Enzymatic process for producing a maltotetraose-rich composition

Abstract

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

Description

Title of the invention: Enzymatic process for producing a maltotetraose-rich composition 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. Prior state of the art

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

[0005] Compositions rich in maltotetraose or maltotetraose syrups can be used in the manufacture of food for human or animal consumption. Advantageously, maltotetraose syrups offer numerous advantages over sucrose or glucose syrups. Indeed, they have a lower sweetness than 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 generally manufactured from starches, such as corn starch, by enzymatic reactions. The starch is first hydrolyzed by an alpha-amylase to produce maltodextrins.

[0007] Maltodextrin compositions are mixtures of different sugars obtained from the hydrolysis of starch and having varying 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 complete starch hydrolysis. The higher the DE, the more extensive the hydrolysis, and therefore the higher the proportion of simple (short-chain) sugars composing the maltodextrin. A DE of zero would represent starch itself, while a DE of 100 would represent pure dextrose, i.e., completely transformed starch.

[0008] The DE limit for a maltodextrin is 20. Beyond that, the product obtained has the legal designation "glucose syrup".

[0009] DE and DP are therefore inversely correlated.

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

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

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

[0013] A maltotetrahydrolase type enzyme is marketed by the company 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 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-type enzyme to form syrups comprising between 40 and 60% by weight of maltotetraose.

[0017] Furthermore, the company Tereos 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. Moreover, it also contains significant amounts 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 then discovered that it was possible, starting from a liquefied starch rich in amylopectin, to produce a composition rich in maltotetraose by enzymatic reaction. The Applicant thus developed a process that uses a specific enzyme, in combination with a tangential filtration step with a cutoff point at IkDa, carried out simultaneously 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 of maltotetraose, said method comprising reacting a substrate with a glucan 1,4-alpha-maltotetrahydrolysase-type enzyme and tangential flow filtration with a cutoff threshold at IkDa 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 dry weight 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 maize 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 dry weight of said substrate.

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

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

[0030] Preferably, this refers to the enzyme marketed by Genencor / DuPont under the name "OPTIMALE® 4G".

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

[0032] In the process according to the invention, a tangential flow filtration step with a cutoff threshold of 1 kDa is carried out simultaneously with the enzymatic reaction. Low molecular weight sugars, and in particular DP4, are thus filtered and collected as the enzymatic reaction progresses. Indeed, the 1 kDa cutoff threshold allows sugars of lower molecular weights (DP1, DP2, DP3, DP4, DP5, and DP6) to pass into the filtrate and retains sugars of higher molecular weight (retentate).

[0033] Without wishing to be bound by any theory, the inventors have proposed that this simultaneous filtration makes it possible to shift the reaction equilibrium in favour of the production of DP4 by eliminating this reaction product as it is done.

[0034] Thus, the enzymatic reaction can continue until the residual substrate is a limiting dextrin, that is to say, consisting essentially of branched alpha 1-6 glycosidic bonds.

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

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

[0037] Thus, according to a particular embodiment, the invention relates to a process 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, - optionally mix and concentrate the fractions rich in maltotetraose, - optionally, demineralize the resulting composition - possibly freeze-dry the composition.

[0038] In a second aspect, the present invention relates to a composition that can be obtained according to the process 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 DPI, DP2 or DP3 relative to the total dry weight.

[0040] Thus, the cumulative weight of DPI, 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 the DPI 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%, 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 maltotetraose-rich syrup

[0047] 1. Reagents used

[0048] 1.1. Maltodextrin having an ED (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 maize starch (GLUCIDEX® 12 marketed by the Applicant Company.

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

[0051] 1.4. Thermo-resistant Alpha amylase enzyme: LIQUOZYME® supra produced by the Novozyme company.

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

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

[0054] 2. Materials used

[0055] The enzymatic reaction was carried out in a 2-litre beaker placed on a regulated heated 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 procedure

[0058] The following operating procedure was carried out:

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

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

[0061] - Place the solution under stirring and regulate this to 50°C.

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

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

[0064] - The decrease in retentate volume must be continuously compensated with semi-dried water neralized (constant volume dialysis mode).

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

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

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

[0068] Depending on the requirements, the syrup can be concentrated until a refractometric reading of 65 to 70 BX is obtained or lyophilized to ensure the preservation of the sample.

[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 was 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 maltotetraose-rich syrup 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 a starting substrate.

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

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

[0075] The analyses are presented in Table 1.

[0076] [Tables 1] Carbohydrate composition expressed as % / dry matter Reference Glucose DPI Maltose DP2 Maltotriose *DP3 Maltotetraose DP4 Oligosaccharide s >DP4 Fl (before enzyme) 1 6.4 10.1 6.7 75.6 F2 0.8 5.0 6.5 57.8 29.9 F3 0.8 4.3 5 71.9 18.0 F4 0.6 2.8 3.8 79.6 13.1 F5 0.8 2.9 3.3 82.4 10.5 F6 0.7 2.9 3.9 84.5 9.1 F7 0.7 2.2 2.5 86.5 8.0 F8 0.8 2.1 24 87.0 7.8 Final mixture F4 to F8 0.7 2.5 3.2 83.7 9.9

[0077] Example 3: preparation of a maltotetraose-rich syrup 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 a starting substrate.

[0079] The different 300 ml fractions of filtrate were recovered 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] [Tables2] Carbohydrate composition expressed as % / dry matter Reference Glucose DPI Maltose DP2 Maltotriose *DP3 Maltotetraose seDP4 Oligosaccharides >DP4 F0 (before enzyme) 1.9 7.9 12.4 10.4 67.4 Fl 1.9 7.7 11.3 29.9 19.3 F2 1.9 6.9 10.1 45.7 35.4 F3 1.9 6.6 9.5 51.5 30.5 F4 1.9 5.7 8.2 61.5 22.9 F6 1.7 4.0 5.9 71.5 16.9 F7 1.5 3.0 5.0 65.5 25.0 F8 1.7 3.1 4.9 57.7 32.6

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

[0083] Example 4: preparation of a maltotetraose-rich syrup from liquefied waxy maize starch

[0084] Unlike the two previous examples, the raw material for the test was a waxy maize starch liquefied in the following way:

[0085] - Preparation of a Waxy corn starch suspension (100 g q.s. to half water) (neralized 400g)

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

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

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

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

[0090] - Dilution of the solution obtained to obtain 1 liter of a 10% solution dried.

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

[0092] [Tables3] Carbohydrate composition expressed as % / dry matter Reference Glucose DPI Maltose DP2 Maltotriose *DP3 Maltotetraose DP4 Oligosaccharides >DP4 Liquefied maize starch 0.1 1.0 2.1 0.9 95.8

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

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

[0095] The following results were obtained:

[0096] [Tables4] Carbohydrate composition expressed as % / dry matter Reference Glucose DPI Maltose DP2 Maltotriose *DP3 Maltotetraose seDP4 Oligosaccharide s >DP4 Fl (before enzyme) 1.1 7.3 15.3 8.8 66.7 F2 0.9 5.2 9.6 53.6 30.6 F3 1.0 3.7 6.6 71.2 17.5 F5 0.8 2.9 2.9 77.4 13.6 F6 0.8 2.2 3.9 80.0 13.1 F7 0.7 2.4 4.1 78.2 14.8

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

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

Claims

Demands

1. A process for preparing a composition for the preparation of food for human or animal consumption comprising at least 60% by weight of maltotetraose, said process comprising the contact of a substrate with a glucan-type enzyme 1,4-alpha-maltotetrahydrolysase and tangential flow filtration with a cutoff point at IkDa carried out simultaneously with the enzymatic reaction, comprising the following steps: - providing a substrate solution having a pH between 5 and 5.5 - starting the filtration, while maintaining the retentate volume constant by adding demineralized water, - adding the enzyme, - discarding the first fractions of filtrate rich in monosaccharides, disaccharides, and trisaccharides, - collecting the subsequent fractions of filtrate enriched in maltotetraose, - optionally mixing and concentrating the fractions rich in maltotetraose, - optionally,Demineralize the resulting composition - optionally freeze-dry the composition.

2. A method 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. Composition for the preparation of food for human or animal consumption which can be obtained according to the process of any one of the preceding claims.

5. Composition for the preparation of food for human or animal consumption comprising at least 60% by weight of maltotetraose and less than 7% of oligosaccharides of DPI, DP2 or DP3.

6. Use of a composition according to any one of claims 4 or 5 for the preparation of food for human or animal consumption.