Enzymatic process for the production of a composition rich in maltotetraose

Abstract

The invention relates to an enzymatic process for preparing a composition rich in maltotetraose.

Description

Description Title: ENZYMATIC PROCESS FOR THE PRODUCTION OF A COMPOSITION RICH IN MALTOTETRAOSE Technical field

[0001] The present invention relates to an enzymatic process for producing a composition rich in maltotetraose.

[0002] The invention also relates to a composition rich in maltotetraose.

[0003] The present invention also relates to the use of a composition rich in maltotetraose for the preparation of food for human or animal consumption. State of the prior art

[0004] Maltotetraose is an oligosaccharide consisting of 4 glucose units linked linearly by alpha 1-4 glucosidic bonds.

[0005] Maltotetraose-rich compositions or maltotetraose syrups can be used in the manufacture of food for human or animal consumption. Advantageously, maltotetraose syrups have many advantages over sucrose syrups or glucose syrups. Indeed, they have a reduced sweetness compared to sucrose syrups, while preserving the taste of the food. Furthermore, they are less susceptible to degradation by the Maillard reaction and have a higher viscosity.

[0006] Maltotetraose syrup is usually made from starches, such as corn starch, through enzymatic reactions. The starch is first hydrolyzed by alpha-amylase to produce maltodextrins.

[0007] Maltodextrin compositions are mixtures of different sugars derived from the hydrolysis of starch and having different degrees of polymerization. This degree of polymerization or "DP" is experimentally reflected by the "dextrose equivalent", or DE, dextrose being D-glucose, it is the result of a total hydrolysis of the starch. The higher the DE, the more advanced the hydrolysis, and therefore the higher the proportion of simple (short-chain) sugars making up the maltodextrin. A DE of zero would represent the starch itself, a DE of 100 would represent pure dextrose, i.e. a totally transformed starch.

[0008] The DE limit for maltodextrin is 20. Beyond this, the product obtained has the legal name “glucose syrup”.

[0009] DE and DP are therefore inversely correlated.

[0010] Maltodextrins are then subjected to further enzymatic reactions to produce maltotetraose-rich compositions, i.e., rich in DP4 oligosaccharides. Certain enzymes, such as maltotetrahydrolase from Pseudomonas saccharophila ("PS-4"), are capable of preferentially hydrolyzing maltodextrins to DP4.

[0011] However, the conventional production of compositions rich in maltotetraose does not allow the production of compositions with maltotetraose contents greater than or equal to 60%.

[0012] For example, US patent 3,654,082 (CPC International Inc) describes a process for producing maltotetraose syrup that uses the enzymatic activity of an amylase from Pseudomonas stutzeri

[0013] A maltotetrahydrolase enzyme is marketed by Genencor / DuPont under the name “OPTIMALT®4G”.

[0014] Furthermore, international application WO2010 / 118269A2 (Danisco / DuPont) describes variants of the PS-4 enzyme from Pseudomonas saccharophila whose exo-alpha-amylase activity is increased compared to to the native enzyme and whose endo-alpha-amylase activity is reduced compared to the native enzyme.

[0015] In application WO2010 / 118269A2, the syrups obtained in the examples have a maximum content of 47% in DP4.

[0016] Document WO2010132157 describes the use of the PS-4 enzyme with a pullulanase enzyme to form syrups comprising between 40 and 60% by weight of maltotetraose.

[0017] Furthermore, Tereos has marketed a syrup called MYLOSE® 351 which is presented as a glucose syrup rich in DP4. However, this product contains between 50 and 60% by weight of maltotetraose. Furthermore, it also contains significant quantities of DP3 (between 6 and 12%

[0018] It is therefore desirable to obtain compositions rich in maltotetraose and poor in mono-, di- and tri-saccharides Detailed description of the invention

[0019] The Applicant company then found that it was possible, from a liquefied starch rich in amylopectin, to produce a composition rich in maltotetraose by enzymatic reaction. The Applicant company thus developed a process which uses a particular enzyme, in combination with a tangential filtration step with a cut-off threshold of 1 kDa, carried out simultaneously with the enzymatic reaction.

[0020] In a first aspect, the present invention relates to a process for preparing a composition comprising at least 60% by weight of maltotetraose, said process comprising bringing a substrate into contact with a glucan 1,4-alpha-maltotetrahydrolysase enzyme and tangential filtration with a cut-off threshold of 1 kDa carried out simultaneously with the enzymatic reaction.

[0021] According to a preferred embodiment of the invention, the process is a process for preparing a composition comprising at least 60%, preferably at least 65%, even more preferably at least 70%, at least 75%, at least 78%, at least 80% by weight of maltotetraose, relative to the total weight of dry matter of the composition.

[0022] The percentage of maltotetraose (also called “DP4”) can be determined by any suitable method known to those skilled in the art. For example, this percentage can be determined by HPLC chromatography, as illustrated in the examples below.

[0023] The substrate according to the process of the invention can be any product resulting from the partial hydrolysis of starch.

[0024] Preferably, the starting starch is an amylopectin-rich starch, such as a waxy corn starch.

[0025] According to one embodiment of the invention, the substrate is chosen from a liquefied starch rich in amylopectin and a weakly hydrolyzed maltodextrin.

[0026] Typically, the substrate has a DE between 2 and 15, preferably between 2 and 12.

[0027] Typically, the substrate comprises at least 90%, preferably at least 92%, at least 95%, even more preferably at least 95% by weight of amylopectin chains relative to the weight of dry matter of said substrate.

[0028] The method of the invention uses a glucan 1,4-alpha-maltotetrahydrolysase enzyme (EC 3.2.1.60). This is an enzyme that hydrolyzes alpha 1-4 glucosidic bonds every four residues, from the non-reducing ends of polysaccharides.

[0029] Preferably, it is a PS-4 variant described in patent application WO2010 / 118269.

[0030] Preferably, it is the enzyme marketed by the company Genencor / DuPont under the name “OPTIMALT® 4G”.

[0031] In one embodiment, the method according to the invention does not comprise the use of a pullulanase.

[0032] In the process according to the invention, a tangential filtration step with a cut-off threshold of 1 kDa is carried out simultaneously with the enzymatic reaction. The low molecular weight sugars, and in particular the DP4, will therefore be filtered and collected as the enzymatic reaction progresses. Indeed, the cut-off threshold of 1 kDa allows the molecular weight sugars (DP1, DP2, DP3, D4, DP5 and DP6) to pass into the filtrate and retains the higher molecular weight sugars (retentate).

[0033] Without wishing to be bound by any theory, the inventors proposed that this simultaneous filtration allows the reaction equilibrium to be shifted in favor of the production of DP4 by eliminating this reaction product as it goes along.

[0034] Thus, the enzymatic reaction can continue until the residual substrate is a limit dextrin, i.e. consisting essentially of branched alpha 1-6 glucosidic bonds.

[0035] In a preferred embodiment, filtration is started before the addition of the enzyme and a first volume of filtrate is discarded. This allows the enzymatic reaction to be freed from any low DP sugars (mono-, dietary tri-saccharides) that may be present in the substrate.

[0036] Subsequently, after adding the enzyme, the filtrate is collected. This mainly contains maltotetraose (or DP4) since the enzyme does not generate DP1, DP2 or DP3 sugars.

[0037] Thus, according to a particular embodiment, the invention relates to a method comprising the following steps: - provide a substrate solution with a pH between 5 and 5.5 - start filtration, while keeping the volume of the retentate constant by the addition of demineralized water, - add the enzyme - discard the first fractions of filtrate rich in monosaccharides, disaccharides and trisaccharides - collect the following fractions of filtrate enriched in maltotetraose, - possibly mix and concentrate the fractions rich in maltotetraose, - possibly, demineralize the composition obtained - possibly freeze-dry the composition.

[0038] In a second aspect, the present invention relates to a composition obtainable according to the method described above.

[0039] The present invention also relates to a composition comprising at least 60% by weight of maltotetraose and less than 12% of oligosaccharides of DP1, DP2 or DP3 relative to the total weight of dry matter.

[0040] Thus, the cumulative weight of DP1, DP2 and DP3 in the composition according to the invention does not exceed 12%, preferably 11%, preferably 10%, preferably 9%, preferably 8%, preferably 7%.

[0041] According to a particular embodiment, the weight of DP1 in the composition according to the invention does not exceed 2%, preferably 1.8%, preferably 1.6%, preferably 1.4%, preferably 1.2%, preferably 1.0%.

[0042] According to a particular embodiment, the weight of DP2 in the composition according to the invention does not exceed 5%, preferably 4%, preferably 3%, preferably 2.9%, preferably 2.8%, preferably 2.7%, preferably 2.6%, preferably 2.5%.

[0043] According to a particular embodiment, the weight of DP3 in the composition according to the invention does not exceed 6%, preferably 5%, of preferably 4%, preferably 3.8%, preferably 3.7%, preferably 3.6%, preferably 3.5%.

[0044] Finally, the present invention relates to the use of a composition according to the invention in the preparation of food for human or animal consumption.

[0045] The invention will be better understood with the aid of the following examples, which are intended to be illustrative and not limiting.

[0046] Example 1: protocol for preparing a syrup rich in maltotetraose

[0047] 1. Reagents used

[0048] 1.1. Maltodextrin having a DE (dextrose equivalent) of 2 to 6 produced from Waxy corn starch (GLUCIDEX® 2 marketed by the Applicant Company).

[0049] 1.2. Maltodextrin having a DE of 12 produced from standard corn starch (GLUCIDEX® 12 marketed by the Applicant Company.

[0050] 1.3. Glucan 1,4-alpha-mltotetraohydrolylase enzyme: OPTIMALT®4G produced by the company Genencor (Dupont).

[0051] 1.4. Thermo-resistant Alpha Amylase Enzyme: LIQUOZYME® supra produced by Novozyme.

[0052] 1.5. Waxy corn starch N200 ® marketed by the Company Applicant.

[0053] Products 1.4 and 1.5 were only used in Example 4.

[0054] 2. Materials used

[0055] The enzymatic reaction was carried out in a 2-liter beaker placed on a regulated heating magnetic stirrer.

[0056] Tangential filtration was carried out simultaneously with the enzymatic reaction using a Centramate PALL system equipped with a 1 KD REF OS001T12 0.1 m2 nanofiltration cassette.

[0057] 3. Operating mode

[0058] The following procedure was carried out:

[0059] - Prepare a maltodextrin solution by diluting 100 g of product in 900 ml of demineralized water in a 2 liter beaker.

[0060] - Check the pH of the solution (5 to 5.5) and adjust it if necessary with NaOH or HCl 0.1 mol / liter.

[0061] - Place the solution under stirring and regulate it at 50°C.

[0062] - Start nanofiltration with the Centramate system by letting the filtrate flow into a beaker. Once the volume of 300ml of filtrate is reached, add 200pl of the OPTIMALT® 4G enzyme to the maltodextrin solution and recycle the filtrate into the retentate for 15 minutes.

[0063] - Take a sample every 300 ml of filtrate. On each sample, take the refractometry reading and analyze the level of glucose (DP1), maltose (DP2), maltotriose (DP3), maltotetraose (DP4) and those above DP4 by HPLC.

[0064] - The decrease in volume of the retentate must be continuously compensated with demineralized water (constant volume dialysis mode).

[0065] - The fractions were mixed and concentrated using a rotary evaporator until a refractometric reading of 30 BX was obtained.

[0066] - The syrup obtained was decolorized using an activated carbon SA usually used for the purification of glucose syrups (contact time: 1 hour at 70°C.

[0067] - The syrup was then demineralized with the anionic and cationic resins usually used for the purification of glucose syrups.

[0068] Depending on requirements, the syrup can be concentrated to a refractometric reading of 65 to 70 BX or freeze-dried to ensure sample preservation.

[0069] 4. Physicochemical analysis

[0070] The carbohydrate composition of the fractions was analyzed by a Waters E2695 HPLC system equipped with a RID 2414 detector. The column used is an AMINEX HPX 87N at a temperature of 85°. Flow rate of the mobile phase (water): 0.3 ml / min.

[0071] Example 2: Preparation of a syrup rich in maltotetraose from a weakly hydrolyzed maltodextrin produced from waxy starch

[0072] According to the protocol described in Example 1, the test described below was carried out with a solution of GLUCIDEX® 2 (100g QSP demineralized water 1000g) as the starting substrate.

[0073] The different fractions of 300 ml of filtrate were collected and the composition analyzed by HPLC. Fraction F1 corresponds to the filtrate before the addition of enzymes.

[0074] Fractions F4 to F8 were mixed for concentration and purification steps.

[0075] The analyses are presented in Table 1.

[0076] [Table 1]

[0077] Example 3: Preparation of a syrup rich in maltotetraose from a DE 12 maltodextrin produced from waxy starch

[0078] According to the protocol described in Example 1, the test described below was carried out with a solution of GLUCIDEX® 12 (100g QSP demineralized water 1000g) as the starting substrate.

[0079] The different fractions of 300 ml of filtrate were collected and the composition analyzed by HPLC. Fraction F0 corresponds to the filtrate before the addition of enzymes.

[0080] The analyses are presented in Table 2.

[0081] [Table 2]

[0082] The aim of the test was to compare the richness of maltotetraose fractions depending on the raw material. The product was not purified.

[0083] Example 4: Preparation of a syrup rich in maltotetraose from liquefied waxy corn starch

[0084] Unlike the two previous examples, the raw material for the test was a waxy corn starch liquefied as follows:

[0085] - Preparation of a suspension of Waxy corn starch (100 g QSP demineralized water 400g)

[0086] - Liquefaction using a heat-resistant alpha-amylase (LIQUOZYME® supra) at a concentration of 0.8g / kg of dry matter.

[0087] - Use a microwave (5 cycles of 1 minute, 1000 W) then maintain at 95°C for 15 min.

[0088] - Inhibition with 1 N HCl up to pH = 3.0

[0089] - Filtration on COFRAM filter ref. BECO KD3

[0090] - Dilution of the solution obtained to obtain 1 liter of solution at 10% dry matter.

[0091] The composition of the liquefied corn starch thus obtained is presented in Table 3.

[0092] [Table 3]

[0093] Based on the carbohydrate composition, it can be estimated that DE (dextrose equivalent) of the resulting solution is approximately 6.

[0094] This solution was then treated according to the conditions described in Example 1.

[0095] The following results were obtained:

[0096] [Table 4] | 0.7 | 2.4 | 4.1 | 78.2 | 14.8 |

[0097] The aim of the test was to compare the richness of maltotetraose fractions depending on the raw material. The product was not purified.

[0098] All of the above examples demonstrate that the process according to the invention makes it possible to obtain compositions rich in maltotetraose and low in DP1, DP2 and DP3, and this from three different types of substrates (maltodextrin of DE2, maltodextrin of DE 12 and liquefied starch of DE approximately 6)

Claims

Demands

1. A process for preparing a composition comprising at least 60% by weight of maltotetraose, said process comprising bringing a substrate into contact with a glucan 1,4-alpha-maltotetrahydrolysase type enzyme and tangential filtration with a cutoff threshold of 1 kDa carried out simultaneously with the enzymatic reaction.

2. A process according to claim 1, wherein the substrate is selected from a liquefied starch rich in amylopectin or a weakly hydrolyzed maltodextrin.

3. A method according to any one of the preceding claims, characterized in that it does not comprise the use of a pullulanase.

4. A method according to any one of the preceding claims, comprising the following steps: - provide a substrate solution with a pH between 5 and 5.5 - Start the filtration process, while maintaining a constant retentate volume by adding demineralized water. - add the enzyme - discard the first fractions of filtrate rich in monosaccharides, disaccharides and trisaccharides, - collect the following fractions of filtrate enriched in maltotetraose, - possibly mix and concentrate the fractions rich in maltotetraose, - if necessary, demineralize the resulting composition - possibly freeze-dry the composition.

5. Composition which can be obtained according to the process of one of the preceding claims.

6. Composition comprising at least 60% by weight of maltotetraose and less than 12% of oligosaccharides of DP1, DP2 or DP3.

7. Use of a composition according to any one of claims 5 or 6 for the preparation of food for human or animal consumption.

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