Method for isolation of oligosaccharides containing sialic acid and compositions produced in this connection that contain oligosaccharides containing sialic acid. RU patent 2496342.

FIELD: food industry.

SUBSTANCE: invention relates to a method for isolation of oligosaccharides containing sialic acid, in particular - sialyllactose, from a milk flow. The method involves a milk flow treatment at the anion exchange stage using an anion exchange resin in the form of a free base with moisture content equal to 30-48% with a subsequent anion exchange stage, preferably - with the help of a macroporous anion exchange resin or an anion exchange resin. Additionally proposed is a composition where sialyllactose content is equal to 4 wt % in terms of dry substances weight with the ratio of sialyllactose to phosphorus exceeding 6. The composition is used in a baby feeding products as well as in products for therapeutic or enteral alimentation.

EFFECT: invention enables production of a product with a high content of sialyllactose and a low content of phosphorus-containing compounds.

16 cl, 1 dwg, 4 tbl, 4 ex

The invention relates to a method of selection of oligosaccharides, which contain Deputy formed from the acid (hereinafter in this description - oligosaccharides containing acid), and preferably, compositions containing , and in particular, such compositions, in which the ratio containing acid oligosaccharides to phosphorus, and preferably, attitude to phosphorus is relatively high. In addition, the invention relates to the production and obtained songs and the use of such compositions, especially in children products.

is a milk oligosaccharide, which is located in the milk of mammals. It is known that the concentration of such a dairy oligosaccharide, such as cow's milk, is very low; in human milk is in concentrations that are certainly much higher.

has several important functions. It is known that she has prebiotic activity, and described her action. In addition, is that enables it to play an important role in dampening or prevention of intestinal infections by linking pathogenic bacteria and/or viruses, and/or released toxins, so that could be prevented, on the one hand, colonization and, on the other hand, can be prevented by the action of toxin. There have also been reports that can promote brain development in children.

Such positive action is also attributed to other containing acid.

Because, as a rule, the objective is to make food for infants and newborns similar to human milk as much as possible, there is a need (especially for the drafters of milk mixtures for infants) enrich such products containing acid, and preferably, . It is also one of the tasks of the present invention.

To get oligosaccharides containing acid in General, and in particular, in the prior art already offered various ways. A large number of these methods aimed at enzymic obtaining compounds containing group, such as , and in particular, the use of enzymes derived from genetically modified organisms, by which means, for example, using , and/or neuraminidase, you can get containing group, which you can then highlight the further processing.

In addition, the known number of ways through which acid and separated from the milk through a combination of chromatographic methods and/or method of membrane filtration.

A significant disadvantage of the existing methods of extraction and acid is that they give the necessary connections in relatively low quantities or concentrations, and, in addition, at such methods in song has a high content of phosphorus. Known publication in which it is reported that the products are phosphorylated to high extent digested bad, at least, worse, it may cause harmful interference to the absorption by the body of iron and calcium, and usually form complexes with divalent cations, necessary for normal physiological processes and the gastrointestinal tract, and, in addition, can be suppressed the action of certain enzymes in the gastrointestinal tract. This can lead to problems, especially for foods for infants and newborns.

In the composition of the milk for baby food phosphorus is determined, in particular, protein supplements. Additional phosphorus through other ingredients are not recommended and there are even recommending against this introduction. Also for this reason it is envisaged to provide compositions oligosaccharides containing acid, and in particular, compositions containing , in which the ratio groups to phosphorus is so high as possible.

The present invention is the way in which use the methods of separation, leading to a product with a relatively small number-containing compounds relatively oligosaccharide containing acid. This makes the product is highly suitable for use in children products.

This method is also described in the US 6623954 B1. This publication describes the way in which the milk flow containing lactose and , enter in contact with and beta-, then milk RSS subjected nanofiltration and/or ultrafiltration, whereby the desired remains in peptide fractions; after the stage of denaturation and the subsequent microfiltration receive faction. Optional front of the stage at which the milk flow is injected into contact with enzymes, you can spend the stage at which removed positively charged materials, for example, using or reverse osmosis.

A significant drawback of this method is that in the result of use of the membranes substantial part , including , cannot be removed, and that may take a long time before you get a product that meets with low phosphorus to .

The present invention is performed without the use of enzymes to obtain a low content of phosphorus. Further, the invention allows to extract with a high degree of efficiency (high yield) and selectivity (high purity).

With this purpose the invention of the first aspect relates to a method of allocating from the stream skim and, preferably, a protein-free milk which milk flow treated at the stage of anionic exchange with the use of anion exchange resin, preferably in the form of free base. Resin also characterized by moisture content 30-48%. At the next stage associated with anion-exchange resin, preferably, a macroporous type or gel type. Macroporous or gel type anion-exchange resin preferably has a moisture content is higher than 45%, and most preferably 50 to 70%.

The first type of anion exchange resin, characterized by moisture content 30-48%, preferably represents gel type, which milk flow, although it is not linked oligosaccharides containing group, and, in particular, that are also present in salt form. In other words, such includes anion-exchange resin which has selectivity for negatively charged inorganic materials, but not in respect of . For this purpose it is necessary, that the moisture content, i.e. water content does not exceed 48%, and preferably not exceed 45%. At a moisture content below 35%, and more so, at a moisture content below 30%, ability begins too decline to process was effective.

The moisture content of the determined in the following way. Before measuring the moisture content in the resin remove the bound water, for example, turning the resin in the fabric and then putting them to the process of centrifuging (centrifuge with a diameter of 30 cm, 3000 rpm); further resin weighed, for example, in ; after what the resin is dried within 4 hours at a constant temperature of 105 C; then pitch cooled in a desiccator for 30 minutes, after which, in turn, determine the mass of dry resin; the percentage moisture content (wt.%)=[(mass after drying (g)/(wet weight resin)]•100%.

With this much is removed a substantial part of negatively charged ions, and through this does not happen removal of significant amounts of (despite the negative charge).

The specified anion-exchange resin is preferred, as a rule, is free base form ( form), as it leads to the greatest possible demineralization plant capacity. Suitable anion-exchange resin are strongly stitched gels polystyrene divinylbenzene, such as Diaion SA20A, Diaion WA20A (Mitsubishi).

The authors found that the moisture content of anion exchange resin, used for desalination, is important for ash removal (negatively charged inorganic substances), on the one hand, and (SL), on the other hand. With this purpose tested a large number of tar in a control.

Water content (%)

Removing SL

Mitsubishi

Diaion SA20A

Mitsubishi

Diaion WA20A

Applexion

Applexion

Applexion

Dowex 1-X8

Marathon A2

Under these definitions, terms of control following.

Collect unit consisting of two columns. The first column contains 15 ml resin Marathon (Dow; form H + ), the second column contains in form. Column check, passing concentrate NF (see also the example 1) at a rate of 0.5 ml/min After the introduction of approximately 5 volumes layer, you can define removing SL. The ratio between moisture content obtained removing SL next plotted in figure 1.

In order to not get any loss during this process, the pH of the resulting product is preferably from pH 3 to pH 9.

In order to do so, in a preferred embodiment of the present cations (in particular, sodium and potassium) are also removed by means of a cation-exchange resin. Cation-exchange resin is in the form of N+. The preferred configuration for first remove cations, and then anions.

After stage desalination flow of demineralized milk enter in contact with the anion-exchange resin, preferably, macroporous or gel type, which is capable of capturing and link . Theoretically can communicate with any resin, as inorganic salts have been removed. To strengthen the efficiency, preferably, the moisture content of the macroporous or gel type anion exchange resin is more than 45%, and preferably, it ranges from 50 to 70%. In these resins have larger pores than in used at the previous stage. allows easy elution of resin small amount of common salt or salt solution, preferably, sour salt solution.

The resulting eluate neutralize preferably by dilute acids like hydrochloric acid to pH pH 3 to pH 9. Preferably, the pH of the final product is a pH of 6.5

Examples of suitable to associate resins are

Diaion SA21A, content water 55-65%;

Diaion HPA25, water content 58-68%;

Diaion WA10, water content 63-69%;

Dowex 1 x 4, the water content of 55 to 63%;

Dowex Marathon And water content 60-72%.

To adsorb , resin should be preferably in the form of free base. The temperature at the stage of anionic exchange is preferably below 20 C and preferably below 10 degrees Celsius

After the elution of resin can get a product with the content over 4 wt.% preferably, more than 5 wt.%, most preferably, more than 8 wt.% for example, around 10%, relatively dry matter. By the way, the content of relatively dry matter can be further increased by using conventional methods, such as, for example, nanofiltration. As a rule the content in the product obtained according to the method according to the invention, varies from 8 to 15%, although it is possible to achieve higher concentrations. One of the ways to achieve even higher concentrations is the leaching of larger amounts of lactose of anion-exchange resins. This can be accomplished by extending the washing of the water column before salt.

Milk flow treated anion-exchange resin contains no fat, and if it does, at least, less than 0.5 preferable to less than 0.2 and most preferably less than 0.1%of mass. Very preferable for such a dairy faction of the lack of protein. A special advantage is the original thread serum, and more on the filtrate serum (whey permeate). By the way, is not critical circumstance of any mammal happens milk, and good results can be obtained when the source material is cow's milk, sheep's milk, goat's milk, camel milk or a horse's milk.

In a preferred embodiment of milk flow, and more on RSS serum, and it is most preferable, RSS filtrate serum, before being subjected to any of the two stages of anionic exchange, . At this stage of softening polyvalent ions, such as calcium ions and phosphate ions are removed from the milk flow. This can be done, for example, precipitation respective ions or through the exchange of these ions, such as sodium ions and chloride ions through the stages of ion exchange. Such an incarnation is illustrated below in example 1.

In another preferred embodiment of the invention refers to the manner in which milk flow, before being subjected to any of the two stages of anionic exchange process stages nanofiltration a concentrate, has in absolute number, the lower (in comparison with the original thread to nanofiltration) the number of single-charged ions and non-protein nitrogen compounds, and in particular, urea. Such an incarnation is illustrated below in example 2.

Using the method according to the invention, receive high-mass ratio to phosphorus. As a rule, such a ratio according to the invention, at least, equal to 6 or more 6, preferably, is 7 or above 7, preferably, equal to 9 or above 9, and more preferably, 10 and above 10.

To calculate the specified relation determine the content as the sum of 3'-SL and 6'-SL per unit mass of the product.

In particular, the concentration of determined by chromatographic method on (column RA-1, Dionex). detect pulsed detector. Contents calculated as the sum of 3'- and 6'-.

The phosphorus content is defined as total phosphorus (after decomposition of nitric acid per unit mass of the product according to «Official method 973.55 AO AU (analysis of phosphorus in the water)».

Then calculate the ratio to phosphorus.

By the way, the known ways, which the isolated of milk, as a rule, lead to products in which the ratio of /phosphorus below 5.

For the second aspect of the invention refers to a composition containing oligosaccharides containing acid, which can be obtained through the method according to the invention, and the content of dry substance is at least 4 wt.% and mass ratio /phosphorus exceeds 6. In such a composition, in addition to are acid, lactose, non-protein nitrogen compounds and, maybe protein.

Preferred embodiment have the above mentioned components mass ratio.

In the third the aspect of the invention refers to the application of composition according to the invention, contains oligosaccharides containing acid, and especially , food for children, and in particular, such that get the contents , appropriate to its content in human milk.

This not in this means that cannot be used in other applications, such as products for therapeutic and enteral nutrition, or, for example, for cosmetic use as an ingredient in air conditioners for skin care, related to the modulation of the activity of pigments and/or restoration of damage to the skin.

Now the present invention will be explained given further examples, are not restrictive. When in the examples in the text above are percentages or parts per million), they are - unless otherwise noted, respectively, represent a massive interest and mass ppm component of the weight of the composition, including this component.

Example 1

RSS filtrate serum, with dry matter content of 6.66% passed successively through anion-exchange resin (3112 (from Applexion); chloride form) and through the cation-exchanging resin (HA 2033 (from Applexion); sodium form). In this process, depending on the applied load, find that a certain amount of phosphate-ions and calcium ions are exchanged for chloride ions and sodium ions, respectively. When contribute about 25 volumes layer (BV) filtrate serum find that you remove about 90% of those present of calcium ions, and approximately 50-60% of attendees phosphate-ions. Experimental data on a representative softening of the filtrate are summarized in table 1.

Experimental data on softening of leachate (the volume of cation-exchange resin =35 l, the volume of anion exchange resin =35 l)

The filtrate serum

product

Volume (l)

Dry matter (%)

(SL) (ppm)

Phosphate (ppm)

Calcium (ppm)

Sodium (ppm)

Chloride (ppm)

Example 2

The product obtained in example 1, concentrate using nanofiltration. performed with the use of membrane NF-2540 (DOW). During the process of filtering use pressure through a membrane in the range of 2,000 to 2,500 kPa (20-25 bar). As a result of the content of dry substances in the concentrate increases. So, the content of dry substances in the concentrate may be 15%, while it is also possible termination of filtering the content of dry substances reaches 20%. So by removing most of the present inorganic substances. Experimental results of a typical stage nanofiltration summarized in table 2.

product

Concentrate NF

Volume (l)

Dry matter (%)

Example 3

The product obtained in example 2 (200 l)with content dry substances of about 20%, successively pass through the column below.

Column 1 contains 25 liters (Applexion 748).

Column 2 contains 25 liters (Diaion SA20).

Resin transfer in the form of H + and Oh - , respectively. Flow rate, which make the product, is 100-125 l/hour. Excluding sweet-on» and including «sweet-off» you can get around 220-230 liters of product.

The product thus obtained is completely and still contains virtually all of the . As a rule, find that the output exceeds 90%. Typical results of this stage are summarized in table 3.

Typical results for desalination concentrate NF

Concentrate NF

Volume (l)

Dry matter (%)

Example 4

At the next stage present in the product sample 3, connected with a variety of . With this purpose the product (220 l) is passed through (the column volume = 5L, resin = 90, from Applexion). As a result, nearly all present associated with anion-exchange resin.

After washing the column associated SL you can extract certain amount of salt (40 l; 0.2 M NaCl solution). During the elution pH closer to neutral using hydrochloric acid up to approximately pH 6.5. Typical results are summarised in table 4.

1. A way of extracting carbohydrates containing acid, and especially from the stream skim and, preferably, a protein-free milk, where the flow of milk processed at the stage of anionic exchange with the use of anion exchange resin in the form of a free the Foundation, with a moisture content of 30-48%, with the subsequent stage of anionic exchange, preferably with the help of macroporous anion exchange resin or anion exchange resin type of gel.

2. The method according to claim 1, where the second anion-exchange resin has a moisture content is higher than 45%, and preferably 50 to 70%.

3. The method according to claim 1, where as the flow of milk to use RSS serum, and preferably filtrate serum.

4. The method of claim 2, where as the flow of milk to use RSS serum, and preferably filtrate serum.

5. The method according to claim 1, where the milk flow before working on any of the two stages of anionic exchange , preferably with the use of ion exchangers in the form of sodium/chloride.

6. The method of claim 2, where the milk flow before working on any of the two stages of anionic exchange , preferably with the use of ion exchangers in the form of sodium/chloride.

7. The method of claim 3, where the milk flow before working on any of the two stages of anionic exchange , preferably with the use of ion exchangers in the form of sodium/chloride.

8. The method according to claim 4, where the milk flow before working on any of the two stages of anionic exchange , preferably with the use of ion exchangers in the form of sodium/chloride.

9. Method according to pp.5-8, where softening carried out by precipitation of calcium and phosphate ions.

10. The method according to claim 5, where the milk flow before working on any of the two stages of anionic exchange subjected nanofiltration a concentrate, has, in absolute amount, a smaller number of single-charged ions and non-protein nitrogen compounds.

11. The method according to claim 6, where the milk flow before working on any of the two stages of anionic exchange subjected nanofiltration a concentrate, has, in absolute amount, a smaller number of single-charged ions and non-protein nitrogen compounds.

12. The method according to claim 7, where the milk flow before working on any of the two stages of anionic exchange subjected nanofiltration a concentrate, has, in absolute amount, a smaller number of single-charged ions and non-protein nitrogen compounds.

13. The method according to item 8, where the milk flow before working on any of the two stages of anionic exchange subjected nanofiltration a concentrate, has, in absolute amount, a smaller number of single-charged ions and non-protein nitrogen compounds.

14. The method of claim 9, where the milk flow before working on any of the two stages of anionic exchange subjected nanofiltration a concentrate, has, in absolute amount, a smaller number of single-charged ions and non-protein nitrogen compounds.

15. Composition containing oligosaccharides, containing acid, and especially that can be obtained using the method on any of the preceding paragraphs with the content on the dry substance, at least 4 wt.% and with a ratio of /phosphorus exceeding 6.

16. Application of the composition according to item 15 of the product infant food, products of medical or enteral nutrition.