with a sweetening power equal to 70% of that of sucrose. Unlike the latter, D-allulose does not cause weight gain because it is not metabolized by humans. It has a very low caloric value (0.2 kcal per gram) and thus prevents fat gain. In addition, studies have shown that D-allulose is non-cariogenic, even anti-cariogenic. These properties have recently generated significant interest from the food and pharmaceutical industries.

[0004] D-allulose is generally obtained enzymatically, by reacting an aqueous solution of D-fructose with a D-psicose epimerase as described for example in application WO2015 / 032761 A1 in the name of the Applicant. Whatever the enzyme used, the reaction is not complete and the quantity of fructose transformed into D-allulose after epimerization is less than 30%.

[0005] Thus, from the composition resulting from the epimerization reaction, it is necessary to carry out a step of separation of D-allulose, in order to isolate it from the other constituents present and in particular from fructose. To carry out this separation, chromatography of the composition resulting from the epimerization reaction is very generally carried out, for example by continuous chromatography of the simulated moving bed type, which makes it possible to isolate a fraction rich in D-allulose.

[0006] Document JP2001354690 A describes a method for purifying a D-allulose composition starting from a mixture of fructose and D-allulose, said method comprising a separation step consisting of a continuous chromatography step using a particular sequence of samples of the different products of the mixture. A fraction rich in D-allulose (whose D-allulose richness can reach 98%) and a fraction rich in fructose are recovered. The recovery yield in the fraction rich in D-allulose is 96%.

[0007] At the end of the separation steps mentioned above, liquid compositions rich in D-allulose are obtained. Thus, these liquid compositions, generally called syrups, are used for the manufacture of food or pharmaceutical products. For example, application WO 2015 / 094342 in the name of the Applicant describes the manufacture of solid food products comprising a D-allulose syrup, comprising from 50 to 98% of D-allulose and a native protein. It is mainly in this form of syrups that various companies have announced the marketing of D-allulose to date.

[0008] Document WO 2016 / 135458 describes syrups comprising, relative to their dry mass, at least 80% allulose and studies their stability over time. No manufacturing protocol for this syrup is described. The composition of the syrups is analyzed by high-performance liquid chromatography, which is presented as the standard method for analyzing this type of syrup. The syrups described in this application are presented as being crystallizable at low temperature.

[0009] However, for some applications, there may be an advantage in having a non-crystallizable or even less crystallizable D-allulose syrup, even at low temperatures. This is particularly the case when D-allulose is to be used as a humectant, since a compound exhibits this humectant function only when it is in solute form, and therefore not crystallized. Other applications also require that the sugar does not crystallize in the final product to obtain the desired properties. For example, to obtain a soft texture, it is necessary that there is no crystallization of D-allulose in sauces, starch jellies, soft caramels, jams or fruit fillings. Similarly, it is preferable that there is no crystalline deposit in liquid solutions such as salad dressings or drinks. Foods in bar form (nutritional bars, cereal bars, fruit and nut bars, etc.)) are more tender if the sugar does not crystallize in the final product.

[0010] It has also been observed that syrups can exhibit storage instability by forming suspended crystals over time, particularly when this storage is done at low temperatures. In this case, and in particular when these crystals grow, the viscosity of the syrup can increase drastically and it may become necessary to reheat the syrup until the suspended crystals have melted in order to be able to handle it easily again. It may therefore be of interest to provide new syrups with improved storage stability, which do not crystallize or crystallize only slightly, even at low temperatures.

[0011] It was by conducting extensive research that the Applicant was able to observe that, in a process for manufacturing D-allulose syrups, specific impurities are formed during the process. These have, to the best of the Applicant's knowledge, never been reported in the literature. They were able to be identified by the Applicant, using a particular gas chromatography technique, as being D-allulose dimers. The Applicant was also able to show that these dimers, unlike other impurities such as glucose or fructose, have a very significant anti-crystallizing effect. These D-allulose dimers are formed during the process and their presence in the D-allulose syrup is systematic. Beyond this observation, the Applicant has also continued its efforts to provide, for a given dry matter and D-allulose content, new syrups having a less crystallizable character than those of the prior art.To achieve this, it has developed a process enabling it to supply syrups with an increased content of D-allulose dimers. This makes the syrup less crystallizable, making it easier to store for later use, particularly at low temperatures. Summary of the invention

[0012] The subject of the invention is thus a D-allulose syrup comprising, in addition to D-allulose, a mass content of D-allulose dimer, determined by gas chromatography (GC), ranging from 2.0 to 15%, as defined in claim 1.

[0013] This syrup has the advantage of being less crystallizable than the syrups of the prior art, or even non-crystallizable under certain temperature conditions, which allows it to be more stable during storage. This syrup can also be used advantageously in the aforementioned applications.

[0014] Another subject of the invention relates to a method for manufacturing the syrup of the invention, as defined in claim 6. This method comprises: a step of providing a D-allulose composition comprising D-allulose dimers; a step of nanofiltration of said D-allulose composition; a step of recovery of the nanofiltration retentate; a step of concentration of this retentate to provide the D-allulose syrup, the nanofiltration step being carried out with a membrane having a cut-off threshold of less than 300 Da, preferably ranging from 150 to 250 Da.

[0015] The use of a nanofiltration step makes it possible to recover a retentate enriched in D-allulose dimer and thus to obtain, after concentration, the syrup of the invention.

[0016] As mentioned above, the Applicant has been able to observe that, systematically, during the manufacture of D-allulose syrups, specific impurities are formed during the process. These have, to the best of the Applicant's knowledge, never been reported in the literature. This is explained by the fact that, using the high-performance liquid chromatography technique conventionally used to measure the purity of D-allulose, these impurities are not detected on the chromatograms (see Figures 4 And 5 ). It was by using a gas chromatography technique that the Applicant was able to detect their presence (see Figures 6 And 7 ).

[0017] With regard to document WO2015 / 094342, this document uses syrups comprising D-allulose. However, no method for preparing this syrup is indicated. However, as appears in the remainder of the description, the choice of conditions for the preparation steps and in particular the concentration have a major impact on the quantity of D-allulose dimers formed, which had never been reported until now. The document therefore does not describe the syrup of the invention. The same is true in document WO2016 / 135458 already mentioned or document WO2015 / 032761, which does not even exemplify such a syrup.

[0018] With regard to the document JP2001354690 A cited above, it describes the separation of fructose and allulose by chromatography of a composition comprising these two constituents. However, it does not describe the preparation of a D-allulose syrup. However, since the choice of the conditions of the stages of preparation of this syrup and in particular of concentration have a major impact on the quantity of D-allulose dimers, this document therefore does not describe the syrup of the invention.

[0019] Documents WO2011119004 A2, WO 2016 / 064087, CN 104447888 A, CN 103333935 A describe the manufacture of crystalline compositions comprising D-allulose but do not describe the manufacture of D-allulose syrups.

[0020] The invention also relates to a D-allulose syrup obtainable by the process of the invention, as defined in claim 9. Brief description of the Figures

[0021] Figure 1 : There Figure 1represents the production circuit of a non-crystallizable allulose syrup. Figure 2 : There Figure 2 represents a production circuit for a non-crystallizable allulose syrup with recycling loops. Figure 3 : There Figure 3 represents the permeation curve relative to the nanofiltration stage, i.e. the flow rate as a function of the volume concentration factor. Figure 4 : There Figure 4 represents an HPLC chromatogram of a composition rich in D-allulose taken in the process of the invention, before nanofiltration. Figure 5 : There Figure 5 represents an HPLC chromatogram of a retentate taken in the process of the invention, i.e. after nanofiltration. Figure 6 : There Figure 6 represents a CPG chromatogram, in the characteristic zone of the dimers, of a composition rich in D-allulose taken in the process of the invention, before nanofiltration. Figure 7 : There Figure 7represents a CPG chromatogram, in the characteristic zone of the dimers, of a retentate taken in the process of the invention, that is to say after nanofiltration. Figure 8 : There Figure 8 represents the dry matter of the supernatant after 1 month of storage at 4 and 15°C depending on the content of D-allulose dimers. Detailed description of the invention

[0022] The D-allulose syrup of the invention is an aqueous solution which is depleted in D-allulose dimers. By "aqueous composition" or "aqueous solution" is generally meant a composition or solution whose solvent consists essentially of water. By "D-allulose dimer" is meant a compound comprising a D-allulose condensed with at least one second identical or different monosaccharide. These dimers are for example dimers of the D-allulose-D-allulose type.

[0023] As described above, the fact that the quantity of D-allulose dimers included in the syrup is enriched, i.e. that its mass content expressed as dry mass is, according to the invention, ranging from 2.0 to 15%, made it possible to obtain a less crystallizable syrup.

[0024] These dimers could be detected by GC and could not be detected during HPLC analysis, as demonstrated by Figures 4 to 7 . It follows that the mass quantities of the different constituents, expressed in dry mass, are systematically determined in the present application by CPG. To determine the quantities of each of the species in the composition, the sample generally undergoes a treatment step in order to transform the different species present into trimethylsilylated methoxime derivatives. The mass quantities of each of the species are expressed in the present Application, unless otherwise stated, relative to the total dry mass.

[0025] The amounts of glucose, fructose and allulose can be determined in a gas chromatograph equipped with an injector heated to 300°C, a flame ionization detector (FID) heated to 300°C and equipped with a 40 meter DB1 capillary column, having an internal diameter of 0.18 mm and a film thickness of 0.4 µm, the column temperature being programmed as follows: from 200°C up to 260°C at a rate of 3°C / min, then from 260°C up to 300°C at 15°C / min, holding at 300°C for 5 min.

[0026] By quantity of D-allulose dimers is meant the difference between the total quantity of dimers in a sample, determined by GC, and the quantity of known dimers possibly present, which are glucose-glucose dimers such as maltose and isomaltose. The quantity of these glucose-glucose dimers may be very low or even non-existent. In the syrup of the invention, the mass quantity of glucose-glucose dimers is generally less than 2%, often less than 1%, or even less than 0.2% or less than 0.1%.

[0027] The possible amount of glucose-glucose dimers can be determined under the same conditions as those described previously for glucose, fructose and D-allulose: by carrying out a hydrolysis of the glucose-glucose dimers of the sample; by determining the quantity of total glucose in the same chromatograph and under the same conditions, said total glucose comprising the initial so-called free glucose and the glucose resulting from the hydrolysis of the glucose-glucose dimers; by subtracting, from this quantity of total glucose, the quantity of initial glucose of the sample.

[0028] The total amount of dimers can be determined in a gas chromatograph under the same conditions as described above, except that the column used is a 30-meter DB1 capillary column with an internal diameter of 0.32 mm and a film thickness of 0.25 µm, and the column temperature is programmed as follows: from 200°C to 280°C at a rate of 5°C / min, held at 280°C for 6 min, then from 280°C to 320°C at 5°C / min, held at 320°C for 5 min.

[0029] The method is described in more detail in the Examples section.

[0030] D-allulose syrup contains, in addition to D-allulose, a mass content of D-allulose dimer, determined by gas chromatography (GC), ranging from 2.0 to 15%, for example from 2.1 to 8%, or even from 2.4 to 5%.

[0031] According to one variant, the syrup of the invention comprises D-fructose and D-allulose in a D-fructose / D-allulose mass ratio of between 50 / 50 and 40 / 60 and is essentially made up of D-fructose, D-allulose and D-allulose dimers. This syrup has the advantage of having the same sweetening power as sucrose.

[0032] According to another preferred variant, the syrup of the invention comprises a D-allulose content expressed in dry mass greater than or equal to 75%. The syrup of the invention has the advantage of being able to have a low crystallizability, even when it reaches this D-allulose content. This is particularly surprising insofar as it is known that the more a syrup comprises a compound in a majority manner (here D-allulose), the more this syrup tends to have a crystallizable character. According to the invention, the presence of D-allulose dimers ranging from 2.0 to 15% by mass, expressed in dry mass, in the syrup makes it particularly less crystallizable.

[0033] The syrup of the invention may have a D-allulose content expressed as dry mass greater than or equal to 80%, for example greater than or equal to 85%, in particular greater than or equal to 90%.

[0034] D-allulose syrup can advantageously comprise, in relation to its dry mass: from 75 to 99% D-allulose; from 0 to 20% D-fructose; from 0 to 10% glucose; from 2.0 to 15%, for example from 2.1 to 8%, or even from 2.4 to 5% of D-allulose dimers.

[0035] D-allulose syrup may have a dry matter content greater than 50%, for example ranging from 65 to 85%, in particular ranging from 70 to 83%, for example from 75 to 82%. The higher the dry matter content, the more easily the syrup crystallises. However, when the dry matter content is high, the viscosity of the syrup may increase, which can lead to difficulties in handling it.

[0036] A D-allulose syrup is conventionally obtained by a process comprising: a step of providing an aqueous composition comprising D-allulose; a step of concentrating said aqueous composition to form D-allulose syrup.

[0037] The syrup of the invention can be produced according to a process described in detail below which comprises, prior to the concentration step, a nanofiltration step.

[0038] This nanofiltration step makes it possible to increase the quantity of D-allulose dimers in the syrup of the invention. This nanofiltration step takes place before the step of concentrating the composition rich in D-allulose. This step therefore makes it possible to provide a D-allulose syrup whose D-allulose dimer content is richer than that obtained from the same process not using this nanofiltration step.

[0039] In the nanofiltration step, which is essential to the process of the invention, two fractions are formed when a D-allulose composition is subjected to nanofiltration: a permeate, which is depleted in D-allulose dimers; as well as a retentate, which is enriched in D-allulose dimers.

[0040] In the Figure 1 which represents a syrup production circuit of the invention, Stream 6 represents the retentate and Stream 9 represents the permeate. For illustrative but non-limiting reasons, unless otherwise indicated, the Streams indicated in the remainder of the description refer to the flows of the production circuit of this Figure 1 .

[0041] The terms "depleted in D-allulose dimers" and "enriched in D-allulose dimers" are obviously relative to the content of D-allulose oligomers in the composition to be nanofiltered.

[0042] The nanofiltration retentate is an intermediate allowing the manufacture of the D-allulose syrup of the invention.

[0043] An object of the invention therefore relates to a method of manufacturing syrup according to claim 6 which comprises: a step of providing an aqueous D-allulose composition comprising D-allulose dimers; a step of nanofiltration of said D-allulose composition to provide a retentate and a permeate; a step of recovering the nanofiltration retentate; a step of concentrating this retentate to provide the D-allulose syrup of the invention.

[0044] To carry out the nanofiltration step useful in the invention, the composition to be nanofiltered is passed over a nanofiltration membrane. It generally has a dry matter content ranging from 5 to 15%.

[0045] The temperature of this composition to be nanofiltered can range from 10 to 80°C, generally from 15 to 50°C, often around 20°C.

[0046] The skilled person will know how to choose the membrane useful for this separation. This nanofiltration membrane has a cut-off threshold of less than 300 Da, preferably ranging from 150 to

[0047] 250 Da. Ideally, the membrane has a MgSO4 rejection rate of at least 98%. This could be a Dairy DK or Duracon NF1 membrane manufactured by GE ®< .

[0048] The pressure applied to the membrane can also vary widely and can range from 1 to 50 bars, preferably from 5 to 40 bars, most preferably from 15 to 35 bars.

[0049] This nanofiltration step can be accompanied by a diafiltration phase.

[0050] Preferably, the volume concentration factor (VCF) of the nanofiltration ranges from 2 to 20. This volume concentration factor is easily adjusted by those skilled in the art.

[0051] This nanofiltration step can be carried out continuously.

[0052] At the end of this nanofiltration step, the recovered retentate may comprise, relative to its dry mass, from 0.8 to 20% of D-allulose dimers, for example from 1.5 to 15%, in particular from 2 to 5%. It may for example comprise, in dry mass: from 75 to 99% D-allulose; from 0 to 20% D-fructose; from 0 to 10% glucose; from 0.8 to 20% D-allulose dimers, for example from 1.5 to 15%, especially from 2 to 5%.

[0053] It goes without saying that the process according to the invention may comprise other steps, such as the other steps appearing in the conventional process described above and which will be described in detail below. The process according to the invention may also comprise additional purification steps and also intermediate dilution or concentration steps in order to adjust the dry matter and thus carry out the different steps of the process of the invention under the best conditions. All of these steps may be carried out continuously.

[0054] The syrup of the invention generally has a dry matter greater than or equal to 50%. To increase the dry matter (the retentate has a dry matter lower than 50%), it is necessary to carry out a concentration step, during which the content of D-allulose dimers can increase. The formation of D-allulose dimers can also occur during this concentration step, in particular when the temperature is high. It is thus possible to select conditions that further increase the quantities formed in these dimers. The concentration step can be carried out under vacuum or at room temperature under vacuum. A vacuum step makes it possible to reduce the temperature necessary for evaporation and to reduce the duration of this concentration step. It can be carried out at a temperature ranging from 30 to 100°C.This concentration step can be carried out in a single-stage evaporator, a multi-stage evaporator, for example a double-stage evaporator. At the end of the concentration step, the D-allulose syrup of the invention can be obtained. It comprises a content of D-allulose dimers ranging from 2.0 to 15%, for example from 2.1 to 8%, or even from 2.4 to 5%.

[0055] The method of the invention further comprises a step of providing an aqueous D-allulose composition comprising D-allulose dimers. The mass contents of the various constituents of the syrup (and in particular D-allulose, D-fructose and optional glucose) are mainly determined by the contents of each of these constituents included in the aqueous D-allulose composition provided. For example, if the aqueous D-allulose composition provided has a high D-allulose content, the permeate and the syrup obtained from this permeate also have a high D-allulose content. A conventional method for manufacturing a D-allulose composition comprising D-allulose dimers comprises: a step of providing a D-fructose solution; a step of epimerizing said solution to form a D-allulose composition, comprising D-fructose and D-allulose; optionally a chromatography step to enrich the D-allulose composition with D-allulose; a step of concentrating the composition, optionally enriched, with D-allulose.

[0056] Thus, according to the method of the invention, to provide the D-allulose composition, a step of chromatography of a composition comprising D-allulose and D-fructose can be carried out. In this case, this composition comprising D-allulose and D-fructose is advantageously obtained by epimerization of a D-fructose solution.

[0057] The D-allulose composition obtained after the chromatography step, which has a higher D-allulose content than the composition obtained after the epimerization step, comprises D-allulose dimers. In addition to this D-allulose composition, a D-fructose-rich composition or "raffinate" is also formed during this chromatography step.

[0058] The D-fructose composition provided (Stream 1) for carrying out the epimerization step may be a D-fructose syrup, which may be obtained by dissolving D-fructose crystals in water or a glucose / D-fructose syrup. Preferably, this composition comprises a glucose / D-fructose syrup which comprises at least 90% by dry weight of D-fructose, preferably at least 94% of D-fructose.

[0059] In a mode represented in the Figure 2, the D-fructose composition provided to carry out the subsequent epimerization step is a mixture (Stream 1') of this D-fructose syrup with at least one recycled fraction which may be the raffinate (all of the raffinate or a part) (Stream 10 or 12), this recycled fraction being able to comprise a higher quantity of D-allulose.

[0060] The composition of D-fructose subjected to the epimerization step may include: 0 to 10% D-allulose; 70 to 100% D-fructose; 0 to 10% glucose; 0 to 15% D-allulose dimer.

[0061] The epimerization step is carried out from the D-fructose composition provided previously, optionally after adjusting the dry matter. This step is generally carried out at a dry matter ranging from 30 to 60%, often from 45 to 55%. An enzyme of the D-psicose epimerase type or a composition comprising this enzyme is introduced into this composition. The composition comprising this enzyme may be a lyophilisate of a host microorganism synthesizing D-psicose epimerase, this being able to be the Bacillus subtilis,in particular that described in application WO2015 / 032761 A1. The pH is adjusted according to the enzyme used, for example at a pH ranging from 5.5 to 8.5. The reaction can be carried out by heating to a temperature ranging from 40 to 70°C, often from 45 to 60°C. The reaction can last from 0.1 to 100 hours, for example from 0.2 to 60 hours. This reaction can for example be carried out on an enzymatic column, which has the advantage of also working continuously on this step. It is also possible to work sequentially with several reactors to operate continuously. To carry out this epimerization step, it is possible in particular to use the teaching of document WO 2015 / 032761 A1.

[0062] At the end of the reaction, a composition comprising D-fructose and D-allulose is formed, generally according to a D-fructose / D-allulose mass ratio ranging from 85 / 15 to 55 / 45, often according to a D-fructose / D-allulose mass ratio ranging from 80 / 20 to 60 / 40. This ratio depends on the epimerization parameters used and, of course, on the quantity of D-allulose and D-fructose in the D-fructose composition provided in the epimerization step; the quantity of D-allulose in this composition may be particularly greater in the case of recycling.

[0063] At the end of this epimerization step, if necessary, a filtration step can be carried out to recover any cellular debris that may be present, particularly when a lyophilisate of a host microorganism is used. This step can consist of a microfiltration step. The microfiltered composition corresponds to Stream 3 and the cellular debris is recovered in Stream 8.

[0064] In the process of the invention, additional purification steps may also be carried out. Generally, before the chromatography step, a step of demineralization of the composition comprising D-fructose and D-allulose (Stream 3) is carried out, which may be carried out by passing through one or more cationic ion exchange resins (for example a cationic resin of the Dowex 88 type), anionic ion exchange resins (for example an anionic resin of the Dowex 66 type) and a cationic-anionic mixture. In the Figure 3 , this composition corresponds to Flow 4. The composition comprising D-fructose and D-allulose obtained is then demineralized and generally has a resistivity greater than 100 kΩ.cm -1< . It is also possible to carry out, before this demineralization step, a step of decolorization of the composition comprising D-fructose and D-allulose, for example by passing it over a column comprising activated carbon.

[0065] The composition subjected to the chromatography step may comprise, in relation to its dry mass: 22-45% D-allulose, usually 23-37%; 45-75% D-fructose, usually 46-70%; 0-10% glucose; 2-10% D-allulose dimer.

[0066] To carry out this chromatography step, any type of continuous chromatography can be used, in particular simulated moving bed chromatography. (Simulated Moving Bed SMB), of type Improved Simulated Moving Bed (ISMB), of type Divide Improved Simulated Moving Bed (DISMB), Sequential Simulated Moving Bed (SSMB) or type Nippon Mitsubishi Chromatography Improved ( NMCI ) .Water is generally used as the eluent. The chromatography may be equipped with several columns in series, for example, 4 to 8 columns. The columns comprise ion exchange resin, for example, a cationic calcium ion exchange resin. The dry matter of the composition comprising D-fructose and D-allulose may range from 40 to 70%, generally around 50%. The temperature of the composition during chromatography generally ranges from 40 to 80°C, preferably from 55 to 65°C. This chromatography lasts until satisfactory separation is obtained and may last several hours.

[0067] At the end of this step, a composition rich in D-allulose (Stream 5) is obtained which may comprise, relative to its dry matter, at least 80% of D-allulose, advantageously at least 90% of D-allulose. This composition rich in D-allulose may have a dry matter ranging from 5 to 15%. At the end of this step, a raffinate (Stream 10) is also obtained, which generally comprises, relative to its dry matter, at least 75% of D-fructose, often at least 80% of D-fructose. This raffinate generally has a dry matter ranging from approximately 15 to 30%.

[0068] The method according to the invention may comprise a step of recycling at least part of the nanofiltration permeate (Stream 9 of the Figure 2 ) and / or raffinate (Stream 10 of the Figure 2). The syrup can be used for the manufacture of food or pharmaceutical products. The syrups of the invention can thus be used in the known applications of D-allulose and, in general, of sweeteners. The syrup of the invention is advantageously used in applications where it is preferable that there is no crystallization. Thus, the syrup of the invention is advantageously used for the manufacture of beverages, caramels, starch jelly, sauces, salad dressings, jams, fruit filling, nutritional bars, cereal bars, pizzas, fruit and nut bars, dairy products such as yogurts or ice creams, sorbets or as a humectant.

[0069] The invention will now be illustrated in the Examples section below. It is specified that these Examples are not limiting of the present invention. Examples Analytical methods Gas chromatography

[0070] The gas chromatograph used is a Varian 3800 type and is equipped with: A split-splitless injector (with or without dividers); A flame ionization detector (FID); A computer system for processing the detector signal; An automatic sampler (type 8400).

[0071] The various quantities are determined by gas chromatography in the form of trimethylsilylated methoxime derivatives, then quantified by the internal calibration method. Determination of D-allulose, D-fructose and glucose contents

[0072] The applied response coefficients are 1.25 for D-allulose and D-fructose and 1.23 for glucose. Other monosaccharides were not detected. Sample preparation

[0073] In a tare box, weigh 100 to 300 mg of the sample to be tested + 10 ml internal standard solution consisting of methyl α-D-glucopyranoside at 0.3 mg / ml in pyridine. In a 2 ml cup, take 0.5 ml from the tare box and evaporate to dryness under a stream of nitrogen. Add 20 mg of methoxylamine hydrochloride and 1 ml of pyridine. Stopper and leave in the Reacti-therm ®< type incubation system at 70°C for 40 min. Add 0.5 ml of N, O Bis (trimethylsilyl) trifluoroacetamide (BSTFA). Heat for 30 min at 70°C. Chromatographic conditions

[0074] Column: DB1 capillary 40 meters, internal diameter 0.18 mm, film thickness 0.4 um, made of 100% dimethylpolysiloxane, non-polar (J&W Scientific ref.: 121-1043) Column temperature: 100°C programable up to 260°C at 3°C / min, then up to 300°C at 15°C / min, maintain 5 min at 300°C. Injector temperature: 300°C Detector temperature: 300°C (Range 10 -12< ) Pressure: 40 psi (constant flow rate) Carrier gas: Helium Injection mode: Split (Split flow rate: 100 ml / min) Injected volume: 1.0 µl

[0075] D-allulose, D-fructose, and glucose were detected in that order. D-allulose, which was unknown, had a retention time under these conditions of between 39.5 and 40 minutes. Determination of D-allulose dimers and -glucose-glucose dimers contents

[0076] The applied response coefficients are 1.15 for D-allulose dimers and maltose, and 1.08 for isomaltose. Other glucose dimers were not detected. Sample preparation:

[0077] In a tare box, weigh 100 to 300 mg of the sample to be tested + 10 ml internal standard solution consisting of Phenyl beta-D-glucopyranoside at 0.3 mg / ml in pyridine.

[0078] In a 2 ml cup, take 0.5 ml from the tare box and evaporate to dryness under a stream of nitrogen.

[0079] Take up with 0.5 ml of the 40g / l hydroxylamine hydrochloride solution in pyridine, stopper, shake and leave for 40 min at 70°C.

[0080] Add 0.4 ml of BSTFA and 0.1 ml of N-Trimethylsilylimidazole (TSIM). Heat for 30 min at 70°C. Chromatographic conditions

[0081] Column: DB1 capillary 30 meters, internal diameter 0.32 mm, film thickness 0.25 µm (J&W Scientific ref.: 123-1032) Column temperature: 200°C programable up to 280°C at 5°C / min (hold 6 min), then up to 320°C at 5°C / min, hold 5 min at 320°C. Injector temperature: 300°C Detector temperature: 300°C (Range 10 -12< ) Pressure: 14 psi (constant flow) Carrier gas: Helium Injection mode: Split (Split flow rate: 80 ml / min) Injected volume: 1.2 µl Expression of results:

[0082] The content of the different constituents is expressed in g per 100 g of raw product and is given by the following equation: % constituant i = Si Se × Pe P × 100 Ki With : Si = area of the peak(s) of constituent i Se = area of the peak of the internal standard Pe = Weight of internal standard introduced into the beaker (in mg) P = weight of weighed sample (in mg) Ki = response coefficient of constituent i

[0083] If the percentage obtained (expressed here as crude) exceeds 20% for one of the constituents, the sample is diluted and the CPG analysis repeated in order to obtain a mass quantity less than 20%.

[0084] The mass quantities expressed in gross are then expressed in dry, by dividing for the dry matter of the sample tested.

[0085] The mass quantities of D-allulose, D-fructose and glucose are readily determined, with none of the characteristic peaks co-eluting.

[0086] The peak of maltose and D-allulose dimers can be co-eluted. It should be noted, however, that in the syrups of the invention and described in the examples below, maltose is never present.

[0087] If the characteristic peaks of maltose are not detected, the area Si of the D-allulose dimers is determined by integration of the unknown peaks, between 10 and 17 minutes. If the characteristic peaks of maltose are detected (which may be the case in the syrups of the invention), the quantities of maltose are determined and this quantity is subtracted from the total quantity of dimers.

[0088] To determine the total quantity of glucose-glucose dimers, the following protocol is carried out on a sample: Hydrochloric hydrolysis In a 15 ml hydrolysis tube with a Teflon screw cap, weigh approximately 50 to 500 mg of sample (adjust the weight according to the expected sugar content), add 2 ml of the internal standard solution (galactitol at 5 mg / ml in reverse osmosis water) with a two-mark pipette, add 3 ml of water and 5 ml of the 4N HCl solution. Cap tightly, shake for 1 minute with a vortex mixer. Place the tube in a thermostatically controlled dry bath set at 100°C for 1 hour, vortexing occasionally. Demineralization and concentration After cooling, place the entire hydrolysis in a 50 ml beaker. Add 6 to 8 g of a 50 / 50 mixture of AG4 X 4 and AG50 W 8 anionic resin. Stir magnetically for 5 minutes. Filter through paper. Collect the juice and repeat the demineralization step until the pH is close to water.Sample preparation In a tare box, weigh 100 to 300 mg of the sample to be tested + 10 ml internal standard solution consisting of methyl α-D-glucopyranoside at 0.3 mg / ml in pyridine. In a 2 ml cup, take 0.5 ml from the tare box and evaporate to dryness under a stream of nitrogen. Add 20 mg of methoxylamine hydrochloride and 1 ml of pyridine. Stopper and leave in Reacti-therm ®< at 70°C for 40 min. Add 0.5 ml of BSTFA. Heat for 30 min at 70°C.

[0089] The total glucose quantity in the solution (which includes the initial so-called "free" glucose and the glucose resulting from hydrolysis and in particular linked to the presence of maltose and isomaltose) is determined by GC analysis of the glucose. The quantity of maltose can easily be deduced and, by difference with the total quantity of dimers attributed to the peaks between 10 and 17 minutes, the quantity of D-allulose dimers. Example 1: Implementation of a continuous industrial process for the manufacture of D-allulose syrup

[0090] Example 1 consists of a method for the continuous production of non-crystallizable D-allulose syrup. The process steps used are detailed in the Figure 1 The composition and flow rate of the Flows of stages 1 to 5 are described in Table 1a. Step 1:

[0091] 17.3 tonnes of Fructamyl D-fructose syrup (Tereos) comprising 95% D-fructose at 50% dry matter (DM) (Stream 1) are introduced into a 14m 3< useful stirred batch reactor. Stream 1 is maintained at 55°C. A lyophilisate of the strain is introduced into the tank. Bacillus subtilis host of the enzyme D-Psicose 3 Epimerase detailed in patent WO2015032761 in sufficient quantity to have 3.3*10 7< units of activity in the reactor. Three reactors are used sequentially so as to provide a syrup essentially composed of fructose and allulose (Stream 2) continuously at a flow rate of 360kg / h.

[0092] The reaction conditions are as follows: Temperature: 55°C pH=7 Reaction time 48h

[0093] At the end of the reaction, Flux 2 is obtained comprising a D-allulose richness approximately equal to 25% and a D-fructose richness approximately equal to 75%. Step 2:

[0094] Stream 2 passes through a microfiltration membrane during a batch operation. A Stream 3 free of cellular debris and a microfiltration retentate (Stream 8) comprising debris from the freeze-dried product are obtained. Bacillus subtilis which is purged from the circuit. The microfiltration parameters are as follows: Transmembrane pressure: 0-3 bar Pore size: 0.1 µm Temperature: 50°C Average flow rate: 15 L / h / m 2< Membrane: Sepro PS35 Volume Concentration Factor: 33 Step 3:

[0095] Stream 3 is demineralized on the strong cationic resin Dowex 88 followed by a weak anionic resin Dowex 66 at an average flow rate of 2BV / h. The cylinders are maintained at a temperature of 45°C and the resistivity of Stream 4 after demineralization remains greater than 100kΩ.cm -1< at the outlet (Stream 4). Otherwise, the resins are regenerated. Step 4:

[0096] Stream 4 feeds the continuous chromatography (SCC ARI ® equipped with 8 columns) of the circuit. The average feed rate is 348 kg / h at 50% DM.

[0097] The chromatography parameters are defined as follows: Volume / column: 2m 3< Resin: Dowex Monosphere 99Ca / 320 Temperature: 60°C Water flow rate / Flow 4 (vol. / vol.): 2.4 Load: 0.09 h -1<

[0098] Two fractions are extracted: the raffinate (Stream 10), the fraction rich in D-allulose (Stream 5) which goes towards stage 5. Stream 10 is purged. Step 5:

[0099] Stream 5 is passed in batches through a nanofiltration membrane. The parameters are as follows: Transmembrane pressure: 30 bar Temperature: 20°C Membrane: GE Duracon NF1 8040C35 Volume Concentration Factor FCV: 10

[0100] Allulose dimers concentrate in the retentate (Stream 6). The permeate (Stream 9) is purged. The figure 6 gives details of the permeation of syrups as a function of FCV. Step 6:

[0101] Stream 6 passes through an evaporator. The second stage reaches 77% dry matter. D-allulose syrup (Stream 7) is obtained at the end of this stage.

[0102] Table 1b shows the characteristics of the syrup of the invention (Flow 7). Table 1a: Flow rates and composition of the Flows of steps 1 to 5 of Example 1 Stage / Characteristics of Flows Flow Step 1 Stream 1 Stream 2 - Mass flow rate (kg / h) 360 360 Dry Matter (%) 50 50 Fructose Richness (%) 94,5 71,5 Glucose Richness (%) 2 2 Allulose Richness (%) 1 24 Di-Allulose Richness (%) 1 1 Other Wealth (%) 1,5 1,5 Step 2 / Step 3 Stream 2 Stream 3 Stream 8 Mass flow rate (kg / h) 360 348 12 Dry Matter (%) 50 50 50 Step 4 Stream 4 Stream 10 Stream 5 Mass flow rate (kg / h) 348 656 429 Dry Matter (%) 50 20 10 Fructose Richness (%) 71,5 93,7 3,6 Glucose Richness (%) 2 2,5 0,4 Allulose Richness (%) 23,7 0,9 93,4 Di-Allulose Richness (%) 1,2 1,1 1,5 Other Wealth (%) 1,5 1,6 1,1 Step 5 Stream 6 Stream 9 - Mass flow rate (kg / h) 67,1 363,3 Dry Matter (%) 29,3 6,4 Fructose Richness (%) 3,5 3,7 Glucose Richness (%) 0,4 0,4 Allulose Richness (%) 91,8 94,8 Di-Allulose Richness (%) 3,1 0,1 Other Wealth (%) 1,2 1 Table 1b: Flow rates and composition of Stream 7 Step 6 Stream 7 - - Mass flow rate (kg / h) 25,5 Dry Matter (%) 77 Fructose Richness (%) 3,5 Glucose Richness (%) 0,4 Allulose Richness (%) 91,8 Di-Allulose Richness (%) 3,4 Other Wealth (%) 1,2

[0103] The syrup obtained is non-crystallizable at room temperature.

[0104] In the same process where no nanofiltration step is carried out, the D-allulose syrup obtained is similar except that the quantity of D-allulose dimers expressed in dry mass is 1.5%. Example 2 : Evaluation of the crystallizability of different D-allulose syrups

[0105] Example 2 consists of preparing different D-allulose syrups with a dry matter content of 77% and a D-allulose content of approximately 95%.

[0106] These syrups are prepared by adjusting the volume concentration factor of nanofiltration step 5 to obtain different Di-Allulose contents and / or by preparing syrups by mixing the permeate or retentate obtained with D-allulose or D-fructose crystals and / or by using nanofiltration membranes with a lower rejection threshold. This made it possible to adjust the contents of the different constituents while maintaining the D-allulose content at around 95%. The composition of the dry matter of the syrups is shown in Table 2.

[0107] In order to evaluate the crystallizability of the syrups, a sieved D-allulose crystal primer with an average particle size of 70 µm is introduced into each of the syrups in a proportion of 0.3% (mass of primer / mass of dry matter).

[0108] Each syrup thus primed is then placed for 4 weeks in a refrigerated chamber at 4°C or 15°C.

[0109] At the end of this period, the dry matter of the supernatant (or mother liquor) of the sample is measured using the Karl Fischer method. The more the syrup has crystallized, the lower the dry matter content of the supernatant (since the dry matter of the syrup has concentrated in D-allulose crystals).

[0110] The priming carried out during this test allows for an accelerated study of storage stability, particularly at low temperatures. Table 2: Composition of the different syrups and dry matter of the supernatant after storage Composition (% CPG) %MS of the supernatant after one month Sample D-allulose D-allulose dimer Glucose Fructose Others 15°C 4°C 1 95,1 0,1 1 3,5 0,3 70,6 67,5 2 95 0,5 0,3 3,5 0,7 71,8 68,7 3 94,9 0,7 0,1 3,2 1,1 72,8 69,7 4 95 1,2 0,2 1,9 1,7 73,4 70,2 5 95 1,5 0,2 2 1,3 73,9 70,8 6 95,2 2,1 0,2 1,8 0,7 74,7 71,3 7 95 2,7 0,2 1,4 0,7 75,3 71,6 8 95 3 0,2 4,1 0,7 75,3 71,5 9 95,1 3,4 0,1 1 0,4 75,6 71,6 10 94,9 4 0 1 0,1 75,3 71,5 11 94,8 4,6 0 0,5 0,1 75,7 71,8 12 94,9 5 0 0,1 0 75,2 71,9

[0111] These results are represented in the figure 8 .

[0112] These tests demonstrate that D-allulose dimers are very specific compounds, which have a considerable influence on the crystallizable character of a D-allulose syrup containing it, unlike, for example, glucose or fructose.

[0113] Thus, the higher the quantity of D-allulose dimer in the syrup, the less crystallizable the D-allulose syrup is, even though the quantity of D-allulose remains similar.

[0114] These tests demonstrate that syrups containing more than 1.5% D-allulose dimers have significantly improved low-temperature storage stability. This is an advantage because the syrups can be more easily handled before use, without the need for reheating the syrup (or by reheating it less significantly) in order to at least partially remelt it. Example 3. Production of short-textured caramels

[0115] A short (soft) textured caramel composition comprising the D-allulose syrup according to the invention (sample 10) was manufactured.

[0116] Table 3 shows the composition of the formulated caramel. Table 3. Caramel formulations using D-allulose syrup Quantity of ingredients (%) in the formulation Ingredients Formulation Whole cream 27,00 Powdered milk 8,00 Allulose syrup 24,40 NUTRIOSE ®< FM06 23,10 Butter 5,60 Salt 0,10 Water 10,55 Lecithin 0,20 Sodium bicarbonate 0,05 Vanilla (2x) 1,00 Total 100,00 Nutritional data a< per 30g of product Calories (kcal) 100,90 Total carbohydrates (g) 18,80 Sugars (g) 8,94 Fiber (g) 8,13 Protein (g) 0,50 Total fat (g) 6,69 a< Nutritional data calculated from product specifications or USDA's National Nutrient Database for Standard Reference Release 27, when these specifications were not available

[0117] The soft caramel obtained from the syrups of the invention has an excellent, very short texture. Example 4. Making ketchup sauce

[0118] A ketchup sauce comprising the D-allulose syrup according to the invention (sample 10) was produced as well as a reference ketchup sauce. Table 4. Ketchup sauce formulations Ingredients Reference Invention Tomato puree 35,12 35,12 Sucrose 14,15 0 D-allulose syrup 0 18,54 Modified starch (CLEARAM CH 2020) 2,44 2,44 Vinegar 13,66 13,66 Salt 0,49 0,49 Onion powder 0,49 0,49 Water 33,66 29,47 Total 100,00 100,00

[0119] The amount of sugars in dry weight of the two sauces is equal, but the amount of calories is reduced by 43% with the sauce of the invention. The sauce of the invention has a less sweet taste and is brighter, which is an advantage for a sauce of this type.

[0120] Ketchup made from the syrup of the invention has a smooth, clump-free texture, which is synonymous with non-crystallization of D-allulose.

[0121] Moreover, the ketchup made from the syrup of the invention has excellent flow, which is due to the fact that the sauce does not crystallize, even after storage for a week in the refrigerator. Example 5. Making barbecue sauce

[0122] A barbecue sauce comprising the D-allulose syrup according to the invention (sample 10) was produced as well as a reference barbecue sauce. Table 5 Barbecue sauce formulations Ingredients Invention Reference Tomato sauce 45,64 45,64 D-allulose syrup 18,76 0,00 Glucose-fructose syrup HFCS 55 (77% MS) 0,00 18,76 Vinegar 18,26 18,26 Modified starch (PREGEFLO CH 20) 2,28 2,28 Liquid smoke 1,62 1,62 Salt 1,50 1,50 Garlic powder 0,63 0,63 Onion powder 0,53 0,53 Mustard powder 0,30 0,30 Paprika 0,20 0,20 Dye 0,13 0,13 Water 10,14 10,14 Total 100,00 100,00 Method

[0123] The dry ingredients are weighed and blended together. In a blender, the tomato sauce and water are mixed, then the dry ingredients are added. After the dry ingredients are incorporated, the syrup and vinegar are added and mixed well. The mixture is placed in a dish and heated until the water content is approximately 74%. Features

[0124] Reference Invention Dry matter 26,5% 27,3% Taste Sugar Slightly less sweet than the reference sauce Viscosity, texture Soft and viscous enough to be “dipping” Soft and viscous enough to be “dipperable” Nutritional information per 35g of product

[0125] Reference Invention Calories (kcal) 26,7 11,4 Carbohydrates (g) 6,31 6,31 Sugars (g) 5,17 5,17 Fiber (g) 0,83 0,83 Protein (g) 0,34 0,34 Fats (g) 0,29 0,29

[0126] The sauce of the invention has a reduced calorie content of 57% while including the same amount of sugars.

[0127] Although viscous, the sauce of the invention has a very soft texture, which is synonymous with non-crystallization of D-allulose. Example 6. Making jam

[0128] A jam comprising the D-allulose syrup according to the invention (sample 10) was produced as well as a reference jam. Table 6. Jam formulations Ingredients Reference Invention Strawberries 49,27 49,27 D-allulose syrup 0,00 49,27 Glucose-fructose syrup HFCS 55 (77% MS) 49,27 0,00 Modified pectin 0,45 0,45 Citric acid (50% solution) 1,01 1,01 Total 100,00 100,00

[0129] The strawberries are washed, mashed, and the seeds are removed. The strawberry puree, syrup, and pectin are mixed and heated in a saucepan. After boiling for a few minutes, the saucepan is removed from the heat to obtain a dry matter content of about 57%, citric acid is added, and the mixture is left to cool. Organoleptic characteristics

[0130] Reference Invention Dry matter 57,4% 57,2% Taste Sugar Slightly less sweet and more acidic than the reference Viscosity, texture in the mouth Soft and spreadable Soft and spreadable Nutritional information per 17g of jam

[0131] Reference Invention Calories (kcal) 34,2 8,8 Carbohydrates (g) 8,62 8,59 Sugars (g) 8,28 8,31 Observations:

[0132] Jam made from D-allulose syrup does not brown during cooking. This is expected due to the fact that the pH is acidic and it does not contain unmodified protein. The calorie content of the jam of the invention has the advantage of having a 74% reduced calorie content. The color of the jam of the invention is slightly more colorful (redder) than the reference jam, which is an advantage for a jam.

[0133] The jam made from the syrup of the invention also has the advantage of having a soft texture, similar to the reference jam using a very low crystallizability syrup (it includes a mixture of glucose and fructose in almost equivalent quantities). Example 7. Use of syrup as an adhesive agent for a frozen pizza topping

[0134] In this example, the D-allulose syrup according to the invention (sample 10) was used.

[0135] Generally, the cheese in frozen pizza toppings tends to detach from the pizza. Therefore, a layer of adhesive, which may be a glucose-fructose syrup, is used to improve adhesion. However, too rapid crystallization of the adhesive can cause the cheese to separate from the pizza, rather than making it adhere. The adhesive would have the advantage of being less sweet. Protocol

[0136] A pizza dough is rolled out and then placed in an oven at 245°C for 8 minutes.

[0137] After baking, the dough is cooled and then covered with pizza sauce. Then, the pizza is optionally sprayed with 4g of syrup (or water), 50g of grated mozzarella is sprinkled on top and then the pizza is optionally sprayed again with 4g of syrup (or water).

[0138] The pizza, covered with aluminum foil, is placed in the freezer for 4 days. It is then uncovered from the aluminum foil. A first test involves turning the frozen pizza over: the cheese that falls off the pizza is collected. A second test involves doing the same, turning the pizza over once it has thawed.

[0139] The pizza is then cooked and a sensory analysis is carried out. Table 7. Results obtained Adhesive agent Concentration Quantity (g) of cheese lost (frozen) Quantity (g) of cheese lost (thawed) Sensory analysis HFCS 42 50% solution 0 0 Too sweet D-Allulose Syrup 95% purity, 75% MS 50% solution 0 0 Slightly sweet D-Allulose Syrup 95% purity, 75% MS 10% solution 0 0 Taste not altered compared to the reference Control -- 8 16 Reference Tap water tap water 5 14 Taste not altered compared to the reference

[0140] No cheese loss was observed for the pizza (thawed or not) using the adhesive agent obtained from the syrup of the invention, just like the HFCS 42 agent, but which has a sweeter taste than the reference pizza. The reference pizza loses 32% of cheese when it is thawed and then turned over. According to the same test, if water is used as an adhesive agent, 28% of the cheese is lost. Example 8. Ice cream composition

[0141] The reduced sugar ice cream composition was made according to the following recipe: Skimmed milk powder 400 g Water 1801.2g Stabilizing 12 g Sucrose 160 g Allulose 440 g Vanilla 28 g Whole cream 1160 g Total 4000 g

[0142] The resulting ice cream has a creamy and soft texture in the mouth.

Claims

1. D-allulose syrup comprising, in addition to D-allulose, a mass content of D-allulose dimer, determined by gas chromatography (GC) according to the method described on page 5, line 22 to page 6, line 24 as well as on pages 13 to 16, ranging from 2.0 to 15%.

2. D-allulose syrup according to claim 1, characterised in that the content of D-allulose dimer, expressed in dry matter, determined by gas chromatography (GC) according to the method described on page 5, line 22 to page 6, line 24 as well as on pages 13 to 16, ranges from 2.1 to 8%, or even from 2.4 to 5%.

3. D-allulose syrup according to any one of the preceding claims, characterised in that it has a D-allulose content that is greater than or equal to 75%.

4. D-allulose syrup according to any one of the preceding claims, characterised in that it comprises, relative to its dry mass: • 75 to 99% D-allulose; • 0 to 20% D-fructose; • 0 to 10% glucose; • 2.0 to 15% D-allulose dimer, for example 2.1 to 8%, or even 2.4 to 5%, the quantities being determined by gas chromatography (GC) according to the method described on page 5, line 22 to page 6, line 24 as well as on pages 13-16.

5. D-allulose syrup according to any one of the preceding claims, characterised in that it has a dry matter content of more than 50%, for example ranging from 65 to 85%, in particular ranging from 70 to 83%, for example from 75 to 82%.

6. Method for producing a D-allulose syrup according to any one of claims 1 to 5, characterised in that it comprises: • a step of providing a D-allulose composition comprising D-allulose dimers; • a step of nanofiltrating said D-allulose composition; • a step of recovering the nanofiltration retentate; • a step of concentrating this retentate so as to provide the D-allulose syrup, the step of nanofiltration being performed with a membrane that has a cut-off threshold of less than 300 Da, preferably ranging from 150 to 250 Da.

7. Method according to claim 6, characterised in that a step of chromatography of a composition comprising D-allulose and D-fructose is performed so as to provide the D-allulose composition.

8. Method according to claim 7, characterised in that the composition comprising D-allulose and D-fructose is obtained by epimerisation of a D-fructose solution.

9. D-allulose syrup that can be obtained by means of the method of any one of claims 6 to 8.