Porous starch

Abstract

The present invention relates to improving the fluidity and compressibility (tensile strength) of starch, increasing the porosity of starch particles by the use of enzymes, and using porous starch particles for direct tableting of tablets.

Description

[Background technology]

[0001] Starch has a long history of use as an excipient in pharmaceutical dosage forms. Natural starches, such as corn starch, are white or grayish-white, odorless, and tasteless, and are one of the most widely used fillers / binders in tablet manufacturing.

[0002] This biomaterial possesses unique physicochemical and functional properties, as well as various advantages such as low cost, relatively easy isolation in its pure form from plant sources, non-toxicity, biodegradability, good biocompatibility, non-hygroscopicity, inertness (no pharmacological activity), and no interaction with living cells.

[0003] Natural starches are used in tablet formulations as fillers / diluents, binders, and disintegrants. They have only acceptable compressibility, very poor fluidity, and exhibit elastic recovery that results in tablet capping and lamination. Therefore, starches are typically not used in direct tableting, which is the preferred route for efficient and cost-effective tablet production. Wet granulation processes are required for starch-based formulations, especially when using active ingredients with high or low compressibility. Various physically and chemically modified starches have been proposed as direct tableting excipients for tablet formulations (Lawal, MV: Modified Starches as Direct Compression Excipients - Effect of Physical and Chemical Modifications on Tablet Properties: A Review. Starch-Staerke 2019, 71 DOI OI:10.1002 / star.201800040). International Publication No. 2021195216 discloses inhibitory porous granular starch products, including hydrolysis of granular starch feed using one or more enzymes. Lacerda et al. describe the synthesis of porous rice starch and the dependence of its physical properties on enzymatic hydrolysis conditions. (Liziane D. Lacerda, Daiani C. Leite, Nadya P. da Silveira, Journal of Cereal Science, Volume 89, 2019, 102819.) International Publication No. 2013017388-A1 describes the encapsulation of anionic or cationic insecticides with porous starch granules.

[0004] These products typically exhibit improved tabletability compared to natural starches. One such modified starch is STARTAB® (Colorcon), which has high fluidity and compressibility in a direct-compression grade. In cases of high-dose formulations and difficulties in formulating active ingredients, additional fillers / drying binders are typically added for optimal formulation performance. Another modified starch is N-Zorbit, produced by partial hydrolysis, designed as a plating agent with high absorption capacity for liquid active ingredients. N-Zorbit has a porous, granular structure and therefore possesses the ability to carry high fragrance concentrations.

[0005] Another way to enhance the tableting performance of natural starch is to combine it with other excipients in so-called "coprocess" excipient formulations. Such coprocess excipients combine individual excipients in physical form without significant chemical changes to achieve synergistic functional properties. An example of a starch-based excipient is StarLac® (Meggle, Germany), which contains 85% α-lactose monohydrate and 15% natural corn starch. This excipient combines lactose (a typical filler for direct tableting) with starch that provides binding and disintegration properties. Such coprocess excipients exhibit tableting performance that cannot be achieved by simple physical mixtures of components. [Overview of the project] [Means for solving the problem]

[0006] The present invention relates to porous starch particles characterized by nitrogen sorption, determination of physicochemical parameters such as particle size, and determination of Δp, which is the permeability of the filled starch material. At least one parameter must be satisfied by enzyme-treated starch. Preferably, a combination of two parameters that must be satisfied by enzyme-treated starch is preferred, and more preferably, a combination of all three parameters is preferred.

[0007] The present invention further relates to the use of enzymatically hydrolyzed porous starch particles having high interparticle porosity, large specific surface area, improved fluidity, and as a result, improved tableting performance.

[0008] Powder fluidity is improved by a spray drying or spray agglomeration process. The individual particles are modified by partial enzymatic hydrolysis and thus exhibit remarkable tableting properties compared to starches known in the prior art. Even though tablets made of this porous starch have surprisingly high tensile strength, the disintegration time remains small as usual for starches. Due to this property, it is possible to formulate fast-disintegrating tablets that enable the rapid onset of action of the active ingredient without adding a disintegrant.

[0009] Pharmaceutically dosage forms include tablets, pellets, mini-tablets, lozenges and other comprimes. <s

[0010] Typically, pharmaceutical dosage forms consist of, in addition to an active pharmaceutical active ingredient (API), pharmaceutical excipients: fillers, binders, disintegrants, and lubricants. The porous starch according to the present invention exhibits the properties of a filler, a binder and a disintegrant, making simple starch-based tablet formulations accessible by direct tableting. The number of excipients in the formulation can be reduced, the direct tableting route is cost-effective and suitable for integration into a continuous manufacturing line, resulting in a very simple, directly tableted pharmaceutical dosage form. The directly tableted pharmaceutical dosage form can be manufactured without a granulation step and contains no or only a small amount of additional fillers, binders, lubricants, and disintegrants.

[0011] In a further embodiment, the present invention relates to the use of porous starch according to the present invention for the manufacture of tablets, pellets, and minitablet capsule fills. Porous starch is suitable as a liquid, as a solution, or as a carrier of active ingredients that can be impregnated during the melting stage. Starch is currently used in pharmaceutical preparations as a binder, disintegrant, and film-forming material. Due to its low fluidity and compressibility, starch is not suitable for direct tableting.

[0012] The present invention relates to improving tablet properties, including the fluidity and compressibility (tensile strength of the tablet), of starch, increasing the porosity of starch particles by using an enzyme, preferably amylase, more preferably α-amylase, and improving fluidity by spray drying and agglomeration of the particles.

[0013] Enzymatic hydrolysis of porous starch Enzymatically hydrolyzed porous starch is granular starch hydrolyzed by one or more amylose-degrading enzymes. It can be produced by enzymatic hydrolysis of natural starch granules, which are not enzymatically or chemically processed, using one or more amylotic enzymes such as α-amylase and amyloglucosidase at a temperature lower than the gelatinization temperature of starch. Enzymatically hydrolyzed porous starch can be produced by the following steps: a) Hydrolysis of starch by one or more amyllyatic enzymes, preferably amylases. b) Separation of enzymatically hydrolyzed porous starch after hydrolysis, preferably by filtration. c) Optionally wash the separated enzymatically hydrolyzed porous starch with water, preferably deionized water. d) The enzymatically hydrolyzed porous starch is dried, preferably by spray drying or freeze-drying, more preferably by spray drying.

[0014] In one embodiment, the porous starch particles obtained by enzymatic hydrolysis are 2 excess ​<10 Relative excess specific surface area (S excess ) has. In a more preferred embodiment, the enzymatically hydrolyzed porous starch particles are 2.5 excess <7, more preferably 2.9 excess Relative excess specific surface area (S) <6.1 excess ) has.

[0015] In another embodiment, the enzymatically hydrolyzed porous starch particles have a polar interaction component (δP2 < 8.8) determined by inverse gas chromatography. 2) It has. In a more preferred embodiment, the enzymatically hydrolyzed porous starch particles have a polar interaction component (δP2 < 8.70, more preferably δP2 < 8.66) determined by inverse gas chromatography. 2) It holds.

[0016] In another embodiment, the enzymatically hydrolyzed porous starch particles have a pressure difference (Δp) of 200 mbar < Δp < 800 mbar across both ends of a GC column packed with a starch sample and purged with helium at a flow rate of 15 mL / min. In a more preferred embodiment, the enzymatically hydrolyzed porous starch particles have a pressure difference (Δp) of 200 mbar < Δp < 700 mbar, more preferably 235 mbar < Δp < 667 mbar across both ends of a GC column packed with a starch sample and purged with helium at a flow rate of 15 mL / min.

[0017] Natural starch granules can be derived from tapioca, waxy, maize, peas, potatoes, glutinous potatoes, wheat, glutinous wheat, glutinous corn, mung mean, ice, glutinous rice, sweet potatoes, glutinous sweet potatoes, millet, sago, sorghum, quinoa, arrowroot, amaranth, lotus root, and buckwheat.

[0018] ​​Typically, starches for pharmaceutical use are derived from corn, rice, wheat, potatoes, millet, barley, peas, and tapioca. Preferably, natural starches are derived from corn.

[0019] amylase The "amylase" (alpha and / or beta) according to the present invention may be of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of alpha-amylases (EC 3.2.1.1). This includes chemically modified mutants or protein-engineered mutants.

[0020] The amylase according to the present invention has "amylose-degrading activity" or "amylase activity" which includes (endo)hydrolysis of glucosidic bonds in polysaccharides. Alpha-amylase activity can be determined by assays for measuring alpha-amylase activity that are known to those skilled in the art. An example of an assay for measuring alpha-amylase activity is:

[0021] Alpha-amylase activity can be determined by a method utilizing Phadebas tablets as a substrate (Phadebas Amylase Test, supplied by Magle Life Science). Starch is hydrolyzed by alpha-amylase, yielding soluble blue fragments. The absorbance of the resulting blue solution, measured by spectrophotometry at 620 nm, is a function of alpha-amylase activity. The measured absorbance is directly proportional to the specific activity (activity / mg of pure alpha-amylase protein) of the alpha-amylase in question under a given set of conditions.

[0022] Alpha-amylase activity can also be determined by a method utilizing ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside (EPS). D-maltoheptaoside is a block oligosaccharide that can be cleaved by endo-amylase. After cleavage, the alpha-glucosidase contained in the kit digests the substrate, releasing free PNP molecules with a yellow color, which can then be measured by visible spectrophotography at 405 nm. The kit containing the EPS substrate and alpha-glucosidase is manufactured by Roche Costum Biotech (catalog number 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per 1 mg of enzyme) of the alpha-amylase under a given set of conditions.

[0023] Amylose-degrading activity can be provided in units per gram of enzyme. For example, one unit of α-amylase can release 1.0 mg of maltose from starch in 3 minutes at pH 6.9 and 20°C.

[0024] Preferred amylases are Bacillus licheniformis having Sequence ID No. 2 as described in International Publication No. 95 / 10603 and at least 95% variants thereof. Preferred variants are described in International Publication No. 95 / 10603 and include one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, and have amylose-degrading activity. The variant is described as Sequence ID No. 4 in International Publication No. 94 / 02597, International Publication No. 94 / 018314, International Publication No. 97 / 043424, and International Publication No. 99 / 019467. An exemplary sequence is added herein as Sequence ID No. 1.

[0025] Other preferred amylases are derived from Aspergillus oryzae, and are described, for example, in Brzozowski et al. Biochemistry 1997. Preferred variants are described, for example, in U.S. Patent Application Publication No. 20110159545. A preferred variant is included as Sequence ID No. 2.

[0026] The amylase may further be derived from B. stearothermophilus having SEQ ID NO. 6 as disclosed in International Publication No. 02 / 10355, or may be an amylase optionally having C-terminal shortening beyond the wild-type sequence. Preferred variants of SEQ ID NO. 6 include deletions at 179 and / or 181 and / or 182 and / or substitution at position 193.

[0027] TVB146 is a variant of G. stearothermophilus (originally named B. stearothermophilus) "Termamyl-like" [1]-amylase presented in PDB entry 1hvx (Suvd et al., 2001). Compared to most deleted residues ([1]181-182; discussed below) and the single-point variant N193F (numbered in 1hvx; corresponding to Phe191 in 4uzu), the TVB146 enzyme shows 97% sequence identity with the sequence present in 1hvx, differing in two residue deletions [1]181-182, as well as A73T, N193F, S217N, M278T, N281D, T304A and V416G.

[0028] Amylase may also be derived from a species of the genus Bacillus (Bacillus sp.) 707 having SEQ ID NO: 6 as disclosed in International Publication No. 99 / 19467, and at least 95% of its variants. Preferred variants of SEQ ID NO: 6 have substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

[0029] The amylase may also be derived from Bacillus halmapalus having SEQ ID NO: 2 or 7 as described in International Publication No. 96 / 23872, also described herein as SP-722. Preferred variants are described in International Publication Nos. 97 / 3296, 99 / 194671 and 2013 / 001078.

[0030] Amylase may also be derived from a species of the genus Bacillus (Bacillus sp.) DSM 12649 having Sequence ID No. 4 as disclosed in International Publication No. 00 / 22103, and at least 95% of its variants.

[0031] The amylase may also be derived from a species of Bacillus (Bacillus sp.) A 7-7 (DSM 12368) having an amino acid sequence in the region of amino acids 32-516, in particular, that is at least 95% identical to Sequence ID No. 2 as disclosed in International Publication No. 02 / 10356.

[0032] The amylase may also be derived from Bacillus strain TS-23 and its variants having Sequence ID No. 2, as disclosed in International Publication No. 2009 / 061380.

[0033] Amylase may also be derived from a species of the genus Cytophaga (Cytophaga sp.) having Sequence ID No. 1 as disclosed in International Publication No. 2013 / 184577, and at least 95% of its variants.

[0034] Amylase may also be derived from Bacillus megaterium. DSM 90 having Sequence ID No. 1 as disclosed in International Publication No. 2010 / 104675, and at least 95% of its variants.

[0035] Amylase may also be derived from a species of the genus Bacillus (Bacillus sp.) containing amino acids 1-485 of Sequence ID No. 2, as described in International Publication No. 00 / 60060, and at least 95% of its variants.

[0036] The amylase may further be derived from Bacillus amyloliquefaciens or a variant thereof, preferably selected from amylases conforming to Sequence ID No. 3 as described in International Publication No. 2016 / 092009.

[0037] The amylase may have an amylase variant containing SEQ ID NO. 12 as described in International Publication No. 2006 / 002643, or the substitutions Y295F and M202LITV within SEQ ID NO. 12.

[0038] Amylase may have amylase variants that include substitutions at one or more positions selected from the group consisting of SEQ ID NO: 6 as described in International Publication No. 2011 / 098531, or 193[G, A, S, T, or M], 195[F, W, Y, L, I, or V], 197[F, W, Y, L, I, or V], 198[Q, or N], 200[F, W, Y, L, I, or V], 203[F, W, Y, L, I, or V], 206[F, W, Y, N, L, I, V, H, Q, D, or E], 210[F, W, Y, L, I, or V], 212[F, W, Y, L, I, or V], 213[G, A, S, T, or M], and 243[F, W, Y, L, I, or V] within SEQ ID NO: 6.

[0039] Amylase may have amylase variants that include modifications at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within Sequence ID No. 1, as described in International Publication No. 2013 / 001078.

[0040] Amylase may have amylase variants that include a deletion at position 181+182, 182+183, or 183+184 in Sequence ID No. 2, which may optionally include one or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476, and G477 in Sequence ID No. 2, as described in International Publication No. 2013 / 001087.

[0041] The amylase may be a hybrid alpha-amylase derived from the amylase described above, for example, as described in International Publication No. 2006 / 066594.

[0042] The hybrid amylase may conform to International Publication No. 2014 / 183920, having A and B domains that are at least 90% identical to Sequence ID No. 2 of International Publication No. 2014 / 183920 and a C domain that is at least 90% identical to Sequence ID No. 6 of International Publication No. 2014 / 183920, and the hybrid amylase has amylose-degrading activity, preferably the hybrid alpha-amylase is at least 95% identical to Sequence ID No. 23 of International Publication No. 2014 / 183920 and has amylose-degrading activity.

[0043] The hybrid amylase may conform to International Publication No. 2014 / 183921, having A and B domains that are at least 75% identical to SEQ ID NO. 2, SEQ ID NO. 15, SEQ ID NO. 20, SEQ ID NO. 23, SEQ ID NO. 29, SEQ ID NO. 26, SEQ ID NO. 32 and SEQ ID NO. 39 as disclosed in International Publication No. 2014 / 183921, and having a C domain that is at least 90% identical to SEQ ID NO. 6 of International Publication No. 2014 / 183921, the hybrid amylase has amylose-degrading activity, preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO. 30 as disclosed in International Publication No. 2014 / 183921, and has amylose-degrading activity; The hybrid amylase may conform to International Publication No. 2021 / 032881, comprising A and B domains derived from alpha-amylase from a species of the genus Bacillus (Bacillus sp.) A 7-7 (DSM 12368) and a C domain derived from alpha-amylase from Bacillus cereus, preferably the A and B domains being at least 75% identical to the amino acid sequence of SEQ ID NO: 42, and the C domain being at least 75% identical to the amino acid sequence of SEQ ID NO: 44 (both sequences as disclosed in International Publication No. 2021 / 032881), and more preferably the hybrid amylase being at least 80% identical to SEQ ID NO: 54 as disclosed in International Publication No. 2021 / 032881.

[0044] A suitable amylase is a variant of the above-mentioned amylase having amylose-degrading activity. In one embodiment, the amylase variant is one that has at least 40-100% identity with the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, the amylase variant having amylose-degrading activity is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.

[0045] In another embodiment, the present invention relates to amylase variants comprising conservative mutations unrelated to the functional domain of each amylase. The amylase variants of this embodiment having amylose-degrading activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to the full-length polypeptide sequence of the parent enzyme.

[0046] In one embodiment, the amylase variant has the amylose-degrading activity according to the present invention if the amylase variant exhibits increased amylose-degrading activity compared to the parent amylase.

[0047] In one embodiment, an amylase variant has the amylose-degrading activity according to the present invention if the amylase variant exhibits at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amylose-degrading activity of the respective parent amylase.

[0048] In one embodiment, at least one amylase is selected from commercially available amylases, including but not limited to products marketed under trade names such as Duramyl™, Teramyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™, Amplify™, Amplify Prime™ (Novozymes A / S) and Rapidase™, Purastar™, Powerase™, Effectenz™ (M100 from DuPont), Preferenz™ (S1000, S110 and F1000; DuPont), PrimaGreen™ (ALL; DuPont), and Optisize™ (DuPont). >Sequence 1 [ka] >Sequence 2 [ka]

[0049] Tensile strength The tensile strength of a tablet is an important attribute because it needs to be mechanically strong enough to withstand further handling such as film coating, packaging, and transportation, as well as end use by the patient, but also weak enough to break down in the human body and release its contents.

[0050] Tensile strength refers to a material's ability to resist fracture or deformation under tension. In the case of tablets, tensile strength refers to the strength of the tablet to withstand forces during handling, packaging, and transportation. Tablets with high tensile strength are less likely to fracture or disintegrate, ensuring their structural integrity and preventing the loss of their active ingredients. This is particularly important for tablets that require further processing, such as film coating, or those that are generally handled and transported on a large scale, such as those commonly used in the pharmaceutical industry. Manufacturers utilize various techniques to increase the tensile strength of tablets. These include the use of appropriate excipients, optimization of compressibility and compressibility during tablet manufacturing, and improvement of tablet hardness and integrity through the incorporation of binders or disintegrants. Tensile strength testing is typically performed using equipment such as hardness testers or tablet tensile strength testers. This helps manufacturers evaluate the tablet's ability to withstand applied forces and ensure its overall quality. Overall, tablets with high tensile strength are preferred because they are less likely to break, thus ensuring the quality and effectiveness of the pharmaceutical product they contain.

[0051] The minimum tensile strength of a tablet can vary depending on the specific tablet formulation and intended use. However, generally, tablets containing active pharmaceutical ingredients (APIs) should have a tensile strength of at least 1–2 MPa (megapascals) to ensure they can withstand handling and transport without breaking. To achieve this, tablets without APIs, consisting only of excipients, should have a tensile strength of at least 4 MPa.

[0052] For tablet manufacturing, various devices can be used, such as hydraulic hand presses or compactor simulators. These various devices have different residence times, which affect the tensile strength of the tablets produced by them. Residence time in compression is the amount of time it takes for the punch to stop moving vertically and achieve maximum penetration into the die under the primary compression rollers. Residence time occurs when the compression rollers are in flat contact with the punch head. Tablet press residence time is calculated by dividing the planar dimension of the punch head by the tangential velocity of the turret. As a core parameter, residence time greatly affects the overall quality of many tablet products and the manufacturing speed of these products. Specifically, it affects the strength of the tablets and facilitates the movement of the product between tablet presses. Subsequently, an increase or decrease in residence time can significantly affect the outcome of the tablet manufacturing process.

[0053] In the examples, a Specac Atlas Manual 15T hydraulic hand press from SPECAC INC, which has a residence time of 10 seconds, or a StylOne Evo compression simulator (Medelpharm, Germany), which has a residence time of less than 10 milliseconds, was used.

[0054] The enzymatically hydrolyzed porous starch according to the present invention, when compressed at a compression pressure of 150 MPa and a residence time of 10 seconds, yields a solid dosage form having a tensile strength of more than 4 MPa. In a preferred embodiment, the tensile strength is more than 5 MPa.

[0055] The enzymatically hydrolyzed porous starch according to the present invention yields a solid dosage form having a tensile strength of more than 3 MPa when compressed at a compression pressure of 150 MPa and a residence time of less than 10 milliseconds. In a preferred embodiment, the tensile strength is more than 4 MPa.

[0056] Directly compressed tablet formulations Novel porous starches are individual excipients that provide multiple functionalities required for tablet formulation: this single component combines the functions of fillers, binders, and disintegrants, eliminating the need for the addition of these materials. Fluids such as silicon dioxide (typically used at concentrations of 0.2–1% w / w) can be optionally added, and lubricants (typically used at concentrations of 0.5–3% w / w) are necessary to reduce the ejection force during tablet manufacturing.

[0057] Therefore, direct-compressed pharmaceutical dosage forms manufactured with such novel porous starches contain little to no additional excipients, such as fillers, lubricants, binders, and disintegrants, thus enabling very simple formulations and helping to minimize the amount of inactive ingredients in the tablets.

[0058] In one embodiment, the directly compressed pharmaceutically acceptable dosage according to the present invention contains a lubricant and a flowing agent at a concentration of 10% or less, preferably 7.5% or less, and more preferably 5% or less, based on the total weight of the dosage form.

[0059] In another embodiment of the present invention, a directly compressed pharmaceutical dosage form comprises enzymatically hydrolyzed porous starch particles according to the present invention. This dosage form may be a tablet, pellet, minitablet, lozenge, or other solid complement. Preferably, the dosage form according to the present invention, which hereby contains neither APIs nor other additional excipients, is prepared by compression at 150 MPa and exhibits a tensile strength greater than 4 MPa.

[0060] In another embodiment of the present invention, the directly compressed pharmaceutical dosage form contains porous starch particles present at a concentration of 1 to 98% by weight, preferably 40 to 95%, and more preferably 49 to 94%, based on the total weight of the dosage form.

[0061] In another embodiment of the present invention, the directly tabletted pharmaceutical dosage comprises one or more active pharmaceutically ingredients present at a concentration of 1 to 80% by weight, preferably 10 to 80, more preferably 25 to 50% based on the total weight of the dosage form.

[0062] The following experiments (relating to relative excess specific surface area, pressure difference, and polar interaction components) were carried out on each of the samples in Tables 1 and 2. Relative excess specific surface area (S excess ) 1) Determination of specific surface area by nitrogen sorption experiment The specific surface areas of porous and non-porous starches were determined by nitrogen sorption techniques, which measure the amount of test gas (here nitrogen) adsorbed onto a defined amount of solid sample at equilibrium under defined conditions. This gives an adsorption isotherm, which can be evaluated using the Brunauer, Emmett and Teller (BET) model [1] or the Langmuir theory [2]. In both cases, values for the surface area available for adsorption per unit mass of the solid material are obtained (S BET and S Langmuir ), and the BET evaluation gives an additional parameter, the interaction constant (C BET , i.e. the affinity of gas molecules for adsorption onto the solid surface).

[0063] For the measurement, approximately 2.5 g of solid sample was packed into a glass column (inner diameter: 9.5 mm) with a known tare weight. Subsequently, to remove volatile components (including water) from the solid sample, the packed column was evacuated to a pressure of 0.1 mbar and adjusted at this pressure for 48 hours at 25°C using the degassing unit of a Micromeritics (Unterschleissheim, Germany) ASAP 2420 Surface Area and Porosity Analyzer. After adjustment, the weight of the residual material in the column was measured with an accuracy of 0.1 mg. The column was then transferred to the sampling port of the ASAP 2420 instrument, and the free volume in the sample container was measured by adding non-adsorbent helium gas. After removing inert He, the N2 adsorption isotherm was recorded at the nitrogen condensation temperature. BET , C BET and S Langmuir However, the isotherms obtained were used to determine the typical partial pressure range of 0.06 ≤ p / p0 ≤ 0.20 over a 5-point method performed with the supplier's software. The protocol described conforms to the procedure defined in DIN ISO 9277:2003-05 norm. References [1] S. Brunauer, PHEmmett, E. Teller, J. Am. Chem. Soc. 1938, 60, 309-319. [2] I. Langmuir, J. Am. Chem. Soc. 1918, 40, 1361-1402.

[0064] 2) Determination of particle size by laser diffraction measurement In both cases, the particle size distribution of porous starch samples was measured using a Mastersizer 2000 equipped with a Scirocco 2000 sample handling unit manufactured by Malvern Panalytical Ltd. (Worcestershire, UK). The instrument measures the angular variation in the intensity of scattered light as a laser beam passes through a sample of dispersed fine particles. Larger particles overwhelmingly scatter light at small angles relative to the laser beam, while smaller particles contribute more strongly to the scattered light at larger angles. The detailed settings below were used for the measurements: • Optical limits: Optical limits from 0.1 to 6.0 • Timeout period: 2 minutes • Result range: 0.02 to 2000 μm • Result calculation: General purpose • Measurement time: 60 seconds • Measurement snap: 60,000 • Background time: 5 seconds • Background snap: 5,000

[0065] Porous starch particles were measured in dry powder form at a dispersion pressure of 0.5 bar for approximately 30–60 seconds. From the collected angular scattering intensity data, a simplified Fraunhofer approximation was applied to the evaluation [3], and the particle size distribution was calculated using manufacturer-provided software (version 6.00). Particle size was obtained as the diameter of the volume-equivalent sphere, and the lower 10% (d) of the overall distribution. 10 ), 50%(d 50 ), and 90%(d 90 This is reported as a characteristic percentile value that includes ). References [3] J.Vargas-Ubera,JFAguilar,DMGale,Appl.Opt.2007,46,124-132.

[0066] 3) Calculation of relative excess specific surface area d obtained by light scattering 50 Based on the value, the envelope volume (V) of the detected species (single starch granule or aggregate thereof) is calculated.Envelope ) and surface area (A Envelope ) was calculated assuming a spherical shape using the following formula:

number

number

[0067] These parameters allow the experimentally measured d 50 The theoretical specific surface area (S) of a dense (i.e., non-porous) object having a value Envelope ) can be obtained according to the following:

number

number

[0068] The dimensionless values ​​obtained in this way represent the surface area (and therefore overall porosity) caused by the pores within the granules due to the previous amylase treatment, as well as the increase in voids between individual granules in the aggregated structure. Both of these contributions alter the properties of the treated starch product and affect its final mechanical properties. Table 1 gives experimentally measured and calculated parameter values ​​for different types of treated and untreated starch samples, but S excess A plot of tensile strength as a function of is shown in Figure 1.

[0069] Inverse gas chromatography (iGC), pressure difference (Δp) To investigate the surface properties of treated and untreated starch materials by iGC, the samples were packed into a stainless steel GC column with an inner diameter of 4 mm and a length of 10 cm, which was closed at one end with a thin layer of silanized glass wool. Homogeneous and reproducible packing of the solid sample was achieved by vibrating the column under defined conditions. In this way, the column was packed with the starch sample to a height of approximately 9.5 cm (see Table 2 for the required sample mass m), and the remaining open end was then closed with silanized glass wool. The packed column was connected to the injector module of a Thermo Fisher Scientific GmbH (Dreieich, Germany) GC 1310 gas chromatograph using 11 Wagelok fittings and placed in the oven of the GC instrument. After equilibrium at 22.7°C, the samples in the column were purged with helium gas (Nippon Oxygen, 6.0 quality) at a set inlet flow rate of 15 mL / min (controlled by the instrument's injection module), which was independently measured at the inlet of the injection module using a calibrated mass flowmeter (Brooks SLA 5800). As a result of the overall differences in porosity of the packed starch columns (due to different particle sizes and degrees of aggregation), the pressure required to maintain the target flow rate varied depending on the sample and was measured at the column inlet. The difference in this pressure value relative to atmospheric pressure at the column outlet (Δp) was used as a parameter describing the permeability of the packed starch material, as listed in Table 2, and correlated with the tensile strength obtained from mechanical tests in Figure 2.

[0070] Determination of polar interaction component (δP2) After determining the Δp value, the packed column was prepared in a dry helium stream of 10 mL / min at 27°C for 12 hours to remove any remaining volatile compounds and thereby equilibrate the starch surface for subsequent iGC measurements under so-called "infinite dilution" (ID) conditions [4]. For this purpose, small amounts of different test molecules were injected into a helium carrier gas (using the PAL3 RSI Injection System) at predetermined times and guided through the thus packed column where interaction with the starch sample occurred. Retention time t R However, for each probe, the measurement was performed using a flame ionization detector (Trace 1300 / 1600 FID Module from Thermo Fisher Scientific GmbH) located at the column outlet. The following probe molecules were selected: n-heptane, n-octane, n-nonane, n-decane, n-undecane, chloroform, acetone, 2-butanone, diethyl ether, tetrahydrofuran, benzene, and toluene. The dead time t0 was determined by the injection of methane as a non-interacting molecule, thereby determining the net retention time t. N However, regarding each test probe, N =t R It was calculated using -t0. Based on that, the specific retention volume V g This was obtained using the following formula [4]:

number

number

[0071] χ 12 ∞ The Hansen solubility parameter (HSP) is related to the squared distance D between interaction partners in space, as follows [5,6]:

number

[0072] In the first embodiment, the enzymatically hydrolyzed porous starch particles are subjected to polar interaction components (δP2 < 8.8) determined by inverse gas chromatography. 2) It holds.

[0073] In the second embodiment, the porous starch particles enzymatically hydrolyzed according to the first embodiment are 2 excess <10 Relative excess specific surface area (S excess ) has.

[0074] ​In the third embodiment, the enzymatically porous starch particles according to Embodiment 1 or 2 have a pressure difference (Δp) of 200 mbar < Δp < 800 mbar at both ends of a GC column packed with the starch sample and purged with helium at a flow rate of 15 mL / min.

[0075] In the fourth embodiment, the enzymatically porous starch particles according to embodiments 1 to 3 are enzymatically hydrolyzed by an enzyme selected from the group of amoylases.

[0076] In the fifth embodiment, the enzymatically porous starch particles according to embodiments 1 to 3 are enzymatically hydrolyzed by an enzyme selected from the group of α-amoylases.

[0077] In the sixth embodiment, the enzymatically porous starch particles according to embodiments 1 to 5 are selected from the group consisting of corn, rice, wheat, potato, and tapioca starch.

[0078] In the seventh embodiment, the enzymatically porous starch particles according to Embodiment 6 are selected from corn starch.

[0079] In the eighth embodiment, the tablets, pellets, and mini-tablet capsules contain enzymatically porous starch particles according to embodiments 1 to 7.

[0080] In the ninth embodiment, tablets, pellets, and mini-tablet capsules according to Embodiment 8, prepared at a compression pressure of 150 MPa, exhibit a tensile strength of more than 4 MPa.

[0081] In the tenth embodiment, the pharmaceutical preparation, pharmaceutical excipient, nutritional supplement, functional food, health food, or cosmetic contains enzymatically porous starch particles according to Embodiments 1 to 3.

[0082] In the eleventh embodiment, the enzymatically porous starch particles according to embodiments 1 to 3 are used for direct tableting of tablets.

Brief Description of the Drawings

[0083] [Figure 1] Plot of the tensile strength of tablets prepared at a compression pressure of 150 MPa, measured as a function of the relative excess specific surface area obtained by the combination of particle size and nitrogen sorption measurement. White circles: Porous starch samples prepared by the method described according to the present invention in the presence of amylase, in which a tensile strength of more than 4 MPa is observed only at 2 < Sexcess < 10. Black circles: Non-porous samples prepared by the method described according to the present invention without amylase. Black squares: Reference substances described in Table 1. [Figure 2] Plot of the tensile strength measured on tablets prepared at a compression pressure of 150 MPa as a function of the pressure difference across a GC column filled with starch sample and purged with helium at a flow rate of 15 mL / min. White circles: Porous starch samples prepared by the method described according to the present invention in the presence of amylase, in which a tensile strength of more than 4 MPa is observed only at 200 mbar < Δp < 700 mbar. Black circles: Non-porous samples prepared by the method described according to the present invention without amylase. Black squares: Reference substances described in Table 2. [Figure 3] Plot of the tensile strength measured on tablets prepared at a compression pressure of 150 MPa as a function of the polar interaction component determined by inverse gas chromatography at infinite dilution within the framework of the HSP theory. White circles: Porous starch samples prepared by the method described according to the present invention in the presence of amylase, in which a tensile strength of more than 4 MPa is observed only at δP2 < 8.8, and there is a more or less linear dependence of σ150 on δP2 (dashed line). Black circles: Non-porous samples prepared by the method described according to the present invention without amylase. Black squares: Reference substances described in Table 2. [Figure 4]Scanning electron microscope images (1st row) and fluorescein microscope images (2nd row) of natural corn starch, N-ZORBIT, and porous starch particles. The highest porosity can be observed in porous starch. Scanning electron microscope image (3rd row) of the destruction of tablets manufactured from the corresponding particles at a compression pressure of 150 MPa. [Modes for carrying out the invention]

[0084] Examples Method for producing porous starch particles (Examples 1-27) Example 1 Add 3185g of deionized water to a 4L glass reactor. Add 702g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 4.8 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moist filter cake to -40°C. Freeze-dry the frozen filter cake for 120 hours to remove the freeze water. Yield: 365g white powder.

[0085] Example 2 Add 3185g of deionized water to a 4L glass reactor. Add 717g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 3 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moistened starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moistened filter cake to -40°C. Freeze-dry the frozen filter cake for 96 hours to remove the freeze water. Yield: 398g white powder.

[0086] Example 3 Add 3185g of deionized water to a 4L glass reactor. Add 717g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 4.6 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moist filter cake to -40°C. Freeze-dry the frozen filter cake for 96 hours to remove the freeze water. Yield: 360g white powder.

[0087] Example 4 Add 3185g of deionized water to a 4L glass reactor. Add 732g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 2 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moistened starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moistened filter cake to -40°C. Freeze-dry the frozen filter cake for 96 hours to remove the freeze water. Yield: 429g white powder.

[0088] Example 5 Add 3185g of deionized water to a 4L glass reactor. Add 732g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 7 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moistened starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moistened filter cake to -40°C. Freeze-dry the frozen filter cake for 96 hours to remove the freeze water. Yield: 335g white powder.

[0089] Example 6 Add 3185g of deionized water to a 4L glass reactor. Add 732g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 3.8 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and cool the moist filter cake to -40°C. Freeze-dry the frozen filter cake for 96 hours to remove the freeze water. Yield: 380g white powder.

[0090] Example 7 Comparative example without amylase 3160g of deionized water is placed in a 4L glass reactor. 720g of corn starch (dry weight, excluding moisture) is added, and the mixture is stirred. The mixture is heated to 61°C for 2.75 hours. The mixture is cooled to ambient temperature, and the starch particles are separated by filtration. The moist filter cake is crushed by pressing it through a 4000μm mesh sieve. The moist starch is transferred to a vacuum dryer and dried at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first pressed through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed through a 50μm mesh sieve, and 592g of starch particles with a particle size fraction of 50-1000μm are collected.

[0091] Example 8 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.75 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pushing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 431 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0092] Example 9 Add 3185g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 3 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 96 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 324 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0093] Example 10 Comparative example without amylase 1585g of deionized water was placed in a 4L glass reactor. 360g of corn starch (dry weight, excluding moisture) was added, and the mixture was stirred. The mixture was heated to 61°C for 1 hour. The mixture was cooled to ambient temperature and spray-dried. Spray drying was performed using a Buechi Mini Spray Dryer B-290 (BUECHI Labortechnik AG) equipped with a 2.2mm bifluid nozzle, Inert Loop B-295, and Dehumidifier B-296 under the following conditions: nitrogen flow rate: 30m³ 3 / h; inlet temperature 145℃±5℃; outlet temperature 80±5℃ and liquid flow rate 8~13g / min. Products were collected using a cyclone.

[0094] Example 11 Add 3170g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 0.5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and isolate the moist filter cake. Repeat washing and filtering with 2000g of deionized water twice. Finally, mix the moist filter cake with 500g of deionized water and spray dry. Spray drying was performed using a GEA Niro MM-PSR Mobile Minor Spray Dryer (GEA Process Engineering Pte. Ltd.) equipped with a 1.0 mm bifluid nozzle: inlet temperature 145°C ± 5°C; outlet temperature 80°C ± 5°C; spray pressure 0.2 bar and liquid flow rate 25-35 g / min. The product was collected using a cyclone.

[0095] Example 12 Add 3170g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture to isolate the moist filter cake. Repeat washing and filtering with 2000g of deionized water twice. Finally, mix the moist filter cake with 500g of deionized water and spray dry. Spray drying was performed using a GEA Niro MM-PSR Mobile Minor Spray Dryer (GEA Process Engineering Pte. Ltd.) equipped with a 1.0 mm bifluid nozzle: inlet temperature 145°C ± 5°C; outlet temperature 80°C ± 5°C; spray pressure 0.2 bar and liquid flow rate 25-35 g / min. The product was collected using a cyclone.

[0096] Example 13 Add 3170g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 2.5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and isolate the moist filter cake. Repeat washing and filtering with 2000g of deionized water twice. Finally, mix the moist filter cake with 500g of deionized water and spray dry. Spray drying was performed using a GEA Niro MM-PSR Mobile Minor Spray Dryer (GEA Process Engineering Pte. Ltd.) equipped with a 1.0 mm bifluid nozzle: inlet temperature 145°C ± 5°C; outlet temperature 80°C ± 5°C; spray pressure 0.2 bar and liquid flow rate 25-35 g / min. The product was collected using a cyclone.

[0097] Example 14 Add 3170g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and isolate the moist filter cake. Repeat washing with 2000g of deionized water and filtering twice. Finally, the moist wet cake was mixed with 500 g of deionized material and spray-dried. Spray drying was performed using a GEA Niro MM-PSR Mobile Minor Spray Dryer (GEA Process Engineering Pte. Ltd.) equipped with a 1.0 mm bifluid nozzle: inlet temperature 145°C ± 5°C; outlet temperature 80°C ± 5°C; spray pressure 0.2 bar and liquid flow rate 25-35 g / min. The product was collected using a cyclone.

[0098] Example 15 Add 3170g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.75 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the wet starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and isolate the wet filter cake. Finally, mix the wet cake with 500g of deionized water and spray dry. Spray drying was performed using a Buechi Mini Spray Dryer B-290 (BUECHI Labortechnik AG) equipped with a 2.2 mm bifluid nozzle, Inert Loop B-295, and Dehumidifier B-296 under the following conditions: nitrogen flow rate: 30 m³ / h; inlet temperature: 145°C ± 5°C; outlet temperature: 80°C ± 5°C; and liquid flow rate: 8-13 g / min. The product was collected using a cyclone.

[0099] Example 16 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Heat the mixture to 61°C for 2.75 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. Press the dried starch particles first through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 550 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0100] Example 17 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 0.5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 431 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0101] Example 18 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate. Add 14.1g of Termamyl and stir the mixture at 61°C for 5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. Press the dried starch particles first through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 344 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0102] Example 19 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 6 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 336 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0103] Example 20 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 6 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 302 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0104] Example 21 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 58°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 58°C for 0.5 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 572 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0105] Example 22 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.8 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moist starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and crush the moist filter cake by pressing it through a 4000μm mesh sieve. Transfer the moist starch to a vacuum dryer and dry at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first passed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles are removed using a 50 μm mesh sieve, and 442 g of starch particles with a particle size fraction of 50 to 1000 μm are collected.

[0106] Example 23 3160g of deionized water is placed in a 4L glass reactor. 720g of corn starch (dry weight, excluding moisture) is added, and the mixture is stirred. The mixture is heated to 61°C for 2.75 hours. The mixture is cooled to ambient temperature, and the starch particles are separated by filtration. The moist filter cake is crushed by pressing it through a 4000μm mesh sieve. The moist starch is transferred to a vacuum dryer and dried at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first pressed through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed through a 50μm mesh sieve, and 610g of starch particles with a particle size fraction of 50-1000μm are collected.

[0107] Example 24 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.8 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moistened starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and store the moistened filter cake at 40°C for 24 hours.

[0108] The moist filter cake is placed in a 4L glass reactor containing 2000g of deionized water, and the mixture is stirred at ambient temperature (20-25°C) for 1 hour. The mixture is filtered to separate the starch particles. The moist filter cake is crushed by pressing it through a 4000μm mesh sieve. The moist starch is transferred to a vacuum dryer and dried at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first pressed through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed through a 50μm mesh sieve, and 588g of starch particles with a particle size fraction of 50-1000μm are collected.

[0109] Example 25 Add 3160g of deionized water to a 4L glass reactor. Add 720g of corn starch (dry weight, no moisture) and stir the mixture. Add 1.23g of calcium acetate monohydrate and heat the mixture at 61°C for 1 hour. Add 14.1g of Termamyl and stir the mixture at 61°C for 1.8 hours. Cool the mixture to ambient temperature and filter to separate the starch particles. Place the moistened starch filter cake into a 4L glass reactor containing 2000g of deionized water and stir the mixture at ambient temperature (20-25°C) for 1 hour. Filter the mixture and store the moistened filter cake at 40°C for 24 hours.

[0110] The moist filter cake is placed in a 4L glass reactor containing 2000g of deionized water, and the mixture is stirred at ambient temperature (20-25°C) for 1 hour. The mixture is filtered to separate the starch particles. The washing and filtering with 2000g of deionized water is repeated twice. The moist filter cake is crushed by pressing it through a 4000μm mesh sieve. The moist starch is transferred to a vacuum dryer and dried at 40°C under reduced pressure of 40-60mbar for 24 hours. The dried starch particles are first pressed through a 2000μm mesh sieve, and then through a 1000μm mesh sieve. Fine particles are removed through a 50μm mesh sieve, and 324g of starch particles with a particle size fraction of 50-1000μm are collected.

[0111] Example 26 Natural corn starch purchased from Sigma-Aldrich was dispersed in deionized water in a 4:6 ratio. Spray drying was performed using a Buechi Mini Spray Dryer B-290 (BUECHI Labortechnik AG) equipped with a 2.2 mm bifluid nozzle, Inert Loop B-295, and Dehumidifier B-296 under the following conditions: nitrogen flow rate: 30 m³ / h; inlet temperature: 140°C ± 5°C; outlet temperature: 80°C ± 5°C; and liquid flow rate: 8-13 g / min. The product was collected using a cyclone.

[0112] Example 27 2570g of deionized water was placed in a 4L glass reactor. 1227g of corn starch (dry weight, excluding water) was added, the mixture was stirred, and the mixture was heated at 61°C for 1 hour. 1.85g of calcium acetate monohydrate was added and dissolved for 5 minutes. 21.1g of amylase (Termamyl 120L) or 5.3g (Termamyl 2X) was added, and the mixture was stirred at 61°C for 2 hours. The mixture was cooled to ambient temperature, filtered, and washed with 1000g of deionized water. Spray drying was performed using a GEA Niro MM-PSR with an inlet temperature of 150°C and an outlet temperature of 75°C. Finally, the sugar content (<5%, w / w) of the dried product was determined. Yield: Approximately 550g of white powder.

[0113] Reference N-Zorbit: I purchased N-Zorbit from Ingredion. N-Zorbit has a 20% sugar content, which results in a long decay time.

[0114] N-Zorbit was used either in its purchased condition or in the cleaned state described below.

[0115] 100 g N-Zorbit was placed in a 1 L beaker containing 250 mL of deionized water, and the mixture was stirred at ambient temperature (20-25°C) for 1 hour, then separated using a Nutsche suction filter. The resulting wet solid was washed three times again with 250 mL of deionized water each time and separated using a Nutsche filter. The wet filter cake was crushed by pressing it through a 4000 μm mesh sieve and stored at 40°C or 20-25°C for 24 hours. The wet product was transferred to a vacuum dryer and dried at 40°C under reduced pressure of 40-60 mbar for 24 hours. The dried particles were first pressed through a 2000 μm mesh sieve, and then through a 1000 μm mesh sieve. Fine particles were removed through a 50 μm mesh sieve, and the product with a particle size fraction of 50-1000 μm was collected.

[0116] Physicochemical characterization of porous starch particles of each sample shown in Tables 1 and 2 and Figure 1. Relative excess specific surface area (S excess The parameters δP2, Δp, and other parameters were determined as described above.

[0117] Scanning electron microscopy Next, starch particles were placed on a stub using Leit-C (conductive carbon cement). After sputtering a 9 nm thick platinum layer onto the surface of the granules, the sample was transferred to a scanning electron microscope (Zeiss Gemini 500). Using an accelerating voltage of 5 kV, images of the starch particles were captured using secondary electron contrast (based on an Eberhart-Thornley detector).

[0118] Fluorescence microscopy Starch particles were dispersed in water, and 100 ppm sodium fluorescein salt was added. After about 1 hour, a stack of fluorescence images of fluorescein staining the starch structure was captured at an excitation wavelength of 488 nm. The emission was integrated from 500 nm to 600 nm using a confocal laser scanning microscope (Leica, SP8).

[0119] Tablet manufacturing a) Tablet manufacturing by hand press (dwell time 10 seconds) For tablet preparation, a Specac Atlas Manual 15T hydraulic hand press manufactured by SPECAC INC. was used. The hand press was equipped with a 10 mm diameter biplane punch. Before the compression process, all powders were adjusted to the same moisture content as natural starch (8.2 ± 0.8%). The matrix was filled with 300 mg of the thus prepared powder. Compression forces of 400, 800, 1200, 1600, and 2000 kg, corresponding to compression pressures of 50, 100, 150, 200, and 250 MPa, and holding times of 10 seconds were used. Eleven tablets were prepared in the same manner at each compression pressure.

[0120] Tablet characterization To characterize the tablets, a Sotax ST 50 from Sotax AG with pre-installed q-doc i software was used. The following five physical parameters were analyzed using the Sotax ST 50: tablet hardness, diameter, thickness, and mass. The measured tablet hardness was converted to tensile strength by applying the following formula:

number

[0121] One sample corresponding to one powder produced at one compression pressure was investigated five times. The calculated average value was used as the result.

[0122] Scanning electron microscopy of a tablet cross-section The tablet was manually divided into two halves. The cross-section was then placed on a stub using Leit-C (conductive carbon cement). After sputtering a 12 nm thick platinum layer onto the surface of the cross-section, the sample was transferred to a scanning electron microscope (Zeiss Gemini 500). Using a 5 kV acceleration voltage, images of the cross-section were captured using secondary electron contrast (based on an Everhart-Thornley detector).

[0123] [Table 1]

[0124] [Table 2]

[0125] [Table 3]

[0126] [Table 4]

[0127] The following experiments were conducted using the porous starch from Example 27: Determination of sugar content in dried porous starch: Determine the solid content of the porous starch sample (e.g., the sample has a solid content of 95%, meaning the sample consists of 9.5 g of product and 0.5 g of water). Place 25 g of water in a beaker and add 10 g of the porous starch sample while stirring, taking into account the solid content of the sample. Stir at room temperature for 1 hour and then vacuum using a blue band filter. Determine the solid content of the filtrate at 120°C (vacuum) for 2 hours.

[0128] Tablet compression experiment without active ingredient (starch) (residence time < 10 milliseconds) Tablet compression experiments were performed using a fully equipped compression simulator, StylOne Evo (Medelpharm, Germany), equipped with a circular, flat punch (10 mm diameter). Each tablet contained 300 mg and was compressed with compressive forces of 4, 8, 12, 16, and 20 kN. Tablet weight, dimensions, and hardness were measured using a Sotax Tabet Hardness Tester (Sotax, Switzerland). 1) The compression pressure was calculated using the following formula:

number

number

[0129] Porous starch particles were measured in dry powder form at a dispersion pressure of 0.5 or 1.0 bar for approximately 30–60 seconds. From the collected angular scattering intensity data, the particle size distribution was calculated using manufacturer-provided software (version 6.00), applying a simplified Fraunhofer approximation to the evaluation [1]. Particle size was obtained as the diameter of the volume-equivalent sphere and reported as characteristic percentile values, including the lower 10% (d10), 50% (d50), and 90% (d90) of the overall distribution. References [3] J.Vargas-Ubera,JFAguilar,DMGale,Appl.Opt.2007,46,124-132.

[0130] 4) Determination of loss on drying To determine the loss on drying, a Sartorius MA 150 IR moisture meter was used. 5-6 g of sample was uniformly distributed in a sample tray and dried at 105°C until the mass was constant.

[0131] [Table 5]

[0132] Tablet compression experiment containing the active ingredient Using propranolol hydrochloride, paracetamol, and diclofenac-Na as model active ingredients, we demonstrated the performance of a novel porous starch as a multifunctional tablet excipient.

[0133] The composition of the compound was as shown in (Table 4).

[0134] [Table 6]

[0135] The active ingredient and starch-based excipient were sieved (800 μm sieve) and mixed in a Turbula mixer for 8 minutes. Sodium stearyl fumarate (pre-sieved at 800 μm) was added to the mixture and mixed for a further 2 minutes.

[0136] Tablet compression experiments were performed using a fully equipped compression simulator, StylOne Evo (Medelpharm, Germany), equipped with a circular, flat punch (10 mm diameter). Each tablet contained 300 mg (e.g., (50% API formulation): 150 mg of active ingredient, 147 mg of starch-based excipients, and 3 mg of sodium stearyl fumarate). The tablets were compressed with compressive forces of 4, 8, 12, 16, and 20 kN (residence time < 10 milliseconds).

[0137] The compression pressure was calculated using the following formula:

number

[0138] The tensile strength of the tablets was calculated using the following formula:

number

[0139] Disintegration time (minutes) was measured using a Sotax ST50 disintegration tester, according to the United States Pharmacopeia Chapter 701 disintegration method. Six tablets were tested in water at 37°C.

[0140] [Table 7]

[0141] [Table 8]

[0142] [Table 9]

[0143] [Table 10]

[0144] [Table 11]

[0145] [Table 12]

[0146] Additional tableting experiments using different residence times, without the active ingredient (starch). N-Zorbit was purchased from Ingredion. N-Zorbit has a sugar content of 20%, resulting in a long decay time. In the following examples, N-Zorbit was used as purchased (N-Zorbit-W0).

[0147] 1. Production of porous starch Example H202-24) Add 2570g of deionized water to a 4L glass reactor. Add 1227g of corn starch (dry weight, excluding water), stir the mixture, and heat at 61°C for 1 hour. Add 1.85g of calcium acetate monohydrate and allow to dissolve for 5 minutes. Add 21.1g of amylase (Termamyl 120L) or 5.3g (Termamyl 2X) and stir the mixture at 61°C for 2 hours. Set the pH of the product medium to 3 with 1N sulfuric acid, stir for another 30 minutes, and then set the pH to 6.5 with 1N NaOH (H202-24-01-W0). Cool the mixture to ambient temperature, filter, and wash with 1000g of deionized water for 30 minutes (1 wash step, 3 wash steps). Spray drying is performed in a B290 Advanced Buechi at an inlet temperature of 135°C and an outlet temperature of 75°C. Yield: Approximately 110g of white powder. H202-24-01-W1 (Has one washing step) H202-24-03-W3 (Has 3 washing steps)

[0148] 2. Tablet manufacturing by hand press (10-second dwell time) For tablet preparation, a Specac Atlas Manual 15T hydraulic hand press manufactured by SPECAC INC. was used. The hand press was equipped with a 10 mm diameter double punch. Prior to the compression process, the entire powder was adjusted to the same moisture content as natural starch (8.2 ± 0.8%). 300 mg of the thus prepared powder was filled into the matrix. Compression forces of 400, 800, 1200, 1600, and 2000 kg and holding times (residence times) of 10 seconds were applied, corresponding to compression pressures of 50, 100, 150, 200, and 250 MPa. Eleven tablets were prepared in the same manner at each compression pressure.

[0149] Tablet characterization To characterize the tablets, a Sotax ST 50 from Sotax AG with pre-installed q-doc i software was used. The following five physical parameters were analyzed using the Sotax ST 50: tablet hardness, diameter, thickness, and mass. The measured tablet hardness was converted to tensile strength by applying the following formula:

number

[0150] One sample corresponding to one powder produced at one compression pressure was investigated five times. The calculated average value was used as the result.

[0151] 3. Tablet manufacturing using a compression simulator (dwell time < 10 milliseconds) Tablet compression experiments were conducted using a fully equipped compression simulator, StylOne Evo (Medelpharm, Germany), equipped with a 10 mm diameter two-sided punch. Prior to the compression process, all powders were adjusted to the same moisture content (8.2 ± 0.8%). Each tablet contained 300 mg and was compressed with compressive forces of 4, 8, 12, 16, and 20 kN, corresponding to 50, 100, 150, 200, and 250 MPa, and with residence times of less than 10 milliseconds. Ten tablets were prepared in the same manner at each compression pressure. Tablet weight, dimensions, and hardness were measured using a Sotax Tabet Hardness Tester (Sotax, Switzerland). 1) The compression pressure was calculated using the following formula:

number

number

[0152] [Table 13]

[0153] [Table 14]

Claims

1. 2 < S ｅｘｃｅｓｓ <10 Relative excess specific surface area (S ｅｘｃｅｓｓ Enzymatically hydrolyzed porous starch particles having ).

2. δP ２ <8.8 Polar interaction component determined by inverse gas chromatography (δP ２） Enzymatically hydrolyzed porous starch particles having [a certain characteristic].

3. Enzymatically hydrolyzed porous starch particles with a pressure difference (Δp) of 200 mbar < Δp < 800 mbar at both ends of a GC column packed with a starch sample and purged with helium at a flow rate of 15 mL / min.

4. 2 < S ｅｘｃｅｓｓ <10 (S ｅｘｃｅｓｓ ) has a relative excess specific surface area of ​​δP ２ <8.8 Polar interaction component determined by inverse gas chromatography (δP ２） Enzymatically hydrolyzed porous starch particles having [a certain characteristic].

5. 2 < S ｅｘｃｅｓｓ < 10 of relative specific surface area (S ｅｘｃｅｓｓ ) having, filled with starch sample, and purged with helium at a flow rate of 15 mL / min, enzyme-hydrolyzed porous starch particles having a pressure difference (Δp) of 200 mbar < Δp < 800 mbar at both ends of the GC column.

6. 2 < S ｅｘｃｅｓｓ <10 Relative excess specific surface area (S ｅｘｃｅｓｓ ) has δP ２ <8.8 Polar interaction component determined by inverse gas chromatography (δP ２） Enzymatically hydrolyzed porous starch particles having a pressure difference (Δp) of 200 mbar < Δp < 800 mbar at both ends of a GC column packed with a starch sample and purged with helium at a flow rate of 15 mL / min.

7. 2 < S ｅｘｃｅｓｓ <7 Relative excess specific surface area (S ｅｘｃｅｓｓ Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 6, having )

8. Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 7, wherein the enzyme is selected from the group of amylases.

9. The enzyme hydrolyzed porous starch particles according to claim 8, wherein the enzyme is selected from the group of α-amylases.

10. Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 9, wherein the porous starch is selected from the group consisting of corn, rice, and potato starch.

11. The enzymatically hydrolyzed porous starch particles according to claim 10, wherein the selected starch is corn starch.

12. Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 11, wherein the porous starch particles preferably contain 5% by weight or less, more preferably 1.5% by weight or less, and even more preferably 0.5% by weight or less of sugars and oligosaccharides.

13. Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 12, which, when compressed at a compression pressure of 150 MPa, give a solid dosage form having a tensile strength of more than 4 MPa.

14. Enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 13, which are compressed at a compression pressure of 150 MPa and a residence time of 10 seconds to give a solid dosage form having a tensile strength of more than 4 MPa.

15. A dosage form comprising enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 14.

16. The directly compressed pharmaceutical dosage form according to claim 15, which is a tablet, pellet, minitablet, lozenge, or other solid complement.

17. The directly compressed pharmaceutical dosage form according to claim 16, comprising porous starch particles present in a concentration of 1 to 98% by weight based on the total weight of the dosage form.

18. A directly compressed pharmaceutical dosage form according to any one of claims 15 to 17, comprising one or more pharmaceutical active ingredients present at a concentration of 1 to 80% by weight based on the total weight of the dosage form.

19. The direct-compressed pharmaceutical dosage form according to claim 17, wherein one or more pharmaceutical active ingredients are mixed with and / or filled in the enzymatically hydrolyzed porous starch particles.

20. A directly compressed pharmaceutical dosage form according to any one of claims 16 to 19, wherein the concentration of the lubricant and the flowing agent based on the total weight of the dosage form is 10% or less, preferably 7.5% or less, and more preferably 5% or less.

21. A pharmaceutical formulation, food formulation, feed formulation, pesticide formulation, or cosmetic formulation comprising enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 15.

22. A directly compressed pharmaceutical dosage form according to any one of claims 15 to 20 having the following characteristics: A directly compressed pharmaceutical dosage form according to any one of claims 16 to 20 having the following characteristics: a content of 10 to 80% by weight of the active pharmaceutical ingredient (API), a tablet tensile strength greater than 1.5 MPa (compressed at 150 MPa), and a disintegration time of less than 5 minutes without the presence of a disintegrant.

23. The directly compressed pharmaceutical dosage form according to claim 22, wherein the API content is 25 to 50% by weight, preferably 25% by weight.

24. A directly compressed pharmaceutical dosage form according to any one of claims 15 to 20 having the following characteristics: A directly compressed pharmaceutical dosage form according to any one of claims 16 to 20 having the following characteristics: API content of 10 to 80% by weight, preferably 25 to 50% by weight, more preferably 25% by weight; tensile strength of tablets greater than 1.5 MPa (compressed at 150 MPa and residence time of less than 10 milliseconds); and disintegration time of less than 5 minutes without disintegrant.

25. Use of enzymatically hydrolyzed porous starch particles according to any one of claims 1 to 14 for direct tableting of tablets, pellets, minitablets, lozenges and other complementary formulations.

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