Enzymatic process for producing a composition rich in maltotetraose

Abstract

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

Description

Technical Field

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

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

[0003] The present invention further relates to the use of a composition rich in maltotetraose for preparing a foodstuff for human or animal consumption.

Background Art

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

[0005] A composition rich in maltotetraose, or maltotetraose syrup, can be used in the production of foodstuffs for human or animal consumption. Maltotetraose syrup offers more advantages than sucrose or glucose syrup. This is because it is not sweeter than sucrose syrup but preserves the taste of food. In addition, they are less susceptible to Maillard degradation and have a higher viscosity.

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

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

[0008] The D.E. limit of maltodextrin is 20. Further, the legal name of the resulting product is "glucose syrup".

[0009] Therefore, D.E. and DP are inversely correlated.

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

[0011] However, in the conventional production of compositions rich in maltotetraose, it has not been possible to obtain a composition in which the proportion of maltotetraose is 60% or more.

[0012] For example, U.S. Patent No. 3,654,082 (CPC Internationale Inc) describes a process for producing maltotetraose syrup using the enzymatic activity of an amylase from Pseudomonas stutzeri.

[0013] The maltotetrahydroylase enzyme is commercially available under the name "OPTIMALT® 4G" by Genencor / DuPont.

[0014] Furthermore, International Publication No. 2010 / 118269 (A2) (Danisco / DuPont) describes variants of the Pseudomonas saccharophila PS-4 enzyme having increased exo-α-amylase activity compared to native enzymes and decreased endo-α-amylase activity compared to native enzymes.

[0015] In International Publication No. 2010 / 118269 (A2), the syrup obtained in the examples has a maximum DP4 content of 47%.

[0016] International Publication No. 2010132157 describes the use of the PS-4 enzyme with a pullulanase enzyme to form a syrup containing 40 to 60% by weight maltotetraose.

[0017] Furthermore, Tereos markets a syrup called MYLOSE® 351, which is offered as a glucose syrup rich in DP4. However, this product contains 50 to 60% by weight maltotetraose. It also contains a significant amount of DP3 (6 - 12%).

[0018] Therefore, it is desirable to obtain a composition rich in maltotetraose and low in monosaccharides, disaccharides, and trisaccharides.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Subsequently, the Applicant's company has found that it is possible to produce a composition rich in maltotetraose from amylopectin-rich liquefied starch by an enzymatic reaction. Therefore, the company has developed a process using specific enzymes in combination with a tangential filtration step with a 1 kDa cut-off carried out simultaneously with the enzymatic reaction.

[0020] In a first aspect, the present invention is a process for preparing a composition comprising at least 60% by weight of maltotetraose, the process comprising contacting a substrate with a glucan 1,4-α-maltotetrahydrolyase enzyme and tangential filtration with a cut-off of 1 kDa carried out simultaneously with the enzymatic reaction.

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

[0022] The percentage of maltotetraose (also known as "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 shown in the following examples.

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

[0024] Preferably, the starting starch is a starch rich in amylopectin, such as waxy maize 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 of 2 to 15, preferably 2 to 12.

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

[0028] This process uses an enzyme of the glucan 1,4-α-maltotetrahydrolase type (EC 3.2.1.60). This enzyme hydrolyzes the α1-4 glucoside bond every 4 residues from the non-reducing end of the polysaccharide.

[0029] Preferably, it is the PS-4 variant described in International Publication No. 2010 / 118269.

[0030] Preferably, it is an enzyme commercially available under the name "OPTIMALT® 4G" from Genencor / DuPont.

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

[0032] In the process according to the invention, a tangential filtration step at a 1 kDa cut-off is carried out simultaneously with the enzyme reaction. Low molecular weight sugars, especially DP4, are filtered and recovered as the enzyme reaction progresses. The 1 kDa cut-off allows sugars of lower molecular weight (DP1, DP2, DP3, D4, DP5 and DP6) to pass through into the filtrate while retaining higher molecular weight sugars (retentate).

[0033] Without wishing to be bound by any theory, the inventors propose that this simultaneous filtration advantageously shifts the reaction equilibrium towards DP4 production by removing the reaction product as it is formed.

[0034] In this way, the enzyme reaction can continue until the residual substrate becomes limit dextrin, i.e., essentially composed of α1-6 glucoside branch linkages.

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

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

[0037] Accordingly, according to certain embodiments, the present invention relates to a process comprising the following steps. - Supplying a substrate solution having a pH of -5 to 5.5; - Initiating filtration and maintaining a constant liquid volume by adding demineralized water; - Adding an enzyme; - Discarding a first filtrate fraction rich in monosaccharides, disaccharides, and trisaccharides; - Collecting the following filtrate fraction enriched in maltotetraose; - Optionally, mixing and concentrating the fraction rich in maltotetraose; - Optionally, desalting the resulting composition; - Optionally, lyophilizing the composition.

[0038] In a second aspect, the present invention relates to a composition obtainable by the above process.

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

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

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

[0042] According to certain embodiments, the weight of DP2 in the composition according to the present 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 certain embodiments, the weight of DP3 in the composition according to the present invention does not exceed 6%, preferably 5%, preferably 4%, preferably 3.8%, preferably 3.7%, preferably 3.6%, preferably 3.5%.

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

[0045] The present invention is better understood with the aid of the following examples, which are intended to be illustrative and non-limiting.

[0046] Example 1: Protocol for preparing syrup rich in maltotetraose. 1. Reagents used 1.1. Maltodextrin having a DE (dextrose equivalent) of 2 to 6 produced from waxy corn starch (GLUCIDEX® 2 commercially available from the applicant's company). 1.2. Maltodextrin having a DE of 12 produced from standard corn starch (GLUCIDEX® 12 commercially available from the applicant's company). 1.3. Glucan 1,4-α-maltotetrahydrolyase enzyme: OPTIMALT® 4G, manufactured by Genencor (Dupont). 1.4. Heat-stable α-amylase enzyme: LIQUOZYME® supra, manufactured by Novozyme. 1.5. N200® which is a waxy corn starch commercially available from the applicant's company.

[0047] Only products 1.4 and 1.5 were used in Example 4.

[0048] 2. Equipment Used The enzymatic reaction was carried out in a 2 - liter beaker placed on a regulated heated magnetic stirrer.

[0049] Cross - flow filtration was carried out simultaneously with the enzymatic reaction using a Centramate PALL system equipped with a 1KD REF OS001T12 0.1m2 nanofilter cassette.

[0050] 3. Procedure The following procedure was used. - Prepare a maltodextrin solution by diluting 100 g of the product with 900 mL of demineralized water in a 2 - liter beaker. - Check the pH of the solution (5 - 5.5) and adjust with 0.1 mol / liter of NaOH or HCl if necessary. - Shake the solution at 50 °C. - Start nanofiltration with the Centramate system and discharge the filtrate into the beaker. When the filtrate volume reaches 300 mL, add 200 μL of OPTIMALT® 4G enzyme to the maltodextrin solution and recirculate the filtrate in the retentate for 15 minutes. - Take grab samples every 300 mL of filtrate. For each sample, take a refractive index measurement reading and analyze, by HPLC, the levels of glucose (DP1), maltose (DP2), maltotriose (DP3), maltotetraose (DP4), and higher levels of DP4. - The decrease in the retentate volume must be continuously compensated with demineralized water (constant - volume dialysis mode). - Mix the fractions and concentrate to a refractive index reading of 30 BX with a rotary evaporator. - Decolorize the resulting syrup using SA activated carbon, a common method for purifying glucose syrup (contact time: 1 hour at 70 °C). - Then, desalt this syrup with anionic and cationic resins commonly used for glucose syrup purification.

[0051] If necessary, the syrup can be concentrated to a refractive index measurement of 65 - 70 BX or lyophilized for sample storage.

[0052] 4. Physical / Chemical Analysis The carbohydrate composition of the fractions was analyzed using a Waters E2695 HPLC system equipped with a RID 2414 detector. The column used was AMINEX HPX 87N at a temperature of 85 °C. Mobile phase flow rate (water): 0.3 mL / min.

[0053] Example 2: Preparation of a maltotetraose-rich syrup from low DE maltodextrin produced from waxy starch. According to the protocol described in Example 1, the following tests were conducted using a solution of GLUCIDEX® 2 (100 g QSP deionized water 1000 g) as the starting substrate.

[0054] Various fractions of 300 mL of the filtrate were collected and the composition was analyzed by HPLC. Fraction F1 corresponds to the filtrate before enzyme addition.

[0055] Fractions F4 - F8 were mixed for concentration and purification.

[0056] The results are shown in Table 1.

[0057] [Table 1]

[0058] Example 3: Preparation of a maltotetraose-rich syrup from DE12 maltodextrin produced from waxy starch. According to the protocol described in Example 1, the following tests were conducted using a solution of GLUCIDEX® 12 (100 g QSP deionized water 1000 g) as the starting substrate.

[0059] Various fractions of 300 mL of the filtrate were collected and the composition was analyzed by HPLC. Fraction F0 corresponds to the filtrate before enzyme addition.

[0060] The results are shown in Table 2.

[0061]

Table 2

[0062] The purpose of this test was to compare the richness of the maltotetraose fraction as a function of the raw material. The product was not purified.

[0063] Example 4. Preparation of a maltotetraose-rich syrup from liquefied waxy corn starch. In contrast to the previous two examples, the raw material for the test was waxy corn starch liquefied as follows. - Preparation of a waxy corn starch suspension (100 g in 400 g of appropriate demineralized water). - Liquefaction with heat-resistant α-amylase at a concentration of 0.8 g / kg dry matter (LIQUOZYMER® as described above). - Microwave (5 cycles of 1 minute, 1000 w), then hold at 95 °C for 15 minutes. - Adjust to pH = 3.0 with 1N HCl. - Filter with a COFRAM filter (refer to BECO KD3). - Dilute the resulting solution to obtain a 10% dry matter solution of 1 liter.

[0064] The composition of the obtained liquefied corn starch is shown in Table 3.

[0065]

Table 3

[0066] From the carbohydrate composition, it can be estimated that the DE (dextrose equivalent) of the resulting solution is approximately 6.

[0067] Subsequently, this solution was treated according to the conditions described in Example 1.

[0068] The following results were obtained.

[0069]

Table 4

[0070] The purpose of this test was to compare the richness of the maltotetraose fraction as a function of the raw material. The product was not purified.

[0071] All of the above examples show that the process according to the invention makes it possible to obtain a composition rich in maltotetraose and low in DP1, DP2 and DP3 from three different types of substrates (maltodextrin having DE2, maltodextrin having DE12 and liquefied starch having DE of about 6).

Claims

1. A process for preparing a composition comprising at least 60% by weight of maltotetraose, the process comprising contacting a substrate with a glucan 1,4-α-maltotetrahydrolase enzyme and tangential filtration with a 1 kDa cutoff carried out simultaneously with the enzymatic reaction.

2. 2. The process of claim 1, wherein the substrate is selected from amylopectin-rich liquefied starch or low DE maltodextrin.

3. 2. The process according to claim 1, characterized in that it does not involve the use of pullulanase.

4. 2. The process of claim 1, providing a substrate solution having a pH of -5 to 5.5; - starting the filtration and maintaining the retentate volume constant by adding demineralized water; - adding said enzyme; - discarding the first filtrate fraction enriched in mono-, di- and trisaccharides; - collecting the filtrate fractions enriched in maltotetraose, - optionally combining and concentrating the maltotetraose-rich fractions; - optionally desalting the composition obtained; - optionally lyophilizing said composition.

5. A composition obtainable by the method according to any one of claims 1 to 4.

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

7. 10. Use of the composition of claim 6 for preparing food for human or animal consumption.