SK7812002A3 - Oligosaccharide mixture - Google Patents

Abstract

An oligosaccharide mixture on the basis of oligosaccharides, having at least two oligosaccharide fractions, built-up by at least two different oligosaccharides, lactose being not among them, wherein the total spectrum of the oligosaccharides present in the oligosaccharide mixture differs from that of the animal milk or animal milks from which the oligosaccharide fractions were obtained, and if the oligosaccharides were obtained from only one animal milk, the ratio of neutral oligosaccharides to acidic oligosaccharides is 90 60 : 10 40 % by weight, or if the oligosaccharides were obtained from at least two animal milks, the oligosaccharides obtained from each animal milk amount to at least 10 % by weight of the oligosaccharides present in the oligosaccharide mixture, wherein the ratio of neutral oligosaccharides to acidic oligosaccharides is 90 60 : 10 40 % by weight. The mixture can be incorporated into an infant formula, a dietetic food or a pharmaceutical preparation, and may be useful in combating disorders of gastrointestinal functions caused by bacterial or viral colonization of the gastrointestinal tract, or for conserving normal gastrointestinal functions.

Description

p r pp 13 / 1-2 igosaccharide mixture

Technical field

The invention relates to an oligosaccharide composition comprising oligosaccharides which are obtained from one or more animal milk species and which are composed of two or more monosaccharide units, and to the use of the oligosaccharide composition against disorders of gastrointestinal function.

BACKGROUND OF THE INVENTION

The concentration of oligosaccharides in mature human (breast) milk is approximately 10 g / l. To date, approximately 80 oligosaccharides differing in structure, consisting of 3 to 13 monomers, have been described in human milk; Newburg, D. S. and Neubauer, S. H. (1995) Carbohydrates in Milk: Analysis, Quantity and Significance, in: Handbook of Milk Composition, Jensen, R.G., editor. Academic Press, 273-349. Structural and positional isomerism plays a major role in the structural diversity of oligosaccharides. The ratio of neutral oligosaccharides to sialized (acidic) oligosaccharides or oligosaccharide structures is about 10: 1 in mature milk.

In human, specific and non-specific defense against infections, 01-gosaccharides play the following important roles:

1. Human milk oligosaccharides are a source of substrate for bifidobacteria. This promotes normal bowel

J flora required for gastrointestinal tract function and suppresses germs; Rose, C.S., Kuhn, R., Zilliken, F., and Gyorgy, P. (1954) Bifidus factor. In the activity of α- and β-methyl-N-acetyl-D-glucosaminides, Ar. Biochem. Biophys. 49, 123-129.

2. Oligosaccharides prevent the adhesion of germs and / or substances, such as bacteria, toxins and eukaryotic parasites, by acting as specific receptor analogs, thereby preventing the first stage of infection. In various in vitro assays, the antiadhesive effect of oligosaccharides against a variety of pathogenic, microorganisms could be demonstrated; Kunz, C. and Rudloff, S. (1993) Biological Functions of Oligosaccharides in Human Milk, Acta Paediatr. 82, 903-912.

3. Oligosaccharides promote defense against infections by influencing various immunologically active cells. The immunomodulatory effect of certain oligosaccharide structures could be demonstrated in an animal model by increasing production of interleukin 10 (Velupillai, P. and Harn, D. A (1994) Oligosaccharide-specific instruction of interleukin 10 production by B 220+ cells from schistosome-infected mice: a mechanism for regulation of CD4 + T-cell subsets, Proc Natl Acad Sci 91, 1-2.

The heterogeneity of oligosaccharides from most mammalian milk species known hitherto differs in many respects from human milk oligosaccharides. Due to the composition of oligosaccharides, there are a large number of both quantitative and qualitative differences. The species differences in oligosaccharide content in mammalian milk relate primarily to differences in the concentration of the individual components as well as structures with similar characteristics and the underlying structure itself. In comparison with oligosaccharides from human milk, the sequence of the monosaccharides and the binding are different. In addition, the amount of most of the individual components in most animal milk species is significantly lower than in human breast milk.

These differences are believed to be conditioned by the neonatal gastrointestinal tract requirements for the oligosaccharide composition in milk. These requirements vary greatly depending on the species. Such differences in terms of requirements now exist not only in comparison of a newborn baby with a newborn animal, but also between different newborn animals. The differences are based on the state of function of the gastrointestinal tract of newborns, on the degree of immune system development, on species-dependent nutrition and also on the very specific environment of the individual of the species concerned.

If it is now attempting to replace human milk with artificial milk or infant formula based on animal milk, then the problem is that the milk of a single animal species, as it is known, does not have a heterogeneous structural diversity with respect to the oligosaccharides that occur in human milk.

It is an object of the present invention to provide an oligosaccharide composition consisting of oligosaccharides which are obtained from one or more kinds of milk of animals whose positive effect, and in particular whose anti-infective effect corresponds as closely as possible to that of oligosaccharides from human milk.

This object is achieved by means of an oligosaccharide mixture according to the wording of the claims.

Summary of the Invention ·

Surprisingly, it has been found that a new mixture of oligosaccharides from one as well as from various animal milk species increases the anti-infective and / or prebiotic effect of the oligosaccharides from animal milk. Therefore, the oligosaccharide composition of the invention is formed from at least two oligosaccharide fractions, each containing at least two different oligosaccharides. These oligosaccharide fractions can be derived from one kind of animal milk or from several kinds of animal milk. While this should be obvious, it is pointed out that animal milk is not human milk.

Each of these oligosaccharide fractions contains at least two different oligosaccharides. Different oligosaccharides are considered to be those which differ by at least one characteristic. Thus, for example, two disaccharides that consist of the same monosaccharide units but are linked differently are considered to be different oligosaccharides. These differences may therefore consist in the type of structure and also in the composition.

It is therefore an essential aspect of the invention that oligosaccharide fractions are produced from animal milk of one species or different species in a novel manner so as to obtain an enhanced biological effect. In this new formation or mixing, naturally, it must be ensured that no mixture is formed which corresponds to the original mixture occurring in one or more animal milk. For this reason, the total spectrum of oligosaccharides contained in the oligosaccharide mixture must be different from that of the animal milk species or animal milk species from or from which the oligosaccharide fractions were obtained. If the oligosaccharide mixture according to the invention is obtained from only one species of animal milk, then the emphasis should be placed on the overall spectrum characteristic so that the new composition or mixture of oligosaccharide fractions obtained from this single species of animal milk does not lead to the original species again. animal milk. If the oligosaccharide mixture is obtained from several types of animal milk, then the new composition or the new mixture cannot result in the obtained oligosaccharide mixture corresponding to the oligosaccharide composition of the animal milk species originally used.

Moreover, if the oligosaccharides of the oligosaccharide composition of the invention are obtained from only one species of animal milk, the ratio of neutral oligosaccharides to acidic oligosaccharides is 90 to 60: 10 to 40 wt. In other words, there is a neutral oligosaccharide fraction alongside the acidic oligosaccharide fraction. Both fractions contain at least two different oligosaccharides. By changing the ratios of the acid oligosaccharide fraction to the neutral oligosaccharide fraction compared to the ratios of the neutral oligosaccharides to the acid oligosaccharides originally existing in animal milk, a marked increase in the efficiency of their physiological effect can be achieved.

If the oligosaccharides are obtained from different, and thus at least two, kinds of animal milk, the new composition, optionally

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the novel mixture can be realized in such a way that the oligosaccharides obtained from two different kinds of animal milk are at least 10% by weight each. from oligosaccharides found entirely in the oligosaccharide mixture. If the oligosaccharides are obtained from two animal milk species, then the oligosaccharides obtained from one animal milk species must comprise at least 10% by weight, while the oligosaccharides obtained from another animal milk species also comprise at least 10% by weight. % and at most 90 wt. and vice versa. If, on the other hand, oligosaccharides are obtained from three or more types of animal milk, the oligosaccharides must comprise at least 10% by weight of only two types of animal milk. Oligosaccharides obtained from other animal milk species may comprise less than 10 wt.

All kinds of animal milk may be used as a source for the preparation of the oligosaccharide composition of the invention, preferably cow, goat, sheep, mare, camel, and buffalo milk fractions.

The isolation of the oligosaccharide fractions is carried out by known separation methods. Defatting can be performed by centrifugation using a centrifuge. Protein separation may be accomplished by ultrafiltration or precipitation with a solvent (e.g., ethanol, acetone). The lactose content may be reduced by one or more of the following methods: crystallization, filtration, chromatographic methods (ligand exchange chromatography, gel filtration, elution chromatography, recycle chromatography and simulated countercurrent chromatography (SMB)). Mineral substances are separated by electrodialysis, nanofiltration, ion exchange and / or reverse osmosis. The fractionation of oligosaccharides to neutral and acidic oligosaccharides is carried out by one of the aforementioned chromatographic methods and / or anion exchange chromatography and also alternatively by filtration.

According to a preferred embodiment, the oligosaccharide mixture according to the invention, the oligosaccharides of which are obtained from at least two kinds of animal milk, is produced in a novel manner such that also in this case the ratio of neutral oligosaccharides to acidic oligosaccharides is 90 to 60: 10 to 40 wt.

By using ratios of the present substrates of 90 to 60: 10 to 40 wt. at least all integer intermediate values are included and published, for example 89:11, 88:12, 87:13, .... 85:15, .... 80:20, 79:21, .... 75:25 74:26, 73:27, 72:28, .... 68:32, 67:33, 66:34, 65:35, 64:36, 63:37, 62:38, 61:39.

Preferably, the oligosaccharides in the oligosaccharide composition of the invention are exposed to one or more of the following monosaccharide units: D-glucose, D-galactose, DN-acetylglucosamine, DN-acetylgalactosamine, L-fucose, D-mannose, sialic acids in D-form (e.g. DN-acetylneuraminic acid and DN-glycolylneuraminic acid) as well as their O-acetylated, sulphated and phosphorylated derivatives.

Moreover, the free lactose present in large amounts in animal milk is not included in the oligosaccharides derived from animal milk which form the oligosaccharide fractions used according to the invention. However, also in the oligosaccharide mixture according to the invention there may be free lactose in addition to the oligosaccharide fractions used according to the invention.

In other words, the oligosaccharide composition of the invention may be formed exclusively from oligosaccharide fractions containing oligosaccharides which are obtained from one or more animal milk species and are composed of two or more monosaccharide units, with no free lactose. However, the oligosaccharide mixture, as mentioned, may contain free lactose, although it now originates from animal milk or not. Further, the oligosaccharide mixture may comprise one or more non-animal oligosaccharides. _f _. Thus, for example, the oligosaccharide composition of the invention may be mixed with conventional carbohydrates which are used in the preparation of nutrition and especially infant formulas. In addition, oligosaccharides which are liberated in the recovery of oligosaccharides or oligosaccharide fractions may also be included in the oligosaccharide mixture according to the invention;

used according to the invention, by chemical, enzymatic and / or technological conversion from glycolipids and / or glycoproteins.

When referring to oligosaccharides within the scope of the present disclosure, the term refers in particular to those oligosaccharides which have been obtained from animal milk. In addition, other oligosaccharides may be present, as mentioned above, and this is mentioned or implied.

A preferred source for the isolation of oligosaccharides is dairy products from which protein fractions have been partially and / or completely removed, as well as fat. The processing and fractionation of products such as the fraction of lactose and minerals resulting from the production of cream, the casein fraction and other components is clearly simplified. The supernatant produced in the manufacture of pharmaceutical lactose also represents a suitable starting source for various oligosaccharides. In this case, the milk or the whey from one animal species or different animal species can be mixed in the desired ratio before the actual fraction isolation is carried out, as necessary for an effective oligosaccharide mixture of individual structures or structural groups.

The oligosaccharide composition of the invention may be used in a high concentration form or in a pure form in disorders of gastrointestinal function in which dysbacteria play a role (ie a disorder of the gastrointestinal flora, e.g. after administration of antibiotics or other medicaments affecting / intestinal flora after food intolerance etc.). or viral infections. It can also be used to prevent such gastrointestinal infections. In addition, the oligosaccharide composition of the invention may be added to infant formulas, dietary foods or pharmaceutical compositions.

The oligosaccharide composition of the invention may also be an additive to otherwise complete nutrition. Such nutrients are, for example, infant formulas or infant formulas and enteric clinical nutrition preparations. In addition, it can be used as an additive to conventional foods to maintain normal (preventive) gastrointestinal functions as well as to improve gastrointestinal functions after disorders in which inappropriate bacterial or viral distribution in the gastrointestinal tract plays a role.

The oligosaccharide composition of the invention may be formulated as capsules, optionally in powder or in diluted form, as an additive to otherwise complete instant nutrition.

The oligosaccharide composition according to the invention is preferably used in an amount of at least 10 mg / kg to 4 g / kg body weight per day, the milligram values refer to the three oligosaccharides in the oligosaccharide composition according to the invention obtained from animal milk. Other oligosaccharides that may be available, such as lactose and non-animal oligosaccharides, are left unnoticed. Particularly preferably about 100 mg / kg per day is administered.

In the following, the invention is explained in more detail by way of examples describing preferably embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

100 l of cow's milk is processed as follows:

The fat is separated by centrifugation at 45 ° C. The resulting skimmed milk is deproteinated by ethanol precipitation (66% ethanol final concentration). The precipitated precipitate is separated by means of a centrifuge. Ethanol and some of the water are evaporated using a gravity evaporator to a volume of 40 L. It contains approximately 4 kg of lactose, 1 kg of minerals and other soluble components, such as water-soluble vitamins, urea or citrates. The proportion of oligosaccharides is approximately 10 g. The oligosaccharide fraction consists of approximately equal parts of neutral and sialylated structures. Separation into neutral (eluent: demineralized water) and acid fraction (eluent: 300 mM ammonium acetate) is carried out by anion exchange chromatography on AG 1 x 2. 60% of the lactose is removed from the neutral fraction by a crystallization technique. By means of gel chromatography on Toyopearl HW 40 (S), 5 g of a neutral oligosaccharide fraction in which the residual lactose content is approximately 10% is obtained. Anion exchange elution buffer (ammonium acetate) is removed from the sialylated oligosaccharide fraction by electrodialysis and the fraction is also lyophilized as a neutral oligosaccharide fraction. In this way 4.5 g of sialylated oligosaccharide fraction can be obtained. A new composition of 1 g acid fraction with 5 g neutral fraction yields a more active mixture than the natural composition.

Example 2

The whey resulting from the production of goat, sheep and cow's cheese is mixed before processing in a ratio of 3: 1: 1 to give 4000 l of total whey from three different animal species. Ultrafiltration with a polysulfone membrane (20 kDa cutoff) separates the residual fat, the glycomacropeptide in solution and whey proteins. The permeate is allowed to concentrate to 900 L by reverse osmosis. Another method is as described above, first by crystallizing lactose and then by anion exchange chromatography to separate the peptides. Fractions from the elution steps with demineralized water and 300 mM ammonium acetate are combined, concentrated, electrodialysed and lyophilized. A mixture of oligosaccharides of three different species in an amount of 570 g can be obtained. After further separation by gel filtration and desalting by electrodialysis, the desired product can be obtained by lyophilization. Analysis by goal filtration, HPAEC-PAD and MALDI-MS shows that, according to the milk species used and the mixing ratio, the ratio of acid oligosaccharides to neutral oligosaccharides is 1: 5. The proportion of galactosyl lactose can be calculated due to the high proportion of goat whey to 120 g. In sheep's milk, N-glycolylneuraminic acid predominates as an important component of acid oligosaccharides compared to N-acetylneuraminic acid. Therefore, the proportion of glycolylneuraminyl oligosaccharides increases compared to goat's and cow's milk.

Example 3

At the same time, grasshopper and goat milk are processed. 50 L are available from each. A fraction containing sugars and minerals is obtained by centrifugation, protein precipitation with ethanol and separation. Using a rotary evaporator, both solutions are concentrated to 8 L. Ligand exchange chromatography on AG 501 x 8 (Ca ²⁺ -form) and elution with demineralized water results in chromatographic separation of lactose and monosaccharides on the one hand and sub-fractionation of oligosaccharides on the other. A fraction is collected from the locust milk between the eluted area of the excluded volume and the tetrasaccharides, which is used for another new oligosaccharide composition. Separation of the fraction containing sugars and minerals from goat's milk in the same phase results in the formation of a total oligosaccharide fraction depleted of lactose and monosaccharides. After desalination and concomitant concentration by nanofiltration of each fraction, lyophilization is performed. The two fractions are mixed in a 2: 5 mixing ratio (cobylia oligosaccharide fraction: goat oligosaccharide fraction).

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Example 4

Goat's milk is processed as described in the example

1. Anion exchange chromatography on AG 1 x 2 yields an acid fraction, the main components of which are 2,3'- and 2,6'-sialyllactose and 2,3'- and 2,6'-N-glycolylneuraminyllactose. These fractions are further separated by chromatography on a reverse phase material to form a fraction consisting of 2,3'- and 2,6'-N-glycolylneuraminyl lactose. This was desalted by electrodialysis, spray dried and made available for further mixtures.

A fraction consisting of six neutral trisaccharides, three isomeric galactosyl lactose, fucosylated lactosamine and two lactose units elongated with N-acetylgalactosamine or N-acetylglucosamine, respectively, is isolated from cow whey by gel filtration.

The acid fraction of goat's milk, the predominant structure of which is the 2,3-isomer, is mixed 1: 7 with the neutral fraction of cow whey trisaccharides in the dry state and made available for feed trials.

Example 5

The structures of lacto-N-neotetrose and lactto-N-neohexose are isolated from locust milk. These oligosaccharides are also found in human milk.

Disialyl lactose is chromatographically isolated from cows' milk by lyophilization and lyophilized.

Two positional isomers of fucosylated tetrasaccharide are isolated from goat's milk by established methods.

Oligosaccharides are mixed in a mixing ratio of 2: 1: 2 (cows 'milk oligosaccharides; cows' milk oligosaccharides; goat's milk oligosaccharides).

Example 6

A fraction of neutral oligosaccharides consisting of the tetroses and other pentoses or hexoses with a hitherto unknown monosaccharide composition (2 hexosamines and 4 hexoses, optionally 2 hexosamines and 4 hexoses as well as 3 hexosamines and 3 hexoses) can be isolated from goat whey. From the same treatment, an acid fraction is obtained which is fractionated by anion exchange chromatography to form a mixture consisting of two different sialyllactoses and two different glycolylneuraminyllactoses. Long-chain acid oligosaccharides and neutral galactosyl lactose are not used to re-create a new oligosaccharide mixture. The isolated acid and neutral fractions were mixed in a 2: 1 ratio.

Example 7

The whey produced in cheese production usually contains, in addition to the free oligosaccharides, numerous bound oligosaccharides in the form of N- and O-glycans. The glycomacropeptide is O-glycosylated and lactoferrin is N-glycosidized. The corresponding glycan structures are largely sialized. The initial ratio of acid glycans to neutral glycans or oligosaccharides is approximately 1: 1. In order to shift this ratio in favor of neutral oligosaccharides, anion exchange chromatography is performed. All sialized and bound acid oligosaccharides (fraction 2) bind to the gel matrix and the neutral oligosaccharides (fraction 1) are eluted with demineralized water. All introduced components are eluted at high ionic strength (e.g. IM NaCl) and further processed.

Due to poor hydrolysis conditions at pH 2.0 and 45 ° C, cleavage of sialic acid is achieved within a few hours. After neutralization with a NaOH solution, this fraction 2 was combined with neutral oligosaccharides (fraction 1). The ratio of neutral oligosaccharides to acidic oligosaccharides is shifted in favor of neutral oligosaccharides.

Claims (9)

PATENT CLAIMS Oligosaccharide mixture based on oligosaccharides obtained from one or more species of animal milk and consisting of two or more monosaccharide units, characterized in that it has at least two oligosaccharide fractions, each consisting of at least two different oligosaccharides which do not include free lactose, that the total spectrum of oligosaccharides contained in the oligosaccharide mixture differs from that of the animal milk species or animal milk species from which or from which the oligosaccharide fractions are derived, and that (a) where oligosaccharides have been obtained from only one species of animal milk, the ratio of neutral oligosaccharides to acidic oligosaccharides shall be 90 to 60: 10 to 40% by weight, or (b) if the oligosaccharides have been obtained from at least two types of animal milk, the oligosaccharides obtained from two different types of animal milk are at least 10% by weight each. of the total oligosaccharides present in the oligosaccharide mixture. Oligosaccharide mixture according to claim 1, characterized in that if the oligosaccharides have been obtained from at least two types of animal milk, the ratio of neutral oligosaccharides to acidic oligosaccharides is 90 to 60: 10 to 10: 40 wt. Oligosaccharide mixture according to claim 1 or 2, characterized in that the oligosaccharides are displayed from one or more of the following monosaccharide units: D-glucose, D-galactose, DN-acetylglucosamine, DN-acetylgalactosamine, L-fucose, D-mannose , sialic acids in the D form (DN-acetylneuraminic acid and DN-glycolylneuraminic acid) as well as their O-acetylated, sulphated and phosphorylated derivatives. Oligosaccharide composition according to one of the preceding claims, characterized in that the oligosaccharides are obtained from one or more of the following types of animal milk: cow, goat, sheep, mare, camel and buffalo milk. Oligosaccharide mixture according to one of the preceding claims, characterized in that the oligosaccharides are obtained from unmixed or mixed milk or whey or unmixed or mixed dairy products from one or more animal species. Oligosaccharide mixture according to one of the preceding claims, characterized in that, in addition to the oligosaccharides or oligosaccharide fractions obtained from animal milk, it also contains lactose, which may originate from one or more animal milk species from which the animal milk is derived. obtained oligosaccharides, one or more non-animal milk oligosaccharides and / or oligosaccharides released in obtaining these oligosaccharides by chemical, enzymatic and / or technological conversion of glycolipids and / or glycoproteins. Oligosaccharide composition according to one of the preceding claims, characterized in that it is incorporated into infant formulas, dietary foods or pharmaceutical preparations or is an additive to otherwise complete nutrition. Use of an oligosaccharide composition according to one of claims 1 to 5 against disorders of gastrointestinal function caused by bacterial or viral occupation of the gastrointestinal tract or for the maintenance of normal gastrointestinal functions. The use of claim 8, wherein the oligosaccharide composition is used in an amount of 10 mg / kg to 4 g / kg body weight per day.