Method for producing slowly digestible branched starch hydrolysate and use thereof

Abstract

The present invention relates to a composition comprising a slowly digestible branched starch hydrolysate, to a process for producing said composition and its uses, especially in the food industry.

Description

[Technical field]

[0001] The present invention relates to a composition comprising a slowly digestible branched starch hydrolysate, to a process for producing said composition and its uses, especially in the food industry. [Background technology]

[0002] Carbohydrates are the major source of energy in human and animal diets and are an important component of a balanced healthy diet.

[0003] Among them, starch represents an important component in human nutrition. Starch is a polysaccharide produced by most green plants as an energy reserve. It is the most common carbohydrate in the human diet and is found in large amounts in staple foods such as potato, maize (corn), rice, wheat, and cassava.

[0004] Starch is a complex carbohydrate composed of amylose and amylopectin molecules.

[0005] Amylose is a polysaccharide consisting of α-D-glucose units linked together through α-1,4 glycosidic bonds with several branches. It constitutes about 20-30% of normal starch, but the ratio varies from 0% to 99%, depending on the plant origin of the starch and on plant genetic variations.

[0006] Amylopectin is a highly branched polymer of α-glucose units linked in a linear chain by α-1,4 glycosidic bonds. The branching occurs at α-1,6 bonds, which account for about 4-5% of the total glycosidic bonds.

[0007] From a nutritional point of view, starch can be classified into three nutritional fractions: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). SDS has recently attracted interest because foods containing SDS are believed to have a low glycemic index (GI) and provide a long-lasting glucose release. This is particularly useful for individuals who wish to control the rate of glucose release into the bloodstream, such as diabetics or pre-diabetics. It can also be used to prevent hyperinsulinemia-induced hypoglycemia and hunger, as well as to improve cognitive function throughout the day.

[0008] The amount of SDS is typically determined using an in vitro method developed by Englyst et al. and published in 1992 in the European Journal of Clinical Nutrition, volume 46, pp. S33-S50.

[0009] In the art, attempts have been made to increase the amount of SDS in starch-based compositions by increasing the branching density, i.e., by increasing the number of α-1,6 glycosidic bonds. The two main enzymes for starch digestion in the human body are α-amylase and glucoamylase. α-1,6 glycosidic bonds cannot be hydrolyzed by α-amylase, and in addition, the branching points prevent the binding of starch substrates to α-amylase. Although glucoamylase can hydrolyze α-1,6 glycosidic bonds, it is an exoenzyme, and its hydrolysis rate is slower than that of α-amylase, which is an intracellular enzyme. Therefore, the higher the number of α-1,6 glycosidic bonds, the slower the digestion rate of starch can be.

[0010] Lee et al. (PLOSOne, 2013, 8(4), e59745, "Enzyme-Synthesized Highly Branched Maltodextrins Have Slow Glucose Generation at the Mucosal α-Glucosidase Level and Are Slowly Digestible In Vivo") describe the sequential action of starch branching enzymes and β-amylase on waxy corn starch. Dialysis was used to remove oligosaccharides and oligosaccharides.

[0011] Shi et al. (Food Chemistry, 2014, 164, pp 317-323 "Pea starch (Pisum sativum L.) with Slow Digestion Property Produced Using β-Amylase and Transglucosidase") describe the treatment of pea starch with β-amylase with or without transglucosidase. Ethanol precipitation was used to remove oligosaccharides and oligosaccharides.

[0012] In both papers, slower digestibility properties were obtained. However, since the starting material was starch, the methods described in these papers are not cost-effective for scale-up to industrial processes, since the high viscosity of starch prevents the use of compositions with sufficient solids content. In addition, the high moisture content leads to high hydrolysis reactions of starch by enzymes, resulting in low product yields.

[0013] Patent document US2011 / 0020496(A1) describes a method for producing branched dextrins by acting on an aqueous dextrin solution with a maltogenic amylase such as β-amylase and a transglucosidase at an enzyme unit ratio of 2:1 to 44:1, respectively. The high amount / ratio of β-amylase (up to 11.9 U / g substrate with an optimal concentration of 6.3 U / g) was intended to hydrolyze the outer α-1,4 bonds of dextrin, reduce the number of α-1,4 bonds, and thus increase the degree of branching. However, based on the in vitro digestion results, there was only a less than 15% reduction in glucose released between the branched dextrins and the control dextrin (substrate) after 2 hours of digestion. In addition, the amount of transglucosidase may be too small for an effective branching reaction, and the high amount / ratio of β-amylase may generate a high amount of free maltose.

[0014] Thus, none of the prior art documents provide a large-scale method for preparing branched slowly digestible starch hydrolysate compositions containing minimal amounts of sugars (or high yield branched starch hydrolysates) with a degree of polymerization (DP) of 1 to 3. There remains a need in the art for sustained release of glucose to prolong satiety, endurance training, and cognitive function.

[0015] The applicant has therefore, to its credit, developed such a composition and a method for its manufacture, which are disclosed in more detail below. Summary of the Invention

[0016] The present invention relates to a method for preparing a composition comprising a branched starch hydrolysate, comprising the step of incubating the composition comprising a starch hydrolysate with a mixture of maltogenic amylase and transglucosidase enzymes in a unit ratio of between 9:5 and 1:20, advantageously between 9:5 and 1:10, preferably between 10:6 and 1:2, even more preferably between about 3:2 and 1:1.

[0017] The present invention also relates to a composition comprising a branched starch hydrolysate obtainable by the process defined above.

[0018] The present invention provides a composition comprising a branched starch hydrolysate, a) having a DE value of 10 to 50, preferably 12 to 30, more preferably 15 to 25; b) having a percentage of α-1,6 bonds of at least 7%, preferably at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, preferably at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, even more preferably at least 30%, c) comprising at least 30% by weight, preferably at least 35%, preferably at least 40% by weight, even more preferably at least 43% by weight of branches of the branched starch hydrolysate having a DP of 10 or more.

[0019] Another object of the invention is the use of a composition comprising a branched starch hydrolysate as defined above in a food formulation.

[0020] The present invention also provides a food formulation containing a composition comprising a branched starch hydrolysate as defined above.

[0021] The present invention also relates to a kit for preparing a branched starch hydrolysate composition comprising a mixture of maltogenic amylase and transglucosidase enzymes in a unit ratio of 9:5 to 1:20, advantageously 9:5 to 1:10, preferably 10:6 to 1:2, even more preferably about 3:2 to 1:1. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 shows in vitro digestion curves of native and branched tapioca maltodextrins according to the method of Yu et al. [Diagram 2] 1 shows total molecular size and chain length distribution results from native and branched tapioca maltodextrins. [Diagram 3] 1 shows total molecular size and chain length distribution results from native and branched tapioca maltodextrins. [Figure 4] The integral of the signal is shown. [Diagram 5] The correlation between the degree of branching and the RDS content is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] A first object of the present invention relates to a method for preparing a composition comprising a branched starch hydrolysate, comprising a step of incubating the composition comprising a starch hydrolysate with a mixture of maltogenic amylase and transglucosidase enzymes in an enzymatic activity unit ratio of between 9:5 and 1:20, advantageously between 9:5 and 1:10, preferably between 10:6 and 1:2, even more preferably between about 3:2 and 1:1.

[0024] The inventors have surprisingly shown that it is possible to implement a process for the large-scale production of branched starch hydrolysates having slow or low digestibility, high solubility, low viscosity, good low temperature stability, and / or low or no dietary fiber.

[0025] The inventors have shown that a specific ratio between β-amylase and transglucosidase can be used to treat a composition comprising a starch hydrolysate to increase the degree of branching in the starch hydrolysate, thereby reducing and / or retarding the digestibility of said composition.

[0026] As used herein, the term "branched" refers to a degree of branching, indicated by the percentage of α-1,6 glycosidic bonds relative to total glycosidic bonds, which is higher than that in native starch molecules (approximately 4-5%).

[0027] Without being bound by theory, it is believed that a certain ratio between the enzymes favors the branching reaction between maltose and maltodextrins. In particular, a small amount of β-amylase leads to a small amount of free maltose being generated through the enzymatic branching process, thereby reducing the transglucosidase reaction between two maltose molecules and reducing the amount of isomaltose, panose, and other isomaltooligosaccharide (IMOS) by-products.

[0028] High solids content also favors transglucosidase branching reactions over other enzymatic reactions (such as hydrolysis), resulting in more efficient production of branched starch hydrolysates with fewer undesirable products.

[0029] Advantageously, the method of the present invention results in the production of small amounts of DP1-3 sugars. Additional molecular separation steps such as ethanol precipitation, membrane filtration, dialysis, and chromatographic separation are not required to remove these undesirable by-products, and the composition can be used as is. However, these small amounts of sugars can be removed by the methods described above.

[0030] For the purposes of the present invention, "food product" is intended to mean a formulation or composition that can be ingested by animals or humans. Examples of food products include foodstuffs for human consumption, for animal feed, and for beverages.

[0031] In the present invention, the term "starch hydrolysate" refers to any product obtained by enzymatic or acid, preferably enzymatic, hydrolysis of starch from legumes, cereals, or tubers. Various hydrolysis processes are known and are generally described in the book Encyclopedia of Chemical Technology by Kirk-Othmer, 3rd Edition, Vol. 22, 1978, pp. 511 and 512. These hydrolysis products are also defined as purified and concentrated mixtures of molecules composed essentially of D-glucose polymers linked by α-1,4 glycosidic bonds. These molecules have about 4-5% branching points formed by α-1,6 glycosidic bonds, have a wide range of molecular weights, and are completely soluble in water. Starch hydrolysates are very well known and are fully described in Encyclopedia of Chemical Technology by Kirk-Othmer, 3rd Edition, Vol. 22, 1978, pp. 499-521.

[0032] In a preferred embodiment, the starch hydrolysate of the present invention is not obtained by acid treatment of starch.

[0033] In one embodiment, the starch hydrolysates useful in the present invention are obtained by enzymatic treatment of starch with α-amylase.

[0034] Thus, in the present invention, the starch hydrolysis product is selected from starch, gelatinized starch, maltodextrin, glucose syrup, maltose, and any mixture thereof.

[0035] The distinction between starch hydrolysis products is mainly based on the measurement of their reducing power, customarily expressed by the concept of "dextrose equivalent" or DE. DE corresponds to the amount of reducing sugars, expressed in dextrose equivalents, per 100 g of dry matter of the product. DE therefore measures the intensity of starch hydrolysis. Because a product is hydrolyzed to a greater extent, the more small molecules it contains (such as dextrose and maltose), the higher its DE value. Conversely, the more larger molecules (polysaccharides) a product contains, the lower its DE value.

[0036] From a regulatory point of view and within the meaning of the present invention, maltodextrins have a DE of 1-20 and glucose syrups have a DE above 20.

[0037] Such products are, for example, the maltodextrins and dehydrated glucose syrups sold by the Applicant under the name GLUCIDEX® (available DE = 1, 2, 6, 9, 12, 17, 19 for the maltodextrins and DE = 21, 29, 33, 38, 39, 40, 47 for the glucose syrup). Mention may also be made of the glucose syrup sold by the Applicant under the name "Roquette glucose syrup".

[0038] In one embodiment the composition comprising a starch hydrolysate is obtained from a starch selected from tapioca, waxy tapioca, maize, waxy maize, rice, glutinous rice, wheat, waxy wheat, barley, waxy barley, sorghum, waxy sorghum, potato, waxy potato, sweet potato, waxy sweet potato, sago, millet, mung bean, pea, fava bean, chickpea, arrowroot, buckwheat, quinoa, lotus root, preferably tapioca, waxy maize, or pea.

[0039] In a preferred embodiment, the starch is tapioca starch.

[0040] In a preferred embodiment, the starch hydrolysate is obtained by incubating the starch source described above with one or more amylases, such as an α-amylase, preferably a thermostable α-amylase.

[0041] The DE of the starch hydrolysate may be between 1 and 30, preferably between 5 and 20, and even more preferably between 8 and 15. Generally, starch hydrolysates with a DE of less than 20 are considered maltodextrins.

[0042] Any suitable method described in the art can be used to determine the DE value of a product. Typically, the DE value can be determined according to the Bertrand method (Bulletin de la Societe Chimique de France, 1906, 35 pp. 1285-1299).

[0043] In a preferred embodiment, the starch hydrolysate contains less than 10% by weight of total dietary fiber, preferably less than 9% by weight, less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, even more preferably less than 3% by weight, less than 2% by weight, or no dietary fiber.

[0044] The free maltose content of the starch hydrolysate may be less than 15% by weight, preferably less than 10% by weight, even more preferably less than 5% by weight. Typically, the maltose content can be measured using chromatography, such as high-performance liquid chromatography (HPLC) and high-performance anion-exchange chromatography (HPAEC). A preferred method for determining the amount of free maltose is HPAEC, as detailed in Examples 4-6 below.

[0045] In one embodiment, the viscosity of the composition comprising the starch hydrolysate prior to the branching reaction is less than 5000 cP, more preferably less than 2000 cP at 50% solids at 25° C. Methods for measuring the viscosity of starch hydrolysate compositions are known in the art. Typically, the viscosity can be measured using a laboratory viscometer (AMETEK Brookfield) or a Rapid Visco Analyser (RVA, Perten Instruments).

[0046] Advantageously, in one embodiment of the invention, the composition comprising the starch hydrolysate has a solids content comprised between 30% and 70% by weight of the total composition, preferably between 40% and 60% by weight of the total composition, and even more preferably between 45% and 55% by weight of the total composition.

[0047] High solids content also favors transglucosidase branching reactions over other enzymatic reactions (such as hydrolysis), resulting in more efficient production of branched starch hydrolysates with fewer undesirable by-products.

[0048] This also allows the method of the present invention to be carried out on an industrial scale in order to obtain high production efficiency.In contrast, many of the methods described in the art, especially those using starch as a substrate rather than starch hydrolysate, are only suitable for laboratory scale experiments.

[0049] According to the method of the present invention, the step of incubating the composition comprising the starch hydrolysate with the mixture of maltogenic amylase and transglucosidase enzymes is carried out at a temperature between 40°C and 70°C, preferably between 50°C and 60°C, and even more preferably at about 55°C.

[0050] This enzymatic branching reaction step is carried out for a time sufficient to allow the production of a suitable amount of branched starch hydrolysate. Typically, the step of incubating the composition comprising the starch hydrolysate with the mixture of maltogenic amylase and transglucosidase enzymes is carried out for a period comprised between 1 and 72 hours, preferably between 2 and 24 hours, even more preferably between 4 and 8 hours.

[0051] Typically, the step of incubating the composition comprising the starch hydrolysate with the mixture of maltogenic amylase and transglucosidase enzymes may be carried out at a pH value comprised between 4 and 8, preferably between 4.5 and 7, and even more preferably between 5 and 6.5.

[0052] After the enzymatic treatment step, the reaction is stopped by enzymatic inactivation, such as incubation at 95° C. for 30 minutes.

[0053] The process of the present invention requires simultaneous treatment with maltogenic amylase and transglucosidase enzymes in a predetermined unit ratio.

[0054] As used herein, one unit of maltogenic amylase is defined as the amount of enzyme that produces 1 μmol of maltose per minute using a 5 wt% aqueous solution of waxy maize starch maltodextrin having a DE of approximately 12 (e.g., GLUCIDEX 12C, Roquette) as a substrate under reaction conditions of pH 5.5 and a reaction temperature of 55°C.

[0055] In one embodiment of the invention, the maltogenic amylase is selected from the group consisting of maltogenic α-amylase and maltogenic β-amylase, preferably β-amylase, even more preferably wheat β-amylase.

[0056] Typically, wheat β-amylase can be used at a concentration of 1.8 units per gram of solids in the composition comprising the starch hydrolysate.

[0057] As used herein, one unit of transglucosidase is defined as the enzyme ability to produce 1 μmol of glucose per minute using a 1 wt % aqueous solution of methyl-α-D-glucopyranoside as a substrate under reaction conditions of pH 5.5 and temperature 55° C.

[0058] According to a preferred embodiment of the invention, the transglucosidase is a D-glucosyltransferase (EC 2.3.1.24). In one embodiment, the transglucosidase is selected from the group consisting of Transglucosidase L "Amano" (registered trademark) (commercially available from Amano Enzyme), Transglucosidase L-2000 (registered trademark) (commercially available from DuPont), and Branchzyme (registered trademark) (commercially available from Novozymes).

[0059] By using maltogenic amylase and transglucosidase simultaneously in a predetermined unit ratio, the amount of free maltose produced throughout the reaction can be precisely controlled. The concentration of free maltose can be monitored during and after incubation. Typically, the concentration of free maltose can be less than 40% by weight, preferably less than 30% by weight, and even more preferably less than 20% by weight of the sugar composition. In a preferred embodiment, the concentration of free maltose is less than 12% by weight, preferably less than 11% by weight, less than 10% by weight, less than 9% by weight, less than 8% by weight, and even more preferably less than 7% by weight, less than 6% by weight, or less than 5% by weight of the composition.

[0060] The present invention also relates to a composition comprising a branched starch hydrolysate obtainable by a process as defined above.

[0061] In one aspect, the present invention provides a composition comprising a branched starch hydrolysate, comprising: a) having a DE value of 10 to 50, preferably 12 to 30, more preferably 15 to 25; and / or b) having a percentage of α-1,6 bonds of at least 7%, preferably at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, preferably at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, even more preferably at least 30%, and / or c) comprising at least 30% by weight, preferably at least 35%, preferably at least 40% by weight, even more preferably at least 43% by weight of branches of the branched starch hydrolysate having a DP of 10 or more.

[0062] Typically, the DE value or "dextrose equivalent" value is determined according to the method of Bertrand (Bulletin de la Societe Chimique de France, 1906, 35 pp. 1285-1299).

[0063] As used herein, the expression "percentage of α-1,6 linkages" refers to the degree of branching of a branched starch hydrolysate, which is calculated as the amount of α-1,6 glycosidic linkages divided by the sum of α-1,4 and α-1,6 glycosidic linkages.

[0064] Any suitable method can be used to determine the percentage of α-1,6 linkages. Typically, the amount of α-1,4 and α-1,6 glycosidic linkages is determined using proton nuclear magnetic resonance (NMR) spectroscopy, as described in Example 4 in the "Examples" section below. 1 1 H NMR) can be used to analyze the

[0065] The compositions according to the invention are also characterized by the percentage (by weight) of branches having a degree of polymerization (DP) of at least 10. This DP is calculated based on the total weight of the linear starch hydrolysate, i.e. after the starch hydrolysate has been debranched by enzymatic treatment with isoamylase (which hydrolyzes α-1,6 glycosidic bonds).

[0066] The DP of the branches (or linear molecules) can be calculated by any suitable method in the art. Typically, the DP of linear starch hydrolysates can be estimated from the hydrodynamic radius using the Marc-Houwink equation according to the method of Liuet al. (Macromolecules, 2010, 43, pp. 2855-2864).

[0067] Advantageously, the inventors have demonstrated that compositions comprising branched starch hydrolysates according to the invention are slowly digestible. As used herein, the term "slowly digestible" refers to a branched starch hydrolysate composition that contains a higher fraction of SDS and RS as measured by the Englyst method than a starch hydrolysate composition that has not been subjected to the enzymatic treatment step of the invention.

[0068] Typically, a composition is considered to be "slowly digestible" if the sum of the SDS and RS fractions, measured according to the method of Englyst et al. (European Journal of Clinical Nutrition, 1992, 46, pp. S33-S50), shows an increase of 50%, preferably an increase of 100%, and even more preferably an increase of 150%, relative to the original sum of SDS and RS of the starch hydrolysate before incubation with transglucosidase and maltogenic amylase.

[0069] Alternatively, a composition is considered to be "slowly digestible" if the digestion rate, as measured according to the method of Yu et al. (Food Chemistry, 2018, 264, pp. 284-292), is less than 80%, preferably less than 60%, and even more preferably less than 50% of the digestion rate of the starch hydrolysate before incubation with transglucosidase and maltogenic amylase.

[0070] The methods of Englyst et al. and Yu et al. are carried out as described in Example 1 of the Examples section below.

[0071] In a preferred embodiment, the compositions comprising the branched starch hydrolysates according to the invention are stable against retrogradation.

[0072] The term "stable to retrogradation" refers to a composition that does not undergo (or undergoes to a lesser extent) the reorganization process known as retrogradation, in which the molecules within a gelatinized starch paste reassociate to form a more ordered structure. This stability to retrogradation is reflected by a smaller increase in viscosity after refrigeration and a smaller change in gel clarity after refrigeration, as compared to starch hydrolyzed compositions that have not been subjected to the enzymatic branching treatment step of the present invention.

[0073] In a preferred embodiment, the composition comprising the branched starch hydrolysate according to the invention contains no or only a small amount of dietary fibre.

[0074] A composition is considered to contain "low dietary fiber" if it contains less than 10% by weight of dietary fiber, preferably less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, and even more preferably less than 3%, less than 2%.

[0075] The amount of dietary fiber can be determined by any suitable method known in the art. Typically, a person skilled in the art can perform AOAC method 2009.01 to determine the amount of total dietary fiber.

[0076] The present invention also relates to a kit for preparing a branched starch hydrolysate composition comprising a mixture of maltogenic amylase and transglucosidase enzymes.

[0077] The invention will be understood more clearly upon reading the following examples, which are intended to be purely illustrative and do not in any way limit the scope of protection. The term "maltodextrins" is used loosely in the examples to denote starch hydrolysates, regardless of their DE. EXAMPLES

[0078] Transglucosidase L-2000® (DuPont): Activity was quantified using the method described above and is equal to 1598 U / mL (approximately 1.6 U / μL).

[0079] Transglucosidase L "Amano" (registered trademark) (Amano Enzyme): Activity was quantified using the method described above and is equal to 1077 U / mL (approximately 1.1 U / μL).

[0080] Wheat β-amylase (Roquette): Activity was quantified using the method described above and is equal to 17906 U / mL (approximately 1.8 U / 0.1 μL).

[0081] Example 1: Enzymatic branching of tapioca maltodextrin using Transglucosidase L-2000® (DuPont) preparation Tapioca Maltodextrin Spezyme® Cassava (thermostable α-amylase from DuPont) was added to a 40% tapioca starch slurry (using deionized water) at a concentration of 0.21 or 0.42 μL / g dry starch. The mixture was autoclaved for 5 min and cooled in the autoclave for an additional 30 min. The enzyme was finally inactivated by adjusting the pH of the slurry to 3.0-3.5 using 5% HCl solution. After 30 min, the pH was neutralized back to 6.0 using 1 M and 0.1 M NaOH solutions. The solution was then spray dried to produce maltodextrin powder. The resulting maltodextrin samples were labeled TM21N and TM42N, respectively, and used for further enzymatic branching reactions according to the present invention.

[0082] Branched Tapioca Maltodextrin A 45% w / v tapioca maltodextrin solution containing 5% w / v glycerol was prepared. Glycerol served as an internal standard for sugar composition analysis using HPLC. Maltodextrin was solubilized by constant stirring in a 55°C water bath for 60 min. Transglucosidase L-2000® (DuPont) and wheat β-amylase (Roquette) were added at concentrations of 1 μL (or 1.6U) and 0.1 μL (or 1.8U) per gram of dry maltodextrin, respectively. The mixture was incubated at 55°C. Samples were collected after 4 hours of reaction (labeled TM21H4 and TM42H4 derived from TM21N and TM42N, respectively) and after 24 hours of reaction (labeled TM21H24 and TM42H24 derived from TM21N and TM42N, respectively). The enzyme was inactivated by heating the samples in boiling water for 20 min.

[0083] analysis Dextrose Equivalent The DE values ​​of native and branched tapioca maltodextrins were analyzed and are presented in Table 1.

[0084] [Table 1]

[0085] Tapioca maltodextrin samples produced using Spezyme Cassava at concentrations of 0.21 and 0.42 μL / g dry starch had DE values ​​of 12 and 17, respectively. The enzymatic branching reaction according to the present invention increased the DE values ​​to 16 and 21, respectively.

[0086] Maltose Content The samples were diluted to approximately 1% solids using ultrapure water and then filtered through a 0.45 μm syringe filter before being injected into an HPLC system (Alliance e2695 Separations Module, Waters) equipped with a refractive index (RI) detector (2414, Waters). The injection volume was 100 μL. The column consisted of a precolumn (TSK guard column PWXL, 60 mm inner diameter, 4 cm length) and two LC columns (TSK-GEL G2500PWXL, 7.8 mm inner diameter, 30 cm length) connected in series. The columns were placed in a column oven at 80° C. Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min.

[0087] Table 1 shows that the amount of maltose in the tapioca maltodextrin samples increased after 4 hours of enzymatic branching using transglucosidase and β-amylase, and then decreased after 24 hours of branching. The increase was due to the hydrolysis reaction of β-amylase, which increased the DE value. The decrease in maltose content after 24 hours of branching was due to the reaction of transglucosidase using maltose as a substrate.

[0088] viscosity The maltodextrin solutions were evaporated to approximately 50% solids. The solutions were heated in a boiling water bath and stored in a refrigerator. On the second day, the viscosity of each solution (25 g) was analyzed using an RVA (RVA 4500, Perten Instruments) with the heating profile summarized in Table 2. The solutions were stored in the refrigerator at 4° C. for an additional month and then reanalyzed using the RVA following the same heating profile.

[0089] [Table 2]

[0090] [Table 3]

[0091] The increase in viscosity between 1 day and 1 month refrigeration is more obvious at low temperature. Table 3 shows the viscosity increase between two refrigeration periods (1 day and 1 month) measured at 20, 30, and 40°C. This difference became smaller after the enzymatic branching reaction according to the present invention. In addition, TM21H24 had a similar DE value as TM42N and TM42H4, which was about 16-17, but the viscosity increase of TM21H24 was smaller. The smaller viscosity increase indicated that the enzymatic branching reaction according to the present invention stabilized the tapioca maltodextrin solution against deterioration during refrigeration. The branched tapioca maltodextrin solution was also clearer than the unmodified tapioca maltodextrin solution after refrigeration (data not shown).

[0092] In vitro starch digestibility Two in vitro digestion methods were used to evaluate the digestibility of branched tapioca maltodextrins.

[0093] The first method was based on the method of Englyst et al. (European Journal of Clinical Nutrition, 1992, 46, pp. S33-S50).

[0094] Acetate buffer (0.1 M) was prepared by dissolving 13.6 g of sodium acetate trihydrate in 250 mL of saturated benzoic acid solution, diluting it to 1 L with deionized water, adjusting the pH to 5.2 using 0.1 M acetic acid, and adding 4 mL of 1 M CaCl2 per liter of buffer.

[0095] Enzyme solutions were freshly prepared prior to the experiment. Four 50 mL centrifuge tubes were prepared, each containing 3.0 g porcine pancreatin (P1750, Sigma) mixed with 20 mL water. The mixtures were stirred for 10 min and centrifuged at 1500×g for 10 min. The supernatants (13.5 mL from each tube) were combined with 2.8 mL amyloglucosidase (A7095, Sigma) and 7.95 mL deionized water and mixed.

[0096] Each sample (1.00 g, dry basis) was mixed with 20 mL of 0.1 M acetate buffer and 50 mg of guar gum in a 50 mL tube. A blank was prepared without sample, using 20 mL of 0.1 M acetate buffer and 50 mg of guar gum. Samples and blanks were equilibrated at 37° C. with shaking in a water bath. 5 mL of enzyme solution was added to the samples and blanks, using one tube per minute. Immediately after mixing, the tubes were returned to the 37° C. water bath with shaking for 120 minutes. Aliquots (0.25 mL at 0 min and 0.20 mL at 20 min and 120 min) were transferred from each tube to a 15 mL tube containing 10 mL of 66% v / v ethanol solution and mixed thoroughly. The ethanol solution was centrifuged at 1,500×g to obtain a clear supernatant, and the glucose content in each supernatant (0.1 mL) was analyzed using the D-Glucose Assay Kit GOPOD Format (Megazyme). The weight percentage of glucose released after 0, 20, and 120 min of in vitro digestion was designated as free glucose (FG), G 20 , and G 120 RDS, SDS, and RS were calculated as follows: RDS=(G 20 -FG)×0.9 SDS=(G 120 -G20 )×0.9 RS = 100% - (FG + RDS + SDS) = 100% - (G 120 ×0.9)

[0097] [Table 4]

[0098] In vitro digestibility of branched tapioca maltodextrins measured by the method of Englyst et al. showed that enzymatic branching treatment with transglucosidase and β-amylase according to the present invention reduced the percentage of RDS while resulting in a greater proportion of SDS and RS combined (Table 4).

[0099] The second in vitro digestion method was based on the method of Yu et al. (Food Chemistry, 2018, 264, pp. 284-292) using fitting by non-linear least-squares (NLLS). Briefly, each sample was accurately weighed to 50 mg in a 15 mL centrifuge tube and mixed with 2 mL of deionized water and 8 mL of enzyme solution. The enzyme solution was freshly prepared on the day by mixing 4 mg of pancreatin (P1750, Sigma) and 0.2 mL of amyloglucosidase (E-AMGDF, Megazyme) in 96 mL of 0.2 M sodium acetate buffer (pH 6.0 containing 200 mM calcium chloride, 0.49 mM magnesium chloride, and 0.02% sodium azide). The samples were incubated in a 37 °C water bath with an agitation speed of 300 rpm. Aliquots (0.1 mL) were collected at different time points from 0 to 300 min and each was transferred to a 1.5 mL microcentrifuge tube containing 0.9 mL of absolute ethanol. The ethanol solution was centrifuged at 1,500 × g to obtain a clear supernatant, and the glucose content in each supernatant (0.1 mL) was analyzed using a D-Glucose Assay Kit GOPOD Format (Megazyme). The weight percentage of glucose released during in vitro digestion was converted to the percentage of starch hydrolysis by multiplying by a factor of 0.9. The digestion profiles were fitted using NLLS to obtain the digestion rate and total digestibility. The former was the slope of the digestion curve, and the latter was obtained by extrapolating the digestion time to infinity.

[0100] [Table 5]

[0101] Figure 1 shows the in vitro digestion curves of native and branched tapioca maltodextrins according to the method of Yu et al., and Table 5 summarizes the digestion rates and total digestibility. The branched tapioca maltodextrins prepared using transglucosidase and β-amylase according to the present invention had lower digestion rates and lower total digestibility than their native tapioca maltodextrin counterparts. The digestion rates of the branched tapioca maltodextrins were less than 50% of the digestion rates of their native tapioca maltodextrin counterparts. In addition, the total digestibility of the branched tapioca maltodextrins was less than 80% of their native tapioca maltodextrin counterparts.

[0102] Dietary fiber content The dietary fiber content of native and branched tapioca maltodextrins was analyzed according to AOAC method 2009.01 including HPLC analysis and compared to resistant dextrin (NUTRIOSE FB06, Roquette).

[0103] The results showed that NUTRIOSE FB06, TM42H4, and TM21H24 had 86.65%, 1.65%, and 1.96% dietary fiber, respectively, while the other branched tapioca maltodextrins contained negligible amounts of dietary fiber. Essentially branched tapioca maltodextrins contained low or no dietary fiber, and the RS fraction observed using the Englyst et al. method in Table 4 and the resistant starch observed using the Yu et al. method in Table 5 may be the fraction of starch that is very slow digestible.

[0104] molecular structure The molecular structure (total molecular size distribution and chain length distribution) of the branched tapioca maltodextrin according to the present invention was compared with that of unmodified tapioca maltodextrin and IMOS (IMO50 and IMO90 from Baolingbao, with DE values ​​of 43 and 42, respectively). IMOS is usually produced from starch using transglucosidase. Total molecular size and chain length distribution analysis was performed according to the method of Gu et al. (Food Chemistry, 2019, 295, pp. 484-492). For total molecular size distribution analysis, 2 mg of sample was directly dissolved in 1 mL of DMSO solution containing 5% LiBr. For chain length distribution, 4 mg of sample was first debranched using isoamylase (E-ISAMY, Megazyme) and then dissolved in 1 mL of DMSO solution containing 5% LiBr. The molecules were separated using a series of SEC columns (GRAM pre-column, GRAM 100 and GRAM 1000, PSS) in series on an LC-20AD system (Shimadzu) coupled with a RID-10A refractive index detector (Shimadzu) using a DMSO solution containing 5% LiBr as the eluent. The SEC columns were maintained at 80° C. inside the column oven. The weight percentages of the molecules were estimated based on the area under the curve.

[0105] Figures 2 and 3 show the total molecular size and chain length distribution results (top and bottom, respectively) from native and branched tapioca maltodextrins. Table 6 shows the hydrodynamic radii (R h ), as well as the weight percentage of branches with DP greater than and less than 10. The DP of the branches (or linear molecules) was estimated from the hydrodynamic radius using the Marc-Houwink equation according to the method of Liu et al. (Macromolecules, 2010, 43, pp. 2855-2864).

[0106] [Table 6]

[0107] Both native and branched tapioca maltodextrins have R values ​​greater than 1 nm. h whereas less than 50% of the molecules in IMO50 and IMO90 had an R greater than 1 nm. h These results are consistent with their DE values.

[0108] The branches of native and branched tapioca maltodextrins were also longer than those of IMOS. More than 40% of the branches in native and branched maltodextrins were larger than DP10, while only less than 15% of the branches in IMO50 and IMO90 were larger than DP10. In addition, IMO50 and IMO90 showed only slight differences in molecular size distribution before and after debranching, indicating that the IMOS samples were mostly linear and / or not susceptible to isoamylase, whereas the branched tapioca maltodextrins still contained some branches that were susceptible to isoamylase hydrolysis as the molecules became smaller after debranching.

[0109] Although there is no direct relationship between the hydrodynamic size of a branched molecule and its molecular weight, it can be assumed that the majority of small molecules (DP<10) in the overall molecular size distribution are linear, and therefore the Rh at 1 nm was close to DP 10. Therefore, the enzymatic branching reaction according to the present invention did not reduce the overall molecular size of tapioca maltodextrin to Rh<1 nm or DP<10, and the product was not the same as IMOS.

[0110] conclusion The enzymatic branching reaction using transglucosidase and β-amylase according to the present invention increased the DE value of tapioca maltodextrin by about 3 after 24 hours of reaction time, which may be attributed to the increased amount of oligosaccharides. The branched tapioca maltodextrin was more stable against deterioration during refrigeration, as indicated by a smaller increase in solution viscosity and opacity after long-term refrigeration. In addition, the branched tapioca maltodextrin showed higher amounts of SDS and RS when analyzed using the method of Englyst et al., and lower digestion rate and total digestibility when analyzed using the method of Yu et al. However, the branched tapioca maltodextrin essentially contained low or no dietary fiber (less than 2%). The branched tapioca maltodextrin still maintained some branches that were susceptible to isoamylase hydrolysis, and most of the molecules were larger than DP10. Thus, branched tapioca maltodextrins are distinct from IMOS, which are also produced using transglucosidase.

[0111] Example 2: Enzymatic branching of waxy maize maltodextrin using Transglucosidase L-2000® (DuPont) preparation Branched Waxy Maize Maltodextrin A 45% w / v waxy maize maltodextrin solution (prepared in deionized water) containing 5% w / v glycerol was prepared using Glucidex 8C (Roquette). Glycerol served as an internal standard for sugar composition analysis using HPLC. Maltodextrin was solubilized by constant stirring in a 55°C water bath for 60 minutes. Transglucosidase L-2000® (DuPont) and wheat β-amylase (Roquette) were added at concentrations of 1 μL (or 1.6U) and 0.1 μL (or 1.8U) per gram of dry maltodextrin, respectively. The mixture was incubated at 55°C. Samples were collected after 1, 2, 4, and 24 hours of reaction (labeled G8CH1, G8CH2, G8CH4, and G8CH24, respectively, and native Glucidex 8C was labeled G8CN). The enzyme was inactivated by heating the samples in boiling water for 20 min.

[0112] analysis Dextrose Equivalent The DE values ​​of native and branched waxy maize maltodextrins were analyzed and are presented in Table 7.

[0113] [Table 7]

[0114] The DE value did not show any obvious change after the enzymatic branching reaction using the transglucosidase and β-amylase according to the present invention.

[0115] Maltose Content The same HPLC method as in Example 1 was used to analyze the maltose content of the native and branched waxy maize maltodextrins.

[0116] Table 7 shows the amount of maltose in the waxy maize maltodextrin samples that increased after enzymatic branching reactions using transglucosidase and β-amylase. This was due to the hydrolysis reaction of β-amylase, but the increase in maltose content did not show a clear increase in the DE value of the waxy maize maltodextrin.

[0117] viscosity Using the same RVA method as in Example 1, the native and branched waxy maize maltodextrins were analyzed for viscosity after refrigeration.

[0118] [Table 8]

[0119] The increase in viscosity between 1 day and 1 month refrigeration became smaller with increasing time of the enzymatic branching reaction of the present invention (Table 8). The smaller viscosity increase indicated that the enzymatic branching reaction stabilized the waxy maize maltodextrin solution against deterioration during refrigeration. The branched waxy maize maltodextrin solutions were also clearer than the unmodified waxy maize maltodextrin solutions after refrigeration (data not shown).

[0120] In vitro starch digestibility The in vitro digestibility of native and branched waxy maize maltodextrins was analyzed using the method of Englyst et al. as described in Example 1.

[0121] [Table 9]

[0122] In Table 9, in vitro digestibility studies of native and branched waxy maize maltodextrins showed that enzymatic branching treatment with transglucosidase and β-amylase according to the present invention increased the percentage of total SDS and RS, while decreasing the total FG and RDS with increasing time of the enzymatic branching reaction.

[0123] conclusion DE values ​​showed no obvious change after the enzymatic branching reaction using transglucosidase and β-amylase according to the present invention, but the amount of maltose in the waxy maize maltodextrin samples increased. The branched waxy maize maltodextrins were more stable against deterioration during refrigeration, as indicated by a smaller increase in solution viscosity and opacity after extended refrigeration storage. In vitro digestibility of branched waxy maize maltodextrins showed that the enzymatic branching treatment with transglucosidase and β-amylase according to the present invention increased the percentage of the sum of SDS and RS, while decreasing the sum of FG and RDS with increasing duration of the enzymatic branching reaction time.

[0124] Example 3: Enzymatic branching of maltodextrin using transglucosidase L "Amano" (registered trademark) (Amano Enzyme) preparation Branched Maltodextrin Maltodextrin solutions (prepared with decarbonated water) with solids concentrations of 57%-67% were prepared using tapioca maltodextrin TM42N as described in Example 1 and Glucidex 8C (Roquette) as described in Example 2. Maltodextrin was solubilized by constant stirring for 30 minutes in a 55°C water bath. Transglucosidase L "Amano" (registered trademark) (from Amano Enzyme) and wheat β-amylase (Roquette) were added at concentrations of 1 μL (or 1.1 U) and 0.1 μL (or 1.8 U) per gram of dry maltodextrin, respectively. The mixture was incubated at 55°C for 6 hours. The enzymes were inactivated by heating the samples in boiling water for 20 minutes. Branched tapioca maltodextrins prepared at solids contents of 57% and 67% were labeled TM42S57 and TM42S67, respectively. On the other hand, branched waxy maize maltodextrins prepared at 57% and 62% were labeled as G8CS57 and G8CS62, respectively.

[0125] analysis Solids and Dextrose Equivalent The solids content of the maltodextrin solution prepared for the enzymatic branching reaction according to the present invention was analyzed based on the weight difference before and after drying overnight in an oven at 110°C.

[0126] The DE values ​​of the resulting branched maltodextrins were analyzed and are presented in Table 10.

[0127] [Table 10]

[0128] The DE values ​​of branched maltodextrins increased after the enzymatic branching reaction using transglucosidase and β-amylase according to the present invention (Table 10). However, the DE values ​​of branched maltodextrins prepared at >60% solids were lower than their counterparts prepared at <60% solids.

[0129] Maltose Content The maltose content of native and branched maltodextrins was analyzed using the same HPLC method as in Example 1. Each sample solution was diluted 50 times using 0.2% glycerol solution, and then filtered through a 0.45 μm membrane filter before being injected into the HPLC system.

[0130] Table 10 shows that the amount of maltose in the branched maltodextrin samples was higher than their native maltodextrin counterparts. This was due to the hydrolysis reaction of β-amylase, which increased the DE value. For waxy maize maltodextrins, the amount of maltose was higher in G8CS57 than in G8CS62. β-amylase appeared to be less effective at higher solids, possibly due to higher viscosity.

[0131] In vitro starch digestibility The in vitro digestibility of native and branched maltodextrins was analyzed using the method of Englyst et al. as described in Example 1.

[0132] [Table 11]

[0133] In Table 11, the in vitro digestibility test of native and branched maltodextrins showed that the enzymatic branching reaction using transglucosidase and β-amylase according to the present invention reduced the amount of RDS while resulting in an increase in the total percentage of SDS and RS. TM42S57 had a higher total percentage of SDS and RS than TM42S67. Furthermore, the change in in vitro digestibility of waxy maize maltodextrin after the enzymatic branching reaction was less than that of tapioca maltodextrin. This may be due to the higher viscosity of waxy maize maltodextrin, which has a lower DE value than tapioca maltodextrin. At higher viscosities, such as lower DE or higher solids, enzymes have lower mobility and are therefore less effective for the branching reaction.

[0134] conclusion The DE values ​​of tapioca and waxy maize maltodextrins increased after the enzymatic branching reaction using the transglucosidase and β-amylase according to the present invention, which may be attributed to the increase in small sugars. The in vitro digestibility results showed that the enzymatic branching reaction using the transglucosidase and β-amylase according to the present invention increased the total SDS and RS, while decreasing the amount of RDS.

[0135] Regarding the effect of maltodextrin concentration on the enzymatic branching reaction, since hydrolysis requires water molecules, a higher concentration of solids should favor the branching (or condensation) reaction of transglucosidase over its hydrolysis reaction. However, there was no clear trend observed with solids content. This may be due to the viscosity of the maltodextrin solution. Higher viscosity reduces the efficiency of the enzyme, since the mobility is reduced. In addition, the change was less evident for waxy maize maltodextrin than for tapioca maltodextrin, which may be due to the higher viscosity of waxy maize maltodextrin, which has a lower DE value than tapioca maltodextrin.

[0136] Example 4: Enzymatic branching of tapioca and pea maltodextrins using transglucosidase L "Amano" (registered trademark) (Amano Enzyme) preparation Tapioca and pea maltodextrin Liquozyme Supra (a thermostable α-amylase from Novozymes) was used to liquefy two types of starch to produce maltodextrins. Tapioca and pea starches were used to prepare 35% and 30% starch slurries with demineralized water (pH 6.0 and 5.5), and the enzyme was added to the slurries at 3.0 and 3.5 μL / g dry starch, respectively. Each mixture was heated in a custom-built laboratory cooker at 110° C. with an average flow rate of about 35 mL / min, which resulted in a liquefaction time of about 17 minutes. The enzyme was then inactivated at 140° C., and the resulting maltodextrin solutions were cooled to room temperature. The tapioca maltodextrin (TM18) had a DE of 18 and the pea maltodextrin (PM19) had a DE of 19. Both maltodextrin solutions had a pH of about 5.5 and were evaporated using a rotary evaporator (Rotavapor R-300, Buchi) to obtain a solid content of >50% and used for further enzymatic branching reactions according to the present invention. The maltodextrins were stored in a refrigerator to prevent microbial growth.

[0137] Branched Maltodextrin The solids content of each maltodextrin solution (pH approx. 5.5) was adjusted and heated at 90°C for 30 minutes in a hot water jacketed beaker. The beaker was covered with aluminum foil to avoid excessive water loss by evaporation. After the maltodextrin solution was cooled to 55°C, it was incubated with Transglucosidase L "Amano" (registered trademark) (Amano Enzyme) and wheat β-amylase (Roquette) at 55°C. The reaction conditions are summarized in Table 12. The resulting branched maltodextrins were heated back to 90°C for 30 minutes to inactivate the enzymes.

[0138] [Table 12]

[0139] analysis Dextrose equivalent and degree of branching The DE values ​​of native and branched maltodextrins were analyzed according to the method of Bertrand (Bulletin de la Societe Chimique de France, 1906, 35 pp. 1285-1299).

[0140] The amount of α-1,4 and α-1,6 glycosidic linkages in native and branched maltodextrins was determined using a 5 mm NMR tube equipped with an Avance III Fourier transform spectrometer (Bruker Spectrospin) operating at 400 MHz and 60 °C. 1 The H NMR techniques were used to analyze the spectra. The procedure is as follows: Each sample (10 mg) was dissolved in 0.75 mL of DO (minimum 99%, Eurisotop) in a water bath. After cooling to room temperature, 50 μL of a solution (10 mg / g) of the sodium salt of 3-trimethylsilyl-1-propanesulfonic acid (Aldrich) was added to each sample. Acquisition was performed without spinning, without solvent suppression, and with a relaxation time of at least 10 seconds. Spectra were collected after Fourier transformation, phase correction, and subtraction of the baseline in manual mode (without exponential multiplication, LB=GB=0).

[0141] The integration of the signals (see FIG. 4 and Table 13) was performed as follows: the S5 signal was normalized to 600. This signal corresponded to the non-exchangeable protons of the anhydroglucose units (H2, H3, H4, H5, and 2H6). Signal S1 corresponded to H1 in α-1,4 bond, signal S2 corresponded to H1 in the reducing end in α configuration, and signal S3 corresponded to H1 in α-1,6 bond. The S4 signal, which corresponds to the reducing end with β configuration, was estimated by multiplying S2 by 1.5. The S6 signal was estimated by the subtraction method S6=100-(S1+S2+S3+S4).

[0142] [Table 13]

[0143] The proportions of α-1,4 and α-1,6 linkages were determined from the surface areas of the S1 and S3 signals and expressed as percentages by normalizing the sum of the two linkages to 100. Thus, the degree of branching was calculated as the amount of α-1,6 glycosidic linkages divided by the sum of α-1,4 and α-1,6 glycosidic linkages.

[0144] [Table 14]

[0145] In Table 14, the DE values ​​of TM(22:1), TM(221:1), and TM(1:10) had high DE values ​​over 30, but the first two samples were only incubated for 3 hours, while TM(1:10) was incubated for 24 hours. This may be explained by the high ratio of β-amylase to substrate used to produce TM(22:1) and TM(221:1), which may release a large amount of maltose and increase the number of reducing sugars in the solution. The DE values ​​of TM(1:1) and PM(1:1) were lower than TM(1:10), which was probably due to the shorter reaction time of TM(1:1) and PM(1:1), resulting in a lower degree of hydrolysis.

[0146] Table 14 also summarizes the degree of branching of unmodified and branched maltodextrins. Only high concentrations of β-amylase increased the degree of branching of TM(22:1) and TM(221:1) from 5% to 8% in unmodified tapioca maltodextrin. When low concentrations of β-amylase were used, the degree of branching of tapioca maltodextrin increased to 39%, but the DE value was lower than that of TM(221:1). The results show that lower amounts of β-amylase with higher amounts of transglucosidase favor the branching reaction, whereas the opposite favors the hydrolysis reaction.

[0147] In vitro starch digestibility The in vitro digestibility of native and branched maltodextrins was analyzed using the method of Englyst et al. as described in Example 1.

[0148] [Table 15]

[0149] Table 15 summarizes the in vitro digestibility results of native and branched maltodextrins. TM(22:1) and TM(221:1) had higher RDS content than native tapioca starch (TM18), suggesting that the high ratio of β-amylase to substrate resulted in smaller molecules that were more rapidly digestible than those in native tapioca maltodextrin (TM18). Meanwhile, TM(1:10) contained the lowest RDS of all treatments. Meanwhile, TM(1:1) and PM(1:1) showed a slight decrease in RDS content compared to their native maltodextrin counterparts. As in other examples, RDS content was negatively correlated with the sum of SDS and RS. The RDS content was also negatively correlated with the degree of branching (Figure 5A), suggesting that the slow digestibility property of branched maltodextrins is due to the higher amount of α-1,6 glycosidic bonds, which cannot be hydrolyzed by pancreatic α-amylase and may result in steric hindrance for the branched maltodextrin substrate to bind with pancreatic α-amylase.

[0150] Free sugar profile The released sugar profile was analyzed using a HPAEC system (Dionex™ ICS-5000, Thermo Scientific™) equipped with pulsed amperometric detection (PAD). The column used for the analysis was CarboPac™ PA1 (4 x 250 mm), preceded by a guard column CarboPac™ PA1 (4 x 50 mm). Each sample was accurately weighed, mixed with 1.0 mL of internal standard (melibiose) and diluted with 20 mL of distilled water. The mixture was stirred for 10 min and filtered through a 0.45 μm membrane. The injection volume was 5 μL. Samples were eluted at 30 °C in a gradient mode of NaOH 100 mM (A) and NaOH 100 mM + CH3COONa 500 mM (B) programmed as follows: 2% B at 0.0 min, 5% B at 60.0 min, 30% B at 65.0 min, 100% B at 65.05 min, 100% B at 75.0 min, 2% B at 75.05 min, and 2% B at 90.0 min.

[0151] [Table 16]

[0152] The free sugar profiles of native and branched maltodextrins are summarized in Table 16. Maltodextrins usually have higher amounts of maltose and maltotriose than glucose, because α-amylase mainly hydrolyzes starch to maltose and maltotriose, not glucose. TM(22:1) and TM(221:1) had the highest amount of maltose, due to the reaction of β-amylase, which hydrolyzes starch from the non-reducing end 0, and each successful hydrolysis releases maltose. This is consistent with their high DE values ​​(Table 14). Meanwhile, the maltose content of TM(1:1) and PM(1:1) was similar to or slightly lower than that of their native maltodextrin counterparts. In addition, TM(1:10) had the lowest amount of maltose. These results confirm that the maltose produced by β-amylase was mostly used by transglucosidase for the branching reaction. The branching reaction of transglucosidase also released glucose, increasing the glucose content in these samples along with isomaltose, another product of transglucosidase.

[0153] conclusion The DE values ​​of tapioca and pea maltodextrins increased after enzymatic branching reactions using transglucosidase and β-amylase, which may be attributed to the increase in small sugars. The high ratio of β-amylase to maltodextrin substrate and the high enzyme unit ratio of β-amylase to transglucosidase favored the hydrolysis of maltodextrin from the non-reducing end, releasing a large amount of maltose. As a result, the DE values ​​increased rapidly in a short time, but the degree of branching only increased slightly. The products of β-amylase hydrolysis appeared to be more rapidly digestible than the molecules in unmodified maltodextrin.

[0154] On the other hand, according to the present invention, a low ratio of β-amylase to maltodextrin substrate combined with a low ratio of enzyme activity units of β-amylase to transglucosidase favored the branching reaction of maltodextrin, as indicated by an increased amount of α-1,6 glycosidic bonds, which cannot be hydrolyzed by pancreatic α-amylase and may result in steric hindrance for the branched maltodextrin substrate to bind with pancreatic α-amylase, resulting in a lower digestion rate or a lower amount of RDS, as shown by the test results obtained using the method of Englyst et al.

[0155] Example 5: Comparison of two transglucosidases for the enzymatic branching modification of tapioca maltodextrin preparation Tapioca Maltodextrin Tapioca maltodextrin (TM17) was prepared similarly to Example 4. Tapioca starch slurry (35% solids in demineralized water, pH 6.0) was liquefied using Liquozyme Supra (Novozymes, 2.9 μL / g dry starch) in a custom-made laboratory cooker at 110° C. and an average flow rate of about 35 mL / min, resulting in a liquefaction time of about 17 min. The enzyme was then inactivated at 140° C. and the resulting maltodextrin solution was cooled to room temperature. The tapioca maltodextrin had a DE of 17 and pH of 5.6 and was then evaporated using a rotary evaporator (Rotavapor R-300, Buchi) to obtain a solids content of >50% and used for further enzymatic branching reactions according to the invention. The maltodextrin was stored in a refrigerator to prevent microbial growth.

[0156] Branched Maltodextrin The solids content of the maltodextrin solution (pH about 5.5) was adjusted to about 47% and heated at 90°C for 30 minutes in a hot water jacketed beaker. The beaker was covered with aluminum foil to avoid excessive water loss by evaporation. The maltodextrin solution was cooled to 55°C and then incubated with two enzyme systems. The first system used Transglucosidase L-2000® from DuPont and the second system used Transglucosidase L "Amano"® from Amano Enzyme. The reactions were carried out at 55°C for 6 or 24 hours and are summarized in Table 17. The resulting branched maltodextrins were heated back to 90°C for 30 minutes to inactivate the enzymes.

[0157] [Table 17]

[0158] analysis Dextrose equivalent and degree of branching The DE values ​​of native and branched tapioca maltodextrins were analyzed according to the Bertrand method described in Example 4. The amounts of α-1,4 and α-1,6 glycosidic bonds in native and branched tapioca maltodextrins were analyzed according to the Bertrand method described in Example 4. 1 It was analyzed by 1 H NMR technique.

[0159] [Table 18]

[0160] Table 18 shows that both the DE value and branching degree of tapioca maltodextrin increased with reaction time, indicating that the enzymatic branching reaction occurred for longer than 6 hours. The increase in DE value was due to the production of maltose by β-amylase, which was then used by transglucosidase for the branching reaction, releasing glucose as a by-product. At the same reaction time, both enzyme systems produced comparable DE values ​​and branching degrees, although transglucosidase L-2000® (DuPont) showed slightly higher DE values ​​and branching degrees. These results suggest that both enzyme systems were effective in branching tapioca maltodextrin.

[0161] In vitro starch digestibility The in vitro digestibility of native and branched tapioca maltodextrins was analyzed using the method of Englyst et al. as described in Example 1.

[0162] [Table 19]

[0163] Table 19 summarizes the in vitro digestibility results of native and branched tapioca maltodextrins. The amount of RDS decreased with reaction time, while the amount of FG and the sum of SDS and RS increased, indicating that the branching reaction reduced the digestibility of maltodextrins. In fact, there is a strong negative correlation between the degree of branching and the RDS content (Figure 5B).

[0164] At the same reaction time, the two enzyme systems produced branched maltodextrins with comparable in vitro digestion profiles, as well as DE and degree of branching (Table 18), indicating that both transglucosidases were effective in branching tapioca maltodextrin and slowing its digestion rate.

[0165] Free sugar profile The free sugar profiles of native and branched tapioca maltodextrins were analyzed as described in Example 4.

[0166] [Table 20]

[0167] The free sugar profiles of native and branched tapioca maltodextrins are summarized in Table 20. Glucose and isomaltose contents increased with incubation time as they are the products of transglucosidase. Maltose, maltotriose, and panose contents decreased between 6 and 24 hours of incubation, indicating that transglucosidase can use these molecules as its substrates. In addition, the content of isomaltose was still below 7% in the branched tapioca maltodextrin samples after 24 hours of incubation, and thus isomaltose was not the main molecule responsible for the slow digestion characteristics. Overall, the amount of these small sugars remained low after 24 hours of incubation, which distinguishes the present invention from IMOS.

[0168] conclusion Tapioca maltodextrin was branched using either Tranglucosidase L-2000® from DuPont or Transglucosidase L “Amano”® from Amano Enzyme in combination with wheat β-amylase. The reaction was carried out for 6 or 24 hours. The DE value, degree of branching, amount of glucose, and sum of SDS and RS of tapioca maltodextrin increased with increasing reaction time, while the amount of RDS decreased. The amounts of glucose, maltose, isomaltose, maltotriose, and panose remained low after 24 hours of incubation. The results showed that increasing the degree of branching decreased the digestion rate of maltodextrin. The increase in DE value was due to the release of maltose by β-amylase, which was then used by transglucosidase for the branching reaction, releasing glucose as a by-product. Also, transglucosidase appears to be able to use maltotriose and panose as its substrates. At the same reaction time, both enzyme systems produced branched tapioca maltodextrins with comparable DE values, branching degrees, and digestion profiles, indicating that both enzyme systems were effective in branching tapioca maltodextrins and slowing their digestion rates.

Claims

1. 1. A method for preparing a composition comprising a branched starch hydrolysate, the method comprising the step of incubating the composition comprising a starch hydrolysate with a mixture of maltogenic amylase and transglucosidase enzymes in an enzyme activity unit ratio of 9:5 to 1:

20.

2. 2. The method of claim 1, wherein the composition comprising a starch hydrolysate is obtained from a starch selected from tapioca, waxy tapioca, maize, waxy maize, rice, glutinous rice, wheat, waxy wheat, barley, waxy barley, sorghum, waxy sorghum, potato, waxy potato, sweet potato, waxy sweet potato, sago, millet, mung bean, pea, fava bean, chickpea, arrowroot, buckwheat, quinoa, or lotus root.

3. The method according to claim 1 or 2, wherein the composition comprising the starch hydrolysate is obtained by incubating a starch source with one or more amylases.

4. The method according to any one of claims 1 to 3, wherein the DE of the starch hydrolysate is from 1 to 30.

5. 5. The method according to any one of claims 1 to 4, wherein the starch hydrolysate has a free maltose content of less than 15% by weight.

6. The method of any one of claims 1 to 5, wherein the viscosity of the composition comprising the starch hydrolysate prior to incubation is less than 5000 cP at 50% solids at 25°C.

7. The method of any one of claims 1 to 6, wherein the maltogenic amylase is selected from the group consisting of maltogenic α-amylases and maltogenic β-amylases.

8. The composition comprising the starch hydrolysate is treated with a maltogenic amylase and a transglucosidase. The method according to any one of claims 1 to 7, wherein the step of incubating with the mixture of cosidase enzymes is carried out at a temperature comprised between 40°C and 70°C.

9. 9. The method according to any one of claims 1 to 8, wherein the step of incubating the composition comprising the starch hydrolysate with a mixture of maltogenic amylase and transglucosidase enzymes is carried out for a period comprised between 1 and 72 hours.

10. A method according to any one of claims 1 to 9, wherein the composition comprising starch hydrolysate has a solids content comprised between 30% and 70% by weight of the total composition.

11. 11. The method according to any one of claims 1 to 10, wherein the step of incubating the composition comprising the starch hydrolysate with a mixture of maltogenic amylase and transglucosidase enzymes is carried out at a pH value comprised between 4 and 8.

12. The method according to any one of claims 1 to 11, wherein the transglucosidase is a D-glucosyltransferase.

13. The method according to any one of claims 1 to 12, wherein the concentration of free maltose is monitored during incubation and is less than 12% by weight of the composition comprising the starch hydrolysate.

14. A composition comprising a branched starch hydrolysate, a) has a DE value of 10 to 50; b) having a percentage of α-1,6 bonds of at least 7%; c) A composition comprising at least 30% by weight of branches of said branched starch hydrolysate having a DP of 10 or more.

15. 15. A food product comprising the composition of claim 14, the food product being a diabetic meal or meal replacement, energy gel, solid drink, or sports drink.

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