CN116528688A

CN116528688A - Oligosaccharide preparation

Info

Publication number: CN116528688A
Application number: CN202180079535.0A
Authority: CN
Inventors: 伊万·德·杰西·盖坦·佩雷斯; 约翰·迈克尔·杰雷米亚; 克里斯蒂娜·哥特舍克; 约瑟夫·霍曼斯珀格; 玛尔塔·帕利亚里; 托尼·斯坦纳布伦纳; 帕特里夏·德里亚; 奥利维亚·布里吉特·维多尼; 亚历山德鲁·扎巴拉
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2020-12-03
Filing date: 2021-12-02
Publication date: 2023-08-01

Abstract

The present invention relates to storage stable formulations of oligosaccharide preparations suitable for use in nutritional compositions such as animal feeds.

Description

Oligosaccharide preparation

Background

Oligosaccharides are a group of heterogeneous carbohydrates with different degrees of polymerization. The oligosaccharide composition may be naturally occurring, for example in milk, or synthesized by enzymatic or chemical processes. Depending on the manufacturing process, the resulting oligosaccharide composition may have different chemical, biological and/or physical properties. Enzymatic hydrolysis of longer chain oligosaccharides and polysaccharides can produce oligosaccharides by specific cleavage under mild reaction conditions. However, the use of enzymes in industrial processes is limited by their thermal stability, and enzymatic methods can produce degradation byproducts that can cause metabolic problems when consumed by poultry, swine, and other livestock. On the other hand, chemical hydrolysis of longer chain oligosaccharides and polysaccharides may require harsh reaction conditions and it is difficult to control the chemical and/or physical properties of the oligosaccharides produced by the chemical hydrolysis process. Thus, there remains a need to make oligosaccharide compositions having desirable properties.

Oligosaccharide preparations (which may typically comprise monosaccharides, oligosaccharides, polysaccharides, functionalized oligosaccharides or combinations thereof) are used as additives in nutritional compositions (e.g. animal feeds). The addition of oligosaccharide preparations may improve animal health and performance.

The oligosaccharide preparation according to the invention is a synthetic oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerisation (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than or equal to 2; and wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides, as measured by relative abundance by mass spectrometry. Preferred oligosaccharide preparations according to the invention are defined hereinafter.

Methods of making oligosaccharide preparations according to the invention are described in WO 2020/097458, WO 2016/007778, characterised by the step of heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerisation.

Because of the physical properties of such oligosaccharide preparations, their addition to animal feed is challenging. The preparation produced by the process disclosed in WO 2020/097458 is in liquid form. Thus, there is a need for a method of developing a solid product form that overcomes the storage and handling problems of liquid feed additives and additionally has good flowability and storage stability, is moisture stable, and can be easily blended with other components commonly used in animal feed products, particularly animal feed products for poultry and swine.

Surprisingly, it has been found that the oligosaccharide preparation according to the invention is efficiently formulated if adsorbed onto silica-based products having an average particle size D (0, 5) of 3000 μm or less, preferably 2000 μm or less, more preferably 1200 μm or less. D (0, 5) means a particle size distribution (Particle Size Distribution, PSD) defined according to standards, and D (0, 5) is hereinafter also referred to as "D".

Thus, in a first embodiment, the present invention relates to a powder formulation characterized in that

(i) At least 20 weight-% (wt-%) of an oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than 2, based on the total weight of the powder formulation; and wherein each fraction comprises from 1% to 90% of anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry,

(ii) 0 to 25% by weight of water and/or additives, based on the total weight of the powder formulation, and

(iii) At least 25% by weight, based on the total weight of the powder formulation, of adsorbates based on silica having an average particle size D < 3000 μm.

It should be fully understood that the composition according to the invention is storage stable, reduces the sensitivity of the oligosaccharide preparation to water absorption and is free-flowing.

The formulation according to the invention is a powder, which depending on the production method and storage conditions may contain some water. The water content is generally less than 25% by weight, preferably less than 10% by weight, based on the total weight of the formulation. Thus, a further embodiment of the invention relates to a formulation as described above, wherein 0 to 21 wt% of water is present, based on the total weight of the formulation.

The formulations according to the invention may also contain small amounts of conventional additives commonly used in the preparation of powder formulations for feed applications. Thus, a further embodiment of the invention relates to a formulation according to the invention, wherein 0 to 5 wt.% of additive is present, based on the total weight of the formulation.

It is evident that in all embodiments of the invention, all weight% added always add up to 100. However, it cannot be excluded that small amounts of impurities or additives, for example in amounts of less than 5% by weight, preferably less than 3% by weight, may be present, which impurities or additives are introduced, for example, via the corresponding raw materials or processes used.

Disclosure of Invention

Provided herein is a solid formulation of a synthetic oligosaccharide preparation as defined above adsorbed onto a silica-based product.

Silica is a well known carrier material in the feed and food industry and refers to white microspheres of amorphous silica (also known as silica) and is available in a variety of particle sizes. Particularly suitable silicas according to the invention are amorphous precipitated silicas (amorphous precipitated silica, AS) having a particle size (particle size). Ltoreq.350. Mu.m, such AS Ibersil D-250 from IQE Group, sipernat 2200 from Evonik or Tixosil 68 from Solvay or Zeofre 5170 from Huber.

Another silica-based product is diatomaceous earth, also known as DE, diatomaceous earth, a naturally occurring soft siliceous sedimentary rock that is easily crushed into fine powder, which is white in nature. It has a particle size ranging from less than 3 μm to more than 1mm. Depending on the particle size (granular), such powders may have an abrasive feel similar to pumice powder and have a low density due to their high porosity. Typical chemical compositions of the oven dried diatomaceous earth are 80-90% silica, 2-4% alumina (mainly attributed to clay minerals) and 0.5-2% iron oxide.

Preferably, in all embodiments of the present invention, the average (median) particle diameter D (0, 5) of the silica-based product according to the present invention is selected from the range of 100 μm to 800 μm, more preferably from 200 μm to 500 μm, most preferably from 200 μm to 350 μm.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described.

Detailed Description

Oligosaccharide preparations and solid compositions comprising such oligosaccharide preparations are described herein. Methods of producing the oligosaccharide formulations and solid compositions are further described herein.

I. Definition of the definition

As used herein, the term "anhydrosubunit" may be a monosaccharide (or monosaccharide subunit) or a reversible thermal dehydration product of a sugar caramelization product. For example, the "anhydrosubunit" may be an anhydromonose, such as anhydroglucose. As another example, a "anhydro subunit" may be linked to one or more conventional or anhydro monosaccharide subunits by glycosidic linkages.

As used herein, the term "oligosaccharide preparation" may refer to a preparation comprising one or more oligosaccharides.

As used herein, "oligosaccharide" or "oligomer" may refer to a monosaccharide, or a compound containing two or more monosaccharide subunits linked by glycosidic linkages. "oligosaccharides" may also refer to dehydrated monosaccharides; or a compound containing two or more monosaccharide subunits, wherein at least one monosaccharide unit is replaced with a anhydro subunit. The "oligosaccharides" may optionally be functionalized. As used herein, the term oligosaccharide encompasses all kinds of oligosaccharides wherein each monosaccharide subunit in the oligosaccharide is independently and optionally functionalized and/or replaced by its corresponding anhydromonose subunit.

As used herein, the term "oligoglucose" may refer to glucose or a compound containing two or more glucose monosaccharide subunits linked by glycosidic linkages. "glucose oligomer" may also refer to anhydroglucose; or a compound containing two or more glucose monosaccharide subunits linked by glycosidic linkages, wherein at least one monosaccharide subunit is replaced with an anhydroglucose subunit. Similarly, "galacto-oligosaccharides" may refer to galactose; or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. "galacto-oligosaccharides" may also refer to anhydrogalactose; or a compound containing two or more galactose monosaccharide subunits linked by glycosidic linkages, wherein at least one monosaccharide subunit is replaced with a anhydrogalactose subunit.

As used herein, "galacto-oligosaccharides" may refer to compounds produced by complete or incomplete sugar condensation reactions of glucose and galactose. For example, the polyglucose-galactose may be an oligoglucose; galactooligosaccharides; or a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. Thus, in some embodiments, the polyglucose-galactose-preparation comprises polyglucose; galactooligosaccharides; and compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic linkages. In some embodiments, the polyglucose-galactose-preparation comprises polyglucose and a compound comprising one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic linkages. In some embodiments, the polyglucose-galactose-preparation comprises galactooligosaccharides and a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic linkages. In some embodiments, the oligoglucose-galactose-preparation comprises a compound comprising one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic linkages.

In addition, the polyglucose-galactose may be an oligoglucose; galactooligosaccharides; or a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic linkages, wherein at least one of the monosaccharide subunits is replaced with its corresponding anhydromonose subunit.

Similarly, an oligoglucose-galactose-xylose may refer to a compound produced by a condensation reaction of glucose, galactose and xylose. An oligosaccharide preparation comprising an oligosaccharide-galactose-xylose may comprise an oligosaccharide-galactose, an oligosaccharide-xylose, and a compound comprising one or more glucose monosaccharide subunits, one or more xylose monosaccharide subunits, and one or more galactose monosaccharide subunits linked by glycosidic linkages.

As used herein, the terms "monosaccharide unit" and "monosaccharide subunit" are used interchangeably unless otherwise indicated. "monosaccharide subunit" may refer to a monosaccharide monomer in an oligosaccharide. For an oligosaccharide having a degree of polymerization of 1, the oligosaccharide may be referred to as a monosaccharide subunit or monosaccharide. For oligosaccharides with a degree of polymerization greater than 1, the monosaccharide subunits are linked via glycosidic linkages.

As used herein, the term "conventional monosaccharide" may refer to a monosaccharide that does not contain a anhydrosubunit. The term "conventional disaccharide" may refer to a disaccharide that does not contain a anhydrosubunit. Thus, the term "conventional subunit" may refer to a subunit that is not a anhydro subunit.

As used herein, the term "relative abundance" or "abundance" can refer to the abundance of a substance in terms of the prevalence or rarity of the presence of the substance. For example, a DP1 fraction comprising 10% anhydrosubunit-containing oligosaccharides by relative abundance may refer to a plurality of DP1 oligosaccharides, wherein 10% by number of the DP1 oligosaccharides are anhydromonosaccharides.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, reference to "the oligosaccharide" includes reference to one or more oligosaccharides (or oligosaccharides) and equivalents thereof known to those skilled in the art, and so forth.

When a range is used herein for a physical property (e.g., molecular weight) or a chemical property (e.g., chemical formula), it is intended to include all combinations and subcombinations of ranges and specific embodiments thereof. When referring to a number or range of values, the term "about" means that the number or range of values of the volume is an approximation within experimental variability (or statistical experimental error), and thus in some cases the number or range of values will vary between 1% and 15% of the number or range of values.

II. preparation of synthetic oligosaccharides

In some embodiments, the disclosed oligosaccharide preparation comprises at least n oligosaccharide fractions, each oligosaccharide having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than or equal to 2. In some embodiments, n is an integer greater than 2. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with its degree of polymerization.

In some embodiments, the relative abundance of the oligosaccharides in at least 5, 10, 20, or 30 DP fractions monotonically decreases with their degree of polymerization. In some embodiments, the relative abundance of the oligosaccharides in each of the n fractions monotonically decreases with its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, at least one fraction comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharides by relative abundance.

In some embodiments, the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharide by relative abundance.

In some embodiments, each fraction comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit-containing oligosaccharides by relative abundance.

In some embodiments, at least one fraction comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharides by relative abundance.

In some embodiments, at least one fraction comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro subunit-containing oligosaccharides by relative abundance. In some embodiments, the oligosaccharide preparation comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70% or 80% anhydro subunit-containing oligosaccharide by relative abundance. In some embodiments, each fraction comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro subunit-containing oligosaccharides by relative abundance.

In some embodiments, at least one fraction comprises greater than 20%, 21%, 22%, 23%, 24%, or 25% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the oligosaccharide preparation comprises greater than 20%, 21%, 22%, 23%, 24% or 25% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction comprises greater than 20%, 21%, 22%, 23%, 24%, or 25% anhydrosubunit-containing oligosaccharides by relative abundance.

In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the anhydro subunit-containing oligosaccharides have only one anhydro subunit.

In some embodiments, the oligosaccharide preparation has a DP1 fraction content of 1% to 40% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP2 fraction content of 1% to 35% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP3 fraction content of 1% to 30% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP4 fraction content of 0.1% to 20% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP5 fraction content of 0.1% to 15% by relative abundance.

In some embodiments, the ratio of DP2 fraction to DP1 fraction is from 0.02 to 0.40 in relative abundance. In some embodiments, the ratio of DP3 fraction to DP2 fraction is from 0.01 to 0.30 in relative abundance.

In some embodiments, the aggregate content of the DP1 fraction and the DP2 fraction in the oligosaccharide preparation is less than 50%, 30% or 10% by relative abundance.

In some embodiments, the oligosaccharide preparation comprises at least 10 ³ Seed, 10 ⁴ Seed, 10 ⁵ Seed, 10 ⁶ Seed or 10 ⁹ Different oligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprise different anhydrosubunits.

In some embodiments, the oligosaccharide preparation comprises one or more anhydrosubunits, which are the product of reversible thermal dehydration of a monosaccharide.

In some embodiments, the oligosaccharide preparation comprises one or more of anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroidose, anhydrotalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, or anhydroxylose subunits. In some embodiments, the oligosaccharide preparation comprises one or more anhydroglucose, anhydrogalactose, anhydromannose, or anhydrofructose subunits.

In some embodiments, the oligosaccharide preparation comprises one or more 1, 6-anhydro- β -D-furanosyl glucose or 1, 6-anhydro- β -D-glucopyranose subunits. In some embodiments, the oligosaccharide preparation comprises both 1, 6-anhydro- β -D-furanose and 1, 6-anhydro- β -D-glucopyranose anhydro-subunits.

In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in the oligosaccharide preparation is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in the oligosaccharide preparation is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in the oligosaccharide preparation is about 2:1.

In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in each fraction is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in each fraction is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio of 1, 6-anhydro- β -D-furanose to 1, 6-anhydro- β -D-glucopyranose in each fraction is about 2:1.

In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the anhydro subunits are selected from the group consisting of: 1, 6-anhydro-beta-D-furanose and 1, 6-anhydro-beta-D-glucopyranose.

In some embodiments, the weight average molecular weight of the preparation is about 300g/mol to 5000g/mol, 500g/mol to 5000g/mol, 700g/mol to 5000g/mol, 500g/mol to 2000g/mol, 700g/mol to 1500g/mol, 300g/mol to 2000g/mol, 400g/mol to 1300g/mol, 400g/mol to 1200g/mol, 400g/mol to 1100g/mol, 500g/mol to 1300g/mol, 500g/mol to 1200g/mol, 500g/mol to 1100g/mol, 600g/mol to 1300g/mol, 600g/mol to 1200g/mol, or 600g/mol to 1100g/mol.

In some embodiments, the number average molecular weight of the preparation is about 300g/mol to 5000g/mol, 500g/mol to 5000g/mol, 700g/mol to 5000g/mol, 500g/mol to 2000g/mol, 700g/mol to 1500g/mol, 300g/mol to 2000g/mol, 400g/mol to 1000g/mol, 400g/mol to 900g/mol, 400g/mol to 800g/mol, 500g/mol to 900g/mol, or 500g/mol to 800g/mol.

Distribution of polymerization Degree (DP)

The degree of polymerization profile of the oligosaccharide preparation may be determined by any suitable analytical method and apparatus including, but not limited to, end-group methods, osmometry (osmometry), ultracentrifugation, viscosity measurement, light scattering, size Exclusion Chromatography (SEC), SEC-MALLS, field flow fractionation (field flow fractionation, FFF), asymmetric flow field flow fractionation (asymmetric flow field flow fractionation, A4F), high-performance liquid chromatography (high-performance liquid chromatography, HPLC) and mass spectrometry (mass spectrometry, MS). For example, the distribution of the degree of polymerization may be determined and/or detected by mass spectrometry (e.g., MALDI-MS, LC-MS, or GC-MS). For another example, the degree of polymerization distribution may be determined and/or detected by SEC, such as gel permeation chromatography (gel permeation chromatography, GPC). As yet another example, the degree of polymerization profile may be determined and/or detected by HPLC, FFF, or A4F. In some embodiments, the distribution of the degree of polymerization is determined and/or detected by MALDI-MS. In some embodiments, the distribution of the degree of polymerization is determined and/or detected by GC-MS or LC-MS. In some embodiments, the distribution of degrees of polymerization is determined and/or detected by SEC. In some embodiments, the distribution of the degree of polymerization is determined and/or detected by HPLC. In some embodiments, the distribution of the degree of polymerization is determined and/or detected by a combination of analytical instruments (e.g., MALDI-MS and SEC). In some embodiments, the degree of polymerization of the oligosaccharide preparation may be determined based on its molecular weight and molecular weight distribution (for a more detailed description, see WO 2020/097458).

Level of anhydro subunit

In some embodiments, each of the n oligosaccharide fractions independently comprises a anhydrosubunit level. For example, in some embodiments, the DP1 fraction comprises 10% anhydro-subunit containing oligosaccharides, and the DP2 fraction comprises 15% anhydro-subunit containing oligosaccharides, relative to the relative abundance. For another example, in some embodiments, the DP1 fraction, DP2 fraction, and DP3 fraction each comprise 5%, 10%, and 2% anhydrosubunit-containing oligosaccharides, respectively, by relative abundance. In other embodiments, two or more oligosaccharide fractions may comprise similar levels of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the DP1 fraction and the DP3 fraction each comprise about 5% anhydrosubunit-containing oligosaccharides, by relative abundance.

The dehydrated subunit level may be determined by any suitable analytical method, such as nuclear magnetic resonance (nuclear magnetic resonance, NMR) spectroscopy, mass spectrometry, HPLC, FFF, A F, or any combination thereof. In some embodiments, the anhydrosubunit level is determined at least in part by mass spectrometry, such as MALDI-MS. In some embodiments, the dehydrated subunit level may be determined at least in part by NMR. In some embodiments, the dehydrated subunit level may be determined, at least in part, by HPLC. For example, in some embodiments, the level of anhydrosubunits may be determined by MALDI-MS, as described in more detail in WO 2020/097458.

Glycosidic bond

In some embodiments, the oligosaccharide preparation used in the methods described herein comprises a plurality of glycosidic linkages. The type and distribution of glycosidic linkages may depend on the source of the oligosaccharide preparation and the method of manufacture. In some embodiments, the type and distribution of the various glycosidic linkages can be determined and/or detected by any suitable method known in the art (e.g., NMR). For example, in some embodiments, glycosidic linkages are determined and/or detected by proton NMR, carbon NMR, 2D NMR (e.g., 2D JRES, HSQC, HMBC, DOSY, COSY, ECOSY, TOCSY, NOESY, or ROESY), or any combination thereof. In some embodiments, the glycosidic linkage is determined and/or detected at least in part by proton NMR. In some embodiments, the glycosidic linkage is determined and/or detected at least in part by carbon NMR. In some embodiments, the glycosidic linkage is determined and/or detected at least in part by 2D HSQC NMR.

In some embodiments, the oligosaccharide preparation may comprise one or more α - (1, 2) glycosidic linkages, α - (1, 3) glycosidic linkages, α - (1, 4) glycosidic linkages, α - (1, 6) glycosidic linkages, β - (1, 2) glycosidic linkages, β - (1, 3) glycosidic linkages, β - (1, 4) glycosidic linkages, β - (1, 6) glycosidic linkages, α (1, 1) α glycosidic linkages, α (1, 1) β glycosidic linkages, β (1, 1) β glycosidic linkages, or any combination thereof.

In some embodiments, the glycosidic bond type distribution of the oligosaccharide preparation is about 0 to 60mol%, 5 to 55mol%, 5 to 50mol%, 5 to 45mol%, 5 to 40mol%, 5 to 35mol%, 5 to 30mol%, 5 to 25mol%, 10 to 60mol%, 10 to 55mol%, 10 to 50mol%, 10 to 45mol%, 10 to 40mol%, 10 to 35mol%, 15 to 60mol%, 15 to 55mol%, 15 to 50mol%, 15 to 45mol%, 15 to 40mol%, 15 to 35mol%, 20 to 60mol%, 20 to 55mol%, 20 to 50mol%, 20 to 45mol%, 20 to 40mol%, 20 to 35mol%, 25 to 60mol%, 25 to 55mol%, 25 to 50mol%, 25 to 45mol%, 25 to 40% or a 1- (6) glycosidic bond.

Molecular weight

The molecular weight and molecular weight distribution of the oligosaccharide preparation may be determined by any suitable analytical means and instruments, such as end-group methods, osmolarity (osmometry), ultracentrifugation, viscosity measurement, light scattering methods, SEC-MALLS, FFF, A4F, HPLC and mass spectrometry. In some embodiments, the molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, the molecular weight and molecular weight distribution are determined by Size Exclusion Chromatography (SEC), such as Gel Permeation Chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined by HPLC. In some embodiments, the molecular weight and molecular weight distribution are determined by MALDI-MS.

In some embodiments of the present invention, in some embodiments, the weight average molecular weight of the preparation is about 100g/mol to 10000g/mol, 200g/mol to 8000g/mol, 300g/mol to 5000g/mol, 500g/mol to 5000g/mol, 700g/mol to 5000g/mol, 900g/mol to 5000g/mol, 1100g/mol to 5000g/mol, 1300g/mol to 5000g/mol, 1500g/mol to 5000g/mol, 1700g/mol to 5000g/mol, 300g/mol to 4500g/mol, 500g/mol to 4500g/mol, 700g/mol to 4500g/mol, 900g/mol to 4500g/mol, 1100g/mol to 4500g/mol, 1300g/mol to 4500g/mol, 1500g/mol to 4500g/mol, 1900g/mol to 4500g/mol, 300g/mol to 4000g/mol 500g/mol to 4000g/mol, 700g/mol to 4000g/mol, 900g/mol to 4000g/mol, 1100g/mol to 4000g/mol, 1300g/mol to 4000g/mol, 1500g/mol to 4000g/mol, 1700g/mol to 4000g/mol, 1900g/mol to 4000g/mol, 300g/mol to 3000g/mol, 500g/mol to 3000g/mol, 700g/mol to 3000g/mol, 900g/mol to 3000g/mol, 1100g/mol to 3000g/mol, 1300g/mol to 3000g/mol, 1500g/mol to 3000g/mol, 1700g/mol to 3000g/mol, 1900g/mol to 3000g/mol, 2100g/mol to 3000g/mol, 300g/mol to 2500g/mol, 500g/mol to 2500g/mol, 700g/mol, 900g/mol to 2500g/mol, 1100g/mol to 2500g/mol, 1300g/mol to 2500g/mol, 1500g/mol to 2500g/mol, 1700g/mol to 2500g/mol, 1900g/mol to 2500g/mol, 2100g/mol to 2500g/mol, 300g/mol to 1500g/mol, 500g/mol to 1500g/mol, 700g/mol to 1500g/mol, 900g/mol to 1500g/mol, 1100g/mol to 1500g/mol, 1300g/mol to 1500g/mol, 2000-2800g/mol, 2100-2700g/mol, 2200-2600g/mol, 2300-2500g/mol, or 2320-2420g/mol. In some embodiments, the weight average molecular weight of the preparation is from about 2000g/mol to 2800g/mol, 2100g/mol to 2700g/mol, 2200g/mol to 2600g/mol, 2300g/mol to 2500g/mol, or 2320g/mol to 2420g/mol.

Types of oligosaccharides

In some embodiments, the type of oligosaccharide present in the oligosaccharide preparation may depend on the type of feed sugar or sugars. For example, in some embodiments, when the feed sugar comprises glucose, the oligosaccharide preparation comprises an oligoglucose. For example, in some embodiments, when the feed sugar comprises galactose, the oligosaccharide preparation comprises galacto-oligosaccharides. For another example, in some embodiments, when the feed sugar comprises galactose and glucose, the oligosaccharide preparation comprises an oligoglucose-galactose.

In some embodiments, the oligosaccharide preparation comprises one or more types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation may comprise oligosaccharides having 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different kinds of monosaccharide subunits.

Method for producing oligosaccharide preparation

A process for manufacturing an oligosaccharide preparation according to the invention is described in detail in WO 2020/097458, said process comprising heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of: (+) -camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; alpha-hydroxy-2-pyridinemethanesulfonic acid; (β) -camphor-10-sulfonic acid; butyl phosphonic acid; diphenyl phosphinic acid; hexyl phosphonic acid; methyl phosphonic acid; phenyl phosphinic acid; phenyl phosphonic acid; t-butyl phosphonic acid; SS) -VAPOL hydrogen phosphate; 6-quinolinesulfonic acid, 3- (1-pyridinyl) -1-propanesulfonic acid salt; 2- (2-pyridyl) ethanesulfonic acid; 3- (2-pyridinyl) -5, 6-diphenyl-1, 2, 4-triazine-p, p' -disulfonic acid monosodium salt hydrate; 1,1 '-binaphthyl-2, 2' -diyl-hydrogen phosphate; bis (4-methoxyphenyl) phosphinic acid; phenyl (3, 5-xylyl) phosphinic acid; l-cysteic acid monohydrate; poly (styrenesulfonic acid-co-divinylbenzene); lysine; ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine; HEPBS (N- (2-hydroxyethyl) piperazine-N' - (4-butanesulfonic acid)); HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid); 2-hydroxy-3-morpholinopropane sulfonic acid; 2- (N-morpholino) ethanesulfonic acid; methanesulfonic acid; a formylhydrazine; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquiniose; trifluoromethanesulfonic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; heptanoic acid; octanoic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; eicosanoids; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan.

In some embodiments, polymerization of the feed sugar is achieved by step-growth polymerization. In some embodiments, polymerization of the feed sugar is achieved by polycondensation.

Feed sugar

The one or more feed sugars used in the methods of making the oligosaccharide preparation described herein may comprise one or more types of sugar. In some embodiments, the one or more feed sugars comprise monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixture thereof.

In some embodiments, the one or more feed sugars comprise glucose. In some embodiments, the one or more feed sugars comprise glucose and galactose. In some embodiments, the one or more feed sugars comprise glucose, xylose, and galactose. In some embodiments, the one or more feed sugars comprise glucose and mannose. In some embodiments, the one or more feed sugars comprise glucose and fructose. In some embodiments, the one or more feed sugars include glucose, fructose, and galactose. In some embodiments, the one or more feed sugars include glucose, galactose, and mannose.

Nutritional compositions comprising oligosaccharide preparations

Provided herein are solid nutritional compositions (powder formulations) comprising oligosaccharide preparations.

As described above, it has surprisingly been found that the oligosaccharide preparation according to the invention is efficiently formulated if adsorbed onto a silica-based product having an average particle size D of 3000 μm or less, for example 800 μm or less, preferably 500 μm or less, more preferably 350 μm or less. It has also been found that the oligosaccharide preparation according to the invention is efficiently formulated if adsorbed onto a silica-based product having an average particle size D of at least 50 μm, preferably at least 100 μm.

(i) At least 20 weight percent (wt-%) of an oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than or equal to 2, based on the total weight of the powder formulation; and wherein each fraction comprises from 1% to 90% of anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry,

In a second embodiment, the present invention relates to a powder formulation characterized in that

(iii) At least 25 wt% of a silica-based adsorbate having an average particle size D of 1200 μm or less, based on the total weight of the powder formulation.

Particle size as given herein may be measured by Malvern Master Sizer 2000 as suggested for particle size analysis via laser diffraction methods (laser diffraction light scattering) as outlined in ISO 13320-1. During such laser diffraction measurements, particles are passed through a focused laser beam. The particles scatter light at an angle inversely proportional to their size. The angular intensity of the scattered light is then measured by a series of photosensitive detectors. The plot of scattering intensity versus angle is the primary source of information for calculating particle size. For measuring the product form according to the invention, a dry powder feeder (Malvern Scirocco) was used.

Advantageously, the silica-based formulation according to the invention reduces the sensitivity of the oligosaccharide preparation to water adsorption (by a factor of 2-2.5 once adsorbed) and is free-flowing (the formulation shows a flowability (s/100/g) of > 4), and thus further improves handling and storage properties.

If desired, the particle size of the product may be analyzed by sieving. For this purpose, at least 50g are used. The product was placed on a sieve column, which was then sieved for 5min, with an amplitude set to 1.00mm. The minimum mesh size is at least 0.1mm.

The term additive as used herein refers to additives commonly used in the preparation of powder formulations for feed applications, such as in particular thickeners, such as in particular gums or cellulose derivatives, such as xanthan gum, karaya gum and/or ethylcellulose. The additive may also be an edible solvent for the oligosaccharide preparation.

A preferred embodiment of the invention is a formulation characterized in that

(i) 25 to 75 wt% of an oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than 2, based on the total weight of the powder formulation; and wherein each fraction comprises from 1% to 90% of anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry,

(iii) 20 to 75 wt%, based on the total weight of the powder formulation, of a silica-based adsorbate having an average particle size of at least 10 μm, preferably at least 50 μm.

A more preferred embodiment of the invention relates to a formulation consisting of:

(i) 30 to 70 wt% of a synthetic oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than or equal to 2, based on the total weight of the powder formulation; and wherein each fraction comprises from 1% to 90% of anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry,

(ii) From 30 to 65% by weight, based on the total weight of the powder formulation, of a silica-based adsorbate having an average particle size of at least 10 μm, preferably at least 50 μm,

(iii) 0 to 21 wt% of water, based on the total weight of the powder formulation.

Typically, to produce a powder according to the invention, the oligosaccharide preparation is optionally diluted in an edible solvent and further optionally blended with additional additives, sprayed onto or blended with the silica according to the invention.

Preferred examples of edible solvents to be used for diluting the oligosaccharide preparation are water, alcohols and mixtures of both, and optionally the additional additive is a preservative, such as sodium benzoate, citric acid.

The powder formulation according to the invention may be additionally coated with a coating commonly used in the art, such as a wax or fat. Such coatings, if present, are typically applied in an amount of 5 to 50 wt% based on the total weight of the powder form. Advantageously, the coating comprises at least one wax and/or at least one fat, the wax and/or fat having a drop point of 30 ℃ to 85 ℃.

The drop point of a material as used herein refers to the temperature (deg.c) at which the material begins to melt under standardized conditions. Thus, the material is heated for a long time until it changes the state of the substance from solid to liquid. The drop point is the temperature at which the first drop is released from the material. The determination of the drop point (tropfpukt) is carried out as described in the standard specification DIN ISO 2176.

Particularly suitable waxes to be used as coating in the context of the present invention include organic compounds consisting of long alkyl chains, natural waxes (vegetable, animal) which are generally esters of fatty acids and long-chain alcohols, and synthetic waxes which are long-chain hydrocarbons lacking functional groups.

Particularly suitable fats to be used as coating in the context of the present invention include a broad group of compounds which are soluble in organic solvents and largely insoluble in water, such as hydrogenated fats (or saturated fats) which are typically triesters of glycerol and fatty acids. Suitable fats may be of natural or synthetic origin. The (poly) unsaturated fat may be hydrogenated to obtain hydrogenated (saturated) fat.

Preferred examples of waxes and fats to be used as coating according to the invention are glycerol monostearate, carnauba wax, candelilla wax, sugar cane wax, palmitic acid, hydrogenated cottonseed oil of stearic acid, hydrogenated palm oil and hydrogenated rapeseed oil, and mixtures thereof.

All the above disclosed formulations can be used as such or in feed products.

In another embodiment, the invention relates to the use of a silica-based product having an average particle size D (v, 0.5) as defined above of ∈800 μm, preferably D (v, 0.5) selected from the range of 200 μm to 500 μm, more preferably in the range of 200 μm to 400 μm, for enhancing the storage stability and handling properties (reduced water adsorption sensitivity and stable flowability) of an oligosaccharide composition.

Preferably, the amount of the formulation in the feed product is selected such that the oligosaccharide preparation is present in the animal feed at a concentration of from about 1ppm to about 10000ppm, from about 1ppm to about 5000ppm, from about 1ppm to about 3000ppm, from about 100ppm to about 2000ppm, from about 100ppm to about 1000ppm, from about 100ppm to about 500ppm, from about 100ppm to about 400 ppm.

The term feed product refers in particular to poultry and swine feed compositions, as well as feed additives.

The invention is illustrated by the following examples.

Examples

Example 1: synthesis of an oligosaccharide-galactose preparation

The synthesis of the galacto-oligosaccharide preparation is performed in a three liter reaction vessel using a catalyst loading, reaction time and reaction temperature selected to enable suitable production on a kilogram scale.

D-glucose monohydrate (825.16 g), D-lactose monohydrate (263.48 g) and 2-pyridinesulfonic acid (1.0079 g, sigma-Aldrich, st. Louis, U.S. Pat.) were charged to a three liter three neck round bottom flask with a center 29/42 ground glass joint and two 24/40 side ground glass joints. A 133mm Teflon stirring blade was fixed to a glass stirring shaft using PTFE tape. A stirring rod was fastened through the center point using a Teflon bearing adapter and attached to an overhead high torque mechanical mixer via a flexible coupler. The flask was secured within a hemispherical electric heating mantle operated by a temperature control unit via a J-shaped rod thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted to reside in the reaction mixture with a gap of a few millimeters above the mixing element. An auxiliary temperature probe connected to an auxiliary temperature monitor is also inserted and fastened by the same means. The second side port of the flask was equipped with a reflux condenser cooled by a water-ethylene glycol mixture maintained below 4 ℃ by a recirculating bath cooler.

The reaction mixture was gradually heated to 130 ℃ while continuously mixing at a stirring rate of 80-100 rpm. When the reaction mixture reached 120 ℃, the reflux condenser was repositioned to a distillation configuration in which the distillate was collected in a 250mL round bottom flask placed in an ice bath. The mixture was maintained at 130 ℃ for 6 hours with continuous mixing after which the thermocouple box was shut off. The distillation apparatus was removed and 390g of distilled water at 60℃was gradually added to the three-necked flask. The resulting mixture was stirred at 40RPM for 10 hours. About 1,250g of a viscous light amber material was collected and its concentration was measured by refractive index to be 71.6Brix.

Example 2: synthesis of an oligoglucose preparation

The synthesis of the oligoglucose preparation is performed in a three liter reaction vessel using a catalyst loading, reaction time and reaction temperature selected to enable suitable production on a kilogram scale.

D-glucose monohydrate (1,150 g) was added to a three liter three neck round bottom flask with a center 29/42 ground glass joint and two side 24/40 ground glass joints. A 133mm Teflon stirring blade was fixed to a glass stirring shaft using PTFE tape. A stirring rod was secured through the central port of the flask using a Teflon bearing adapter and attached to an overhead high torque mechanical mixer by a flexible coupling. The flask was secured within a hemispherical electric heating mantle operated by a temperature control unit via a J-shaped rod thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted to reside in the reaction mixture with a gap of a few millimeters above the mixing element. An auxiliary temperature probe connected to an auxiliary temperature monitor is also inserted and fastened by the same means. The second side port of the flask was equipped with a reflux condenser cooled by a water-ethylene glycol mixture maintained below 4 ℃ by a recirculating bath cooler.

The reaction mixture was gradually heated to 130 ℃ while continuously mixing at a stirring rate of 80-100 rpm. When the reaction temperature was raised to between 120 ℃ and 130 ℃, (+) -camphor-10-sulfonic acid (1.16 g, sigma-Aldrich, st.louis) was added to the three-necked flask and the apparatus was switched from the reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. This setting was maintained for 1 half hour, after which the thermocouple box was turned off, the distillation apparatus was taken out, and 390g of distilled water at 23 ℃ was gradually added to the three-necked flask. The resulting mixture was stirred at 40rpm for 10 hours until the moment of collection. About 1300g of viscous deep amber material was collected and measured to a concentration of 72.6brix.

Example 3: synthesis of an oligosaccharide-mannose preparation

The gluco-mannose oligomer preparation was prepared as two separate components synthesized in separate reaction vessels that were collected separately. Each synthesis uses a different starting reactant, but is completed following the same procedure and method. The final oligosaccharide-mannose preparation is a homogeneous syrup formed by mixing the two synthetic products.

For the synthesis of the first component, 1264.80g of glucose monohydrate was added to a three liter three-necked round bottom flask with one central 29/42 break-in joint flanked by two 24/40 break-in joints. A133 mm Teflon stirrer blade was secured to a 440mm glass stirrer shaft using PTFE tape. A stirring rod was fastened through the center point using a Teflon bearing adapter and attached to an overhead high torque mechanical mixer via a flexible coupler. The flask was placed in a hemispherical electric heating mantle operated by a temperature control unit via a J-shaped rod thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted to reside in the reaction mixture with a gap of a few millimeters above the mixing element. An auxiliary temperature probe connected to an auxiliary temperature monitor is also inserted and fastened by the same means. The second side port of the flask was equipped with a reflux condenser cooled by a water-ethylene glycol mixture maintained below 4 ℃ by a recirculating bath cooler.

The reaction mixture was gradually heated to 130 ℃ while continuously mixing at a stirring rate of 80-100 rpm. Once a temperature control box reading between 120 ℃ and 130 ℃ was observed, 1.15g of (+) -camphor-10-sulfonic acid was added to the three-necked flask and the apparatus was switched from the reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. This setting was maintained for about 1 hour, after which the thermocouple box was turned off, the distillation apparatus was taken out, and 390g of distilled water at 23 ℃ was gradually added to the three-necked flask. The resulting mixture was stirred at 40rpm for 10 hours until the moment of collection. About 1350g of viscous light amber material was collected and measured at a concentration of 71.8brix.

For the synthesis of the second component, 949.00g of glucose monohydrate, 288.00g of pure mannose from wood, 27.94g of distilled water and 1.15g of 2-pyridinesulfonic acid were added to a three liter three-necked round bottom flask with one central 29/42 ground joint flanked by two 24/40 ground joints. The remainder of the second component synthesis followed the same procedure and method as the first component until the moment of collection, except that no (+) -camphor-10-sulfonic acid was added when switching the reflux condenser to the distillation configuration, and the resulting set-up was maintained for about 6 hours. About 1350g of viscous deep amber material was collected and measured at a concentration of 72.0brix.

All of the first and second components were transferred to HDPE containers of appropriate size and thoroughly mixed manually until homogeneous. The final syrup mixture was about 2.7kg, dark amber in color, viscous, and had a concentration of about 72Brix as measured by refractive index.

Example 4: synthesis of an oligosaccharide-mannose preparation

Kilogram scale production of the oligosaccharide preparation was performed in a three liter reaction vessel using catalyst loading, reaction time and reaction temperature found to be suitable for 1kg scale production.

For the synthesis of the first component, 1261.00g of glucose monohydrate and 1.15g of 2-pyridinesulfonic acid were added to a three liter three neck round bottom flask with one central 29/42 ground joint flanked by two 24/40 ground joints. A133 mm Teflon stirrer blade was secured to a 440mm glass stirrer shaft using PTFE tape. A stirring rod was fastened through the center point using a Teflon bearing adapter and attached to an overhead high torque mechanical mixer via a flexible coupler. The flask was secured within a hemispherical electric heating mantle operated by a temperature control unit via a J-shaped rod thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted to reside in the reaction mixture with a gap of a few millimeters above the mixing element. An auxiliary temperature probe connected to an auxiliary temperature monitor is also inserted and fastened by the same means. The second side port of the flask was equipped with a reflux condenser cooled by a water-ethylene glycol mixture maintained below 4 ℃ by a recirculating bath cooler.

The reaction mixture was gradually heated to 130 ℃ while continuously mixing at a stirring rate of 80-100 rpm. Once a temperature control box reading between 120 ℃ and 130 ℃ was observed, the apparatus was switched from the reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. This setting was maintained for about 6 hours, after which the thermocouple box was turned off, the distillation apparatus was taken out, and 390g of distilled water at 23 ℃ was gradually added to the three-necked flask. The resulting mixture was stirred at 40rpm for 10 hours until the moment of collection. About 1250g of viscous light amber material was collected and measured to a concentration of 73.5brix.

For the synthesis of the second component, 949.00g of glucose monohydrate, 288.00g of pure mannose from wood, 28.94g of distilled water and 1.15g of 2-pyridinesulfonic acid were added to a three liter three-necked round bottom flask with one central 29/42 ground joint flanked by two 24/40 ground joints. The remainder of the synthesis of the second component follows the same procedure and method as the first component until the moment of collection. About 1250g of viscous deep amber material was collected and measured to a concentration of 73.3brix.

All of the first and second components were transferred to HDPE containers of appropriate size and thoroughly mixed manually until homogeneous. The final syrup mixture was about 2.5kg, dark amber in color, viscous, and measured at a concentration of about 73brix.

Example 5: synthesis of an oligosaccharide-galactose preparation

The 3L three-necked flask was equipped with an overhead mixer connected to a 14cm crescent shaped mixing element via a 10mm diameter glass stirring shaft. The mixing element was positioned at about 5mm spacing from the flask wall. The flask was heated by a hemispherical electric heating mantle powered by a temperature control unit connected to a rod thermocouple probe inserted into the reaction flask. The thermocouple probe was placed to provide a spacing of 5-10mm above the mixing element. The flask was charged with 576 grams of food-grade dextrose monohydrate and 577 grams of food-grade D-galactose monohydrate and heated to about 115 ℃ to obtain a molten syrup. Once syrup was obtained, the flask equipped with a jacketed reflux condenser was cooled to 4 ℃ by recirculating cooled glycol/water and temperature. 31 g of Dowex Marathon C (H with a moisture content of 0.48 g) ₂ O/g resin) is added to the mixture to form a stirred suspension. The condenser was repositioned to the distillation configuration and the suspension was heated to 145 ℃.

The mixing rate of about 80RPM and a temperature of 145 ℃ were maintained for 3.8 hours after which the set point on the temperature control unit was reduced to 80 ℃ and 119mL of 60 ℃ deionized water was gradually added to the flask to obtain a deep amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60Brix, cooled to room temperature, and vacuum filtered through a 0.45 μm filter to remove the resin. A light amber syrup with a concentration of 60Brix of 1, 200 g was obtained.

Example 6: synthesis of an oligoglucose preparation

The 3L three-necked flask was equipped with an overhead mixer connected to a 14cm crescent shaped mixing element via a 10mm diameter glass stirring shaft. The mixing element was positioned at about 5mm spacing from the flask wall. The flask was heated by a hemispherical electric heating mantle powered by a temperature control unit connected to a rod thermocouple probe inserted into the reaction flask. The thermocouple probe was placed to provide a spacing of 5-10mm above the mixing element. The flask was gradually filled with 1,148 grams of food grade glucose monohydrate and heated to about 115 ℃ to obtain a molten syrup. Once syrup was obtained, the flask fitted with a jacketed distillation condenser was cooled to 4 ℃ by recirculating cooled glycol/water. The reaction temperature was gradually increased to 145 ℃. Once the temperature reached and stabilized, 31 grams of Dowex Marathon C (H2O/g resin with moisture content of 0.48 g) was added to the mixture and the mixing rate of about 80RPM and temperature of 145 ℃ was maintained for 3.8 hours.

After 3.8 hours, the set point on the temperature control unit was lowered to 80 ℃ and 119mL of 60 ℃ deionized water was gradually added to the flask to obtain a dark amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60Brix, cooled to room temperature, and vacuum filtered through a 0.45 μm filter to remove the resin. A deep amber color of the oligoglucose syrup was obtained at a concentration of 1,113 g of 60 Brix.

Example 7: synthesis of solid oligosaccharide preparations

Particle size measurement: the method described below follows the recommendations outlined in ISO13320-1 for diffractive light scattering techniques.

Particle sizes of the various silica grades can be measured by Malvern Master Sizer according to the recommendations of ISO13320-1 regarding diffractive light scattering techniques. An aliquot of a minimum 5 grams of material tempered at 25-35% rh to 55% rh was sampled into the shaker hopper of a dry dispersion unit (sicco). The flow aperture of the distributor sluice was set such that the product was flowed through the measurement zone for 30 seconds at 50% vibratory feed rate using polyethylene tubing. Sample measurements were taken at a dispenser pressure of 0.1 bar for 30 seconds, then taken 30000. The sample passes through a focused beam of light (helium neon laser for red light and solid state light source for blue light) and scatters light, allowing measurement of particles between 0.02 microns and 2000 microns. The volume median particle diameter d (0.5) was determined using the Fraunhofer approximation.

Preparation of the preparation:as listed in table 1, the optionally preheated heat is applied according to examples 1 to 9The different types of oligosaccharide preparations (syrups) are added to the silica-based adsorbate with gentle stirring, mixed in a mixer (standard mixer such as, for example, a conical screw mixer, a paddle mixer or a screw mixer) until adsorption is completed and a free-flowing powder is obtained.

TABLE 1

1: silica-based double adsorbates: de=diatomaceous earth/as=amorphous precipitated silica

D=average particle diameter (D0.5)

2: syrup optionally preheated prior to mixing

3: mixing time (after addition)

4: mixing under vacuum or non-vacuum

5: added carrier/syrup ratio

Physical properties are shown in table 2.

The silica-based formulation according to the invention reduces the sensitivity of the oligosaccharide preparation to water adsorption (by a factor of 2-2.5 once adsorbed) and is free-flowing (the formulation shows a flowability (s/100/g) of > 4), and thus further improves handling and storage properties.

TABLE 2

1: oligosaccharide content as claimed

Example 8: evaluation of physical stability of oligosaccharide adsorbates

The storage behavior of two adsorbates containing fixed levels of oligosaccharides has been evaluated. Materials as described in table 3 were used for the test. The description is performed at room temperature.

TABLE 3 manufacturing parameters for producing the materials tested

AS = amorphous precipitated silica

Method

The physical stability of the powder was tested using a climatic chamber set at 52.5 ℃ and 60% rh. The evaluation was performed visually.

Results

Samples were stored in a climatic chamber at 52.5 ℃ and 60% rh for 3 days. After an elapsed period of time, an assessment of the powder state (molten/viscous or powdery) was performed. The results can be observed in fig. 1.

Figure 1 shows the material after exposure to test conditions. The T1 material showed evidence that release of adsorbed fluid resulted in a doughy consistency. The particles stick together, forming a soft mass that is difficult to handle. Some of the fluid appears to be released and forms a ring (arrow) around the bolus. T2 materials exposed to the same conditions exhibited unchanged properties. The powder remains flowable and the individual particles do not change structure nor melt/adhere to each other. Surprisingly, by using the same carrier with the same amount of fluid, the stabilizing effect can be observed by varying the conditions under which the powder is manufactured.

Example 9: heating the solid oligosaccharide preparation by in-process solid synthesis

The oligosaccharide preparation was adsorbed onto diatomaceous earth under various process conditions and loadings. It was determined that certain process conditions and loadings resulted in a stable, flowable powder form, while certain process conditions resulted in an unacceptable product form.

Preparation of the preparation: the oligosaccharide preparation was formulated onto powdered diatomaceous earth (Perma-Guard EGP-DE-50C fossil flakes) having an average particle size d=50 microns and an initial moisture content of less than 5% by weight. A predetermined mass of carrier material was loaded into a 10L, 600W overhead planetary mixer unit. The mixing vessel is equipped with an external heat trace and thermocouple to control the solids temperature to a predetermined temperature set point throughout the adsorption process. The oligosaccharide preparation is provided as an aqueous syrup having a concentration of between about 60% to about 70% by weight dissolved solids as determined by the calibrated refractive index. The aqueous oligosaccharide syrup was gradually added to the mixer with a peristaltic pump at a predetermined pump flow rate. The temperature of the syrup is maintained at a predetermined temperature set point using an in-line heat exchanger.

Five batches were run under different process conditions of syrup temperature, mixer solids temperature, syrup concentration, and syrup addition rate as described in table 4.

TABLE 4 batch formulation parameters

/>

Determination of oligosaccharide Loading: the mass loading of the oligosaccharide preparation onto the solid support was determined by extraction and refractive index as follows. An aliquot of about 1 gram (mass recorded as + -0.1 mg) was dispensed into a 15mL conical centrifuge tube and suspended in 10.00mL deionized water. The suspension was mixed by vortex stirring for 30 seconds and allowed to stand for 10 minutes. The vortex stirring process was repeated two more times. The resulting extract was centrifuged at 2,500RPM for 10 minutes. The supernatant was removed by pipette and its carbohydrate content was determined by refractive index using a calibrated meter (Hanna Instruments HI96801, digital refractometer). Determination of the solubilized oligosaccharide preparation with reference to a standard calibration curve The mass, and the oligosaccharide content of the adsorbate was determined as the mass ratio of the extracted oligosaccharide preparation to the mass of the initial solid aliquot. Refractive index measurements were performed up to five replicates and the individual replicates were averaged to obtain a measurement result.

Determination of moisture content: the moisture content of the formulated adsorbate is determined gravimetrically by heating to a constant weight or by moisture balance. For the moisture balance determination, about 5 gram aliquots of the solid adsorbate were added to the tray of a halogen heated meter (Mettler Toledo HE moisture balance), with the final temperature set at 120 degrees celsius.

Determination of moisture absorption stability: the moisture absorption stability of the adsorbate is determined by placing an aliquot of the solid adsorbate into an environmental chamber having a fixed temperature and water activity atmosphere. The four chambers are configured to measure hygroscopicity at 20 degrees celsius and water activity of 0.378, 0.576, 0.753, and 0.843 using saturated water reservoirs of magnesium chloride, sodium bromide, sodium chloride, and potassium chloride, respectively. The moisture exchange of the form with its atmosphere was determined gravimetrically and the final moisture content of each sample was determined after 14 days of equilibration. The final material was visually assessed for tackiness, clumping, and flowability through a 6 mm circular orifice.

Stability results were obtained as described in table 5.

TABLE 5 adsorbate loading and stability Properties

Verification of chemical stability: the representative chemical structure properties of the oligosaccharide preparation were confirmed to be unchanged by the adsorption process. The number average molecular weight (Mn) and weight average molecular weight (Mw) of the source oligosaccharide syrup and the oligosaccharides extracted from the adsorbate were determined by size exclusion high performance liquid chromatography (SEC/HPLC). Injecting 1Brix aqueous solution into Gel Permeation Chromatography (GPC) column equipped with temperature of 40deg.C and isocratic elution at 0.625mL/min using 0.05% trifluoroacetic acid aqueous solution as mobile phaseAgilent PL aquagel-OH, 300X 7.5mm, # PL1120-6520, and corresponding guard columns) and Refractive Index (RI) measurements were performed at 40 ℃. The molecular weight is determined from a calibration curve obtained with known Mn and Mw using a true amylopectin standard. The results of HPLC analysis of representative extracts are provided in table 6. Furthermore, by 2D ¹ H- ¹³ The C HSQC NMR spectroscopy determined the glycosidic bond distribution of the oligosaccharide preparation and confirmed that the bond distribution of the oligosaccharide preparation extracted from the adsorbate did not change measurably from the bond distribution of the preparation prior to adsorption.

TABLE 6 Polymer Properties of adsorbed oligosaccharide preparations

Example 10: synthesis of solid oligosaccharide preparations by spray adsorption using two-fluid nozzle

The oligosaccharide preparation was adsorbed onto diatomaceous earth using a two-fluid spray nozzle without the need to heat the solids during adsorption as in the method of example 9. With two different sized carriers, a moisture-absorbing stable, flowable powder form with an oligosaccharide loading of more than 40 wt.% was obtained.

Preparation of the preparation: two different qualities were used to formulate oligosaccharide preparations onto powdered diatomaceous earth: (1) EGP-DE-50C fossil shell powder (Perma-Guard, US) having an average particle diameter d=50 microns, and (2) having a particle diameter distribution of about 200 to 600 micronsDI10KF particulate clay (Imerys, france). A predetermined mass of carrier material was loaded into a 10L, 600W overhead planetary mixer unit. The oligosaccharide preparation was delivered as a 70 wt% aqueous syrup atomized with pressurized air as the atomizing fluid using an external mixing two-fluid nozzle (bite XAEF 100,BETE Fog Nozzle Inc,US) by continuous mixing at ambient temperature. The gas stream was used to provide 10kg of air at a pressure of 40psi (276 kPa) in the nozzleGas/kg syrup. The syrup addition was continued until the desired loading was achieved. The oligosaccharide loading and moisture content of the resulting powder adsorbate formulation were determined using the method of example 9, as described in table 7.

TABLE 7 solid adsorbate preparation

The flowability and moisture absorption stability of the two solid forms, example 10.1 and example 10.2, were confirmed using the method described in example 9.

EXAMPLE 11 comparative example Using calcium carbonate

The process of example 9 and example 10 was repeated using calcium carbonate (FGCC 50, FCC grade powder, 325 mesh, duda Energy, US) as carrier material. None of the set of process conditions evaluated with syrup temperatures between ambient temperature and 80 ℃, solids mixing temperatures between ambient temperature and 90 ℃, air flow rates between 0.1cfm and 4cfm, and air pressure between 5psig and 50psig allowed for a stable, flowable powder with oligosaccharide loadings of greater than 18 wt%.

Claims

1. A storage stable powder formulation comprising

2. A storage stable powder formulation comprising

(i) At least 20 wt-%, preferably 25 wt-% to 75 wt-%, based on the total weight of the powder formulation, of an oligosaccharide preparation comprising at least n oligosaccharide fractions, each oligosaccharide fraction having a different degree of polymerization (DP 1 fraction to DPn fraction) selected from 1 to n, wherein n is an integer greater than or equal to 2; and wherein each fraction comprises from 1% to 90% of anhydrosubunit-containing oligosaccharides, as measured by mass spectrometry,

(iii) 30 to 75% by weight, based on the total weight of the powder formulation, of a silica-based adsorbate having an average particle size D < 1200 μm.

3. A storage stable powder formulation comprising

(ii) 30 to 70 wt%, based on the total weight of the powder formulation, of a silica-based adsorbate having an average particle size of at least 50 μm,

4. The storage-stable powder formulation of any one of the preceding claims, wherein the silica-based adsorbate is amorphous precipitated silica (AS).

5. The storage-stable powder formulation of any one of the preceding claims, wherein the silica-based adsorbate is Diatomaceous Earth (DE).

6. The storage-stable powder formulation according to any one of the preceding claims, wherein the particle size D of the silica-based adsorbate is selected from the range of 100 μιη to 500 μιη, preferably 200 μιη to 500 μιη.

7. The storage-stable powder formulation according to any one of the preceding claims, wherein the particle size D of the silica-based adsorbate is preferably selected from the range of 200 μιη to 300 μιη.

8. The storage stable powder formulation of any one of the preceding claims, wherein the relative abundance of oligosaccharides in each of the n fractions monotonically decreases with its degree of polymerization.

9. The oligosaccharide preparation of any one of claims 1-8, wherein n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

10. The oligosaccharide preparation of any one of claims 1-9, wherein at least one fraction comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit-containing oligosaccharides by relative abundance.

11. The oligosaccharide preparation of any one of claims 1-10, wherein at least one fraction comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharides by relative abundance.

12. The oligosaccharide preparation of any one of claims 1-11, wherein each fraction comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro subunit-containing oligosaccharides by relative abundance.

13. The oligosaccharide preparation of any one of claims 1-12, wherein the weight average molecular weight of the preparation is about 300g/mol to 5000g/mol, 500g/mol to 5000g/mol, 700g/mol to 5000g/mol, 500g/mol to 2000g/mol, 700g/mol to 1500g/mol, 300g/mol to 2000g/mol, 400g/mol to 1300g/mol, 400g/mol to 1200g/mol, 400g/mol to 1100g/mol, 500g/mol to 1300g/mol, 500g/mol to 1200g/mol, 500g/mol to 1100g/mol, 600g/mol to 1300g/mol, 600g/mol to 1200g/mol, or 600g/mol to 1100g/mol.

14. The oligosaccharide preparation of any one of claims 1-13, wherein the weight-average molecular weight of the preparation is about 2000g/mol to 2800g/mol, 2100g/mol to 2700g/mol, 2200g/mol to 2600g/mol, 2300g/mol to 2500g/mol, or 2320g/mol to 2420g/mol.

15. The oligosaccharide preparation of any one of claims 1-14, wherein the number average molecular weight of the preparation is about 1000g/mol to 2000g/mol, 1100g/mol to 1900g/mol, 1200g/mol to 1800g/mol, 1300g/mol to 1700g/mol, 1400g/mol to 1600g/mol, or 1450g/mol to 1550g/mol.