Enzymatic process for production of maltotetraose-rich compositions

Abstract

The present invention relates to an enzymatic process for the preparation of a maltotetraose enriched composition.

Description

Technical Field

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

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

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

[0004] Maltotetraose is an oligosaccharide composed of four glucose units linearly linked by α1-4 glycosidic bonds.

[0005] Compositions rich in maltotetraose or maltotetraose syrup can be used to make food for human or animal consumption. Maltotetraose syrups offer many advantages over sucrose syrup or glucose syrup. This is because they are not as sweet as sucrose syrup while maintaining the taste of the food. In addition, they are not susceptible to Maillard degradation and have a higher viscosity.

[0006] Maltotetraose syrup is usually prepared from starch such as corn starch by an enzymatic reaction. The starch is first hydrolyzed by α-amylase to produce maltodextrins.

[0007] Maltodextrin compositions are mixtures of different sugars derived from starch hydrolysis and having different degrees of polymerization. This degree of polymerization or "DP" is reflected experimentally by the "dextrose equivalent" or DE (dextrose is D-glucose), the result of total starch hydrolysis. The higher the DE, the more complete the hydrolysis, and therefore the higher the proportion of simple (short chain) sugars in the maltodextrin. A DE of 0 represents starch itself, while a DE of 100 represents pure dextrose (i.e., fully converted starch).

[0008] The DE limit for maltodextrin is 20. Beyond this, the legal name of the resulting product is "glucose syrup".

[0009] DE and DP are therefore anti-correlated.

[0010] The maltodextrin is then subjected to further enzymatic reactions to produce a composition enriched in maltotetraose (i.e., enriched in DP4 oligosaccharides). Certain enzymes, such as maltotetrahydrolase ("PS-4") from Pseudomonas saccharophila, are able to preferentially hydrolyze maltodextrin to DP4.

[0011] However, conventional production of compositions enriched in maltotetraose does not allow us to obtain compositions having a maltotetraose proportion greater than or equal to 60%.

[0012] For example, patent US Pat. No. 3,654,082 (CPC Internationale Inc) describes a method for producing maltotetraose syrup using the enzymatic activity of an amylase derived from Pseudomonas stutzeri.

[0013] A malt tetrahydrolase is produced by Genencor / DuPont under the name " 4G” sales.

[0014] Furthermore, International Application WO 2010 / 118269 A2 (Danisco / DuPont) describes variants of the Pseudomonas saccharophila PS-4 enzyme having increased exo-alpha-amylase activity compared to the native enzyme and reduced endo-alpha-amylase activity compared to the native enzyme.

[0015] In application WO 2010 / 118269 A2, the syrups obtained in the examples have a maximum DP4 content of 47%.

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

[0017] In addition, Tereos has marketed The syrup of 351, which appears to be a glucose syrup enriched in DP 4. However, the product contains between 50% and 60% maltotetraose by weight. It also contains a significant amount of DP3 (between 6% and 12%).

[0018] It is therefore desirable to obtain a composition rich in maltotetraose and low in mono-, disaccharides and trisaccharides. DETAILED DESCRIPTION

[0019] The applicant company subsequently discovered that a composition rich in maltotetraose can be produced from liquefied starch rich in amylopectin by an enzymatic reaction. Therefore, the company has developed a method that uses a specific enzyme combined with a tangential filtration step with a cutoff value of 1 kDa (concurrently with the enzymatic reaction).

[0020] In a first aspect, the present invention relates to a method for preparing a composition comprising at least 60% by weight maltotetraose, the method comprising contacting a substrate with glucan 1,4-α-maltotetrahydrolase and performing tangential filtration with a cut-off of 1 kDa simultaneously with the enzymatic reaction.

[0021] According to a preferred embodiment of the invention, the method is a method 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% maltotetraose by weight relative to the total dry weight of the composition.

[0022] The percentage of maltotetraose (also referred to as "DP4") can be determined by any suitable method known to those skilled in the art. For example, the percentage can be determined by HPLC chromatography, as shown in the following examples.

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

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

[0025] According to one embodiment of the present invention, the substrate is selected from liquefied starch rich in amylopectin and low DE 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 uses the glucan 1,4-α-maltotetrahydrolase type (EC 3.2.1.60). This enzyme hydrolyzes α1-4 glycosidic bonds every four residues from the non-reducing end of the polysaccharide.

[0029] Preferably, it is the PS-4 variant described in patent application WO 2010 / 118269.

[0030] Preferably, it is manufactured by Genencor / DuPont under the name " 4G” enzymes sold.

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

[0032] In the method according to the invention, a tangential filtration step with a cutoff of 1 kDa is carried out simultaneously with the enzymatic reaction. As the enzymatic reaction proceeds, low molecular weight sugars, in particular DP4, are filtered out and collected. The 1 kDa cutoff allows lower molecular weight sugars (DP1, DP2, DP3, D4, DP5 and DP6) to enter the filtrate while retaining higher molecular weight sugars (retentate).

[0033] Without wishing to be bound by any theory, the inventors have proposed that this simultaneous filtration shifts the reaction equilibrium in favor of DP4 production by removing this reaction product as it is produced.

[0034] In this manner, the enzymatic reaction can be continued until the remaining substrate is the limited dextrin, ie, consisting essentially of alpha 1-6 glycosidic branching bonds.

[0035] In a preferred embodiment, filtration is initiated before the enzyme is added and the first volume of filtrate is discarded. This cleans the enzymatic reaction of any low DP sugars (monosaccharides, disaccharides and trisaccharides) that may be present in the substrate.

[0036] Subsequently, once the enzyme has been added, the filtrate is collected. This filtrate contains mainly maltotetraose (or DP4) since the enzyme does not produce DP1, DP2 or DP3 sugars.

[0037] Therefore, according to a specific embodiment, the present invention relates to a method comprising the following steps:

[0038] - providing a substrate solution having a pH between 5 and 5.5,

[0039] - Start filtration, keeping the retentate volume constant by adding demineralized water,

[0040] - adding said enzyme,

[0041] - discarding the first filtrate fraction rich in monosaccharides, disaccharides and trisaccharides,

[0042] - collecting the latter filtrate fractions enriched in maltotetraose,

[0043] - if necessary, the maltotetraose-enriched fractions are combined and concentrated,

[0044] - if necessary, demineralizing the composition obtained,

[0045] - If necessary, freeze-drying the composition.

[0046] In a second aspect, the present invention relates to a composition obtainable by the above process.

[0047] The invention also relates to a composition comprising at least 60% by weight of maltotetraose and less than 12% of DP1, DP2 or DP3 oligosaccharides relative to the total weight of dry matter.

[0048] Therefore, 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%.

[0049] 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%.

[0050] 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%.

[0051] According to a particular embodiment, the amount of DP3 in the composition according to the invention does not exceed 6%, preferably 5%, preferably 4%, preferably 3.8%, preferably 3.7%, preferably 3.6%, preferably 3.5% by weight.

[0052] Finally, the invention relates to the use of a composition according to the invention for the preparation of a food for human or animal consumption.

[0053] The invention will be better understood with the aid of the following examples, which are intended to be illustrative and non-limiting.

[0054] Example 1.: Protocol for the preparation of a syrup rich in maltotetraose

[0055] 1. Reagents used

[0056] 1.1. Maltodextrin with DE (dextrose equivalent) of 2 to 6, which is made from waxy corn starch (sold by the applicant company) 2) Produce.

[0057] 1.2. Maltodextrin with a DE of 12, which is made from standard corn starch (sold by the applicant company) 12) Produce.

[0058] 1.3. Glucan 1,4-α-maltotetrahydrolase: produced by Genencor (Dupont) 4G.

[0059] 1.4. Thermostable α-amylase: produced by Novozyme

[0060] 1.5. Sold by the Applicant Company Waxy corn starch.

[0061] Products 1.4 and 1.5 were used in Example 4 only.

[0062] 2. Equipment used

[0063] The enzymatic reaction was performed in a 2-liter beaker placed on a regulated heated magnetic stirrer.

[0064] Cross-flow filtration was performed simultaneously with the enzymatic reaction using a Centramate PALL system equipped with a 1KD REF OS001T12 0.1 m2 nanofiltration cassette.

[0065] 3. Procedure

[0066] Use the following procedure:

[0067] - A maltodextrin solution was prepared by diluting 100 g of the product in 900 ml of demineralized water in a 2 litre beaker.

[0068] - Check the pH of the solution (5 to 5.5) and if necessary, adjust with NaOH or HCl

[0069] (0.1 mol / L) to adjust.

[0070] - Shake the solution at 50°C.

[0071] - Start nanofiltration with the Centramate system, allowing the filtrate to drain into a beaker. Once a filtration volume of 300 ml is reached, add 200 μL of 4G enzyme and the filtrate was recirculated to the retentate for 15 minutes.

[0072] - Grab samples were taken every 300 mL of filtrate. Each sample was read for refractometry and analyzed by HPLC for levels of glucose (DP1), maltose (DP2), maltotriose (DP3), maltotetraose (DP4) and higher levels of DP4.

[0073] - The reduction in retentate volume must be continuously compensated with demineralized water (constant volume dialysis mode).

[0074] - The fractions were combined and concentrated on a rotary evaporator to a refractive index reading of 30 BX.

[0075] - The resulting syrup was decolorized using SA activated carbon, which is a common method for purifying glucose syrups (contact time at 70°C: 1 H).

[0076] - The syrup is then demineralized using anionic and cationic resins commonly used for glucose syrup purification.

[0077] If desired, the syrup can be concentrated to a refractive index reading of 65-70 BX or freeze-dried to preserve the sample.

[0078] 4. Physical / chemical analysis

[0079] The carbohydrate composition of each fraction was analyzed using a Waters E2695 HPLC system equipped with a RID 2414 detector. The column used was an AMINEX HPX 87N at 85°C. Mobile phase flow rate (water): 0.3 mL / min.

[0080] Example 2. Preparation of maltotetraose-rich syrup from low-DE maltodextrin produced from waxy starch

[0081] According to the protocol described in Example 1, 2 solution (100 g QSP demineralized water 1000 g) was used as the starting substrate for the following tests.

[0082] Various fractions of 300 mL of the filtrate were recovered and analyzed for composition by HPLC. Fraction F1 corresponds to the filtrate before addition of the enzyme.

[0083] Fractions F4 to F8 were combined for concentration and purification.

[0084] The results are presented in Table 1.

[0085] [Table 1]

[0086]

[0087] Example 3. Preparation of maltotetraose-enriched syrup from DE 12 maltodextrin produced from waxy starch

[0088] According to the protocol described in Example 1, 12 solution (100 g QSP demineralized water 1000 g) was used as the starting substrate for the following tests.

[0089] Various fractions of 300 mL of the filtrate were recovered and the composition was analyzed by HPLC. Fraction F0 corresponds to the filtrate before the addition of the enzyme.

[0090] The results are presented in Table 2.

[0091] [Table 2]

[0092]

[0093] The aim of this experiment was to compare the abundance of the maltotetraose fractions as a function of the starting material. The product was not purified.

[0094] Example 4. Preparation of maltotetraose-rich syrup from liquefied waxy corn starch

[0095] In contrast to the previous two examples, the raw material used for the test was waxy corn starch liquefied as follows:

[0096] -Preparation of waxy corn starch suspension (100 g of appropriate amount of demineralized water 400 g)

[0097] - with a concentration of 0.8 g / kg dry matter of thermostable α-amylase ( As above)

[0098] liquefaction.

[0099] - Microwave (5 1 minute cycles, 1000w) followed by 15 minutes at 95°C.

[0100] - Inhibit with 1N HCl to pH = 3.0

[0101] - Filtering on COFRAM filter (ref.BECO KD3)

[0102] - Dilute the resulting solution to obtain 1 liter of a 10% dry matter solution.

[0103] The composition of the obtained liquefied corn starch is shown in Table 3.

[0104] [Table 3]

[0105]

[0106] From the carbohydrate composition, we can estimate the DE (dextrose equivalent) of the resulting solution to be about 6.

[0107] The solution was then treated according to the conditions described in Example 1.

[0108] The following results are obtained:

[0109] [Table 4]

[0110]

[0111] The aim of this experiment was to compare the abundance of the maltotetraose fraction as a function of the starting material. The product was not purified.

[0112] All the above examples show that the process according to the invention makes it possible to obtain compositions rich in maltotetraose and low in DP1, DP2 and DP3 from three different types of substrates (maltodextrin with DE 2, maltodextrin with DE 12 and liquefied starch with DE of about 6).

Claims

1. A method for preparing a composition comprising at least 60% by weight of maltotetraose, the method comprising contacting a substrate with glucan 1,4-α-maltotetrahydrolase and performing tangential filtration with a cut-off of 1 kDa simultaneously with the enzymatic reaction.

2. The method according to claim 1, wherein the substrate is selected from liquefied starch rich in amylopectin or low DE maltodextrin.

3. The method according to any one of the preceding claims, characterized in that The method does not include the use of pullulanase.

4. The method according to any one of the preceding claims, comprising the steps of: - providing a substrate solution having a pH between 5 and 5.5, - Start filtration, keeping the retentate volume constant by adding demineralized water, - adding said enzyme, - discarding the first filtrate fraction rich in monosaccharides, disaccharides and trisaccharides, - collecting the latter filtrate fractions enriched in maltotetraose, - if necessary, the maltotetraose-enriched fractions are combined and concentrated, - if necessary, demineralizing the composition obtained, - If necessary, freeze-drying the composition.

5. A composition obtainable by a method according to any one of the preceding claims.

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

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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