preparation

The subject of the present invention is a method of producing a lectin preparation meant for use as feed additive for swine.

An invention known from the Polish application description P. 375476„A method of producing a lectin preparation, a lectin preparation as well as a method of administering a lectin preparation for mammals" assumed the use of preservatives in order to protect the product in the form of a powered, did not indicate the need for use of a preservative during extraction and extract condensation, and in particular during the storage period of the extract as well as the concentrated extract in order to perform necessary activity analyses as well as during the intervening period before the initiation of further stages of production. In certain situations, the known and abovementioned description No. P.375476 allowed the apportionment of the extract of a defined biological activity directly into dosing containers, bypassing the drying stage. This, however, required standard methods of introducing a preservative prior to apportionment, and did not affect either the extraction method nor its results. Other lectin production methods known from literature made use of multistage extraction procedures, pH changes of the extract and multiple filtration, salt precipitation, dialysis and lyophilisation as the basis of a process for the production of a solid product. The abovementioned method facilitates the production of lectin preparations of a predefined activity limited by the presence of ballast substances in the extraction raw materials. There is thus still a need to deliver such a method of extracting that would make it possible to produce a product with an increased activity. The goal of the present invention is to deliver a method of producing highly active finished product in powder form from bean seeds for use as a feed additive for swine. Unexpectedly, the abovementioned problem was solved by the present invention.

The subject the present invention is a method of the industrial production of a lectin preparation from bean seeds based on that the bean seeds are broken up, the bean pulp is extracted in an acidic environment in the presence of antioxidants and a preservative, characterised in that the extraction is performed at a lowered temperature, preferentially from 4°C to 8°C, wherein only in the initial phase of the extraction are the phases mixed and then the extract is concentrated, preferentially on membranes, and a poorly volatile preservative is added to the thusly produced PHA fraction and it is then dried, preferentially by spraying, resulting in a lectin preparation with an active lectin content no lower than 128 HU/100 mg of preparation. Preferentially a method according to the present invention is characterised in that the extraction is performed using an extraction system containing acetic acid, an antioxidant, as well as a highly volatile short- chained organic acid or its salt at a lowered temperature. Equally preferentially, a method according to the present invention is characterised in that the solid and liquid phases of the extract are mixed only during the first stage of extracting, preferentially within 60 minutes. Preferentially, a method according to the present invention is characterised in that the second part of the extraction process occurs without phase mixing, preferentially within 7 hours.

The second subject the present invention is a feed additive for swine produced from seeds of the common bean, characterised in that the lectin activity is no less than 128 HU/100 mg of preparation with a decreased level of ballast substances. Preferentially, a feed additive according to the present invention is characterised in that it is obtained using a method according to any of Claims 1 to 4.

Unexpectedly, it turned out that a great reduction in the mixing time during the extraction, change in temperature range (and thus a change in the dynamics and characteristics of this process) greatly improves the efficiency and selectivity of PHA extraction (Latin: Phytohaemagglutinin), which in effect facilitates and guarantees the production of a product with a higher biological activity and lower level of ballasting substances in relation to methods of producing PHA lectins known from and described in literature. The nature of the new method of producing a lectin preparation in powder form is to create a novel extraction system, wherein the changes introduced into the extraction system, omission of mixing and the parallel decrease in process temperature, significantly influence the selectivity of the extracting process, expressed in terms of a decreased ballast substance content in the extract, with an unchanged lectin level, and thus an unchanged activity level both of the extract and the lectin preparation made of it. The present invention solves the problem of the efficiency of extracting PHA from legume seeds, here the common bean, and ensures a decreased level of ballast substances in the individual phases of the production of the lectin preparation. The novel method of producing a lectin preparation particularly relates to the extraction phase of all forms of lectin from bean seeds, and facilitates the production of highly active lectin preparations in powder form. As a consequence, the activity of the final powdered product can be increased from 128 HU/100 mg to 256 HU/100 mg, and, by the same token, significantly lower the cost of production per 1000 HU. The described method leads to the production of a novel, different lectin preparation with an increased mass content of all five forms of lectins in relation to the remaining organic substances contained and isolated with the extract using the methods of the invention in patent application P. 375476 entitled "A method of producing a lectin preparation, a lectin preparation as well as a method of administering the lectin preparation to mammals", as well as the invention under application number PL 384706 "A lectin feed additive as well as an industrial method of producing it". Both the product and intermediate phases are controlled through the determination of hemagglutinin activity (HU), defined as the amount of material expressed in mg/ml in the final dilution causing 50% agglutination of a 2% erythrocyte suspension at a temperature of +25° C according to the method of Pusztai and Watt, 1974 , with a modification. Another method of indirect control of the extraction and concentration processes, particularly for technical-technological purposes, is to determine dry mass. During work on the improvement of the effectiveness of the production of lectin preparations as well as technologies for variable conditions of extraction, it unexpectedly turned out that the use of technological regimes of extraction, alternate to extant ones, not only improves the conditions of PHA extraction from leguminous plant seed masses, here bean seeds, which is evidenced by the increased hemagglutinin activity of the extract and the powder made from it during subsequent processes in relation to dry mass determinations, thus confirming the decrease of ballast substance content in the extract and product, and facilitates the production of a lectin product with an activity greater than 128 HU/100 mg. The possibility of producing the biological activity of the powder over 128 HU/100 mg not only decreases the costs of producing the same amount of powder, but also facilitates the design of novel lectin product formulations. Example 1 . Extraction of PHA from bean seeds and production of the final product using the method according to the present invention.

Preparation of common bean seeds.

Seeds of the common bean were milled in a mill with internal cooling and milling temperature control. The milling process temperature varied from +34°C to 40°C. The milled particle diameter was set to grains with a diameter of 1 mm, which in practice yields 98% of a fraction with grain diameters of 0.98 - 1 .02 mm.

Preparation of the extraction solution:

An acetic acid solution is prepared for the extraction using a slow agitator, where glacial acetic acid is mixed into water at a rate of 1 .1 g acetic acid in 1000 ml solution. This solution is supplemented with 0.1 g ascorbic acid per every litre of solution well as propionic acid at 0.1 g per litre of solution. The amount of extraction solution is made dependent on the mass of milled bean seeds. The recommended ratio is 10 : 1 .

Extraction process:

For the extraction, a measured amount of ground common bean seeds are added to the extraction solution and mixing is initiated. After 10 minutes, the pH control of the extraction process begins. The recommended pH for the extraction process is in the range 5.0±0.07. Any possible pH corrections should be made after a minimum 30 min of extracting, because after that point one can start to speak of the pH stabilisation of the extract system. Should a pH correction be needed, after 20 minutes the correction result should be checked. The extraction temperature should be +4°C - +8°C. The mixing should last 1 hours altogether, whereafter the mixture is set aside without mixing for another 7 hours.

Centrifuqation of the extract:

The separation of the solids occurs on high-RPM drum centrifuges designed for the separation of protein precipitates. Drum diameter is 200 mm, and rotational speed is 18 000RPM. The process is periodic, and the cycle is determined by drum volume.

The separated post-extraction residue, and the protein-rich product, are processed separately in order to prepare it for use as an animal feed.

Further extract processing:

The clarified extract is concentrated using reverse osmosis. The initial parameters of the extract are: dry mass 1 .242 %, activity 64 HU/100 ml. The concentrated solution is analysed for hemagglutinin activity and dry mass content. Immediately prior to the spray drying, the concentrated extract is supplemented with an appropriate, calculated amount of maltodextrin and alternatively sodium N-propyl p-hydroxybenzoate or sodium N-ethyl p-hydroxybenzoate. The spray drying process is performed at an inlet temperature not higher than +180°C. The product is analysed for biological activity, and a portion of material is formulated, which is analysed according to EU requirements for feed additives. The resulting product is a pale (white to cream-coloured) powder, highly pourable, a tendency to clump and easily forming a suspension in water. The powder is fully compatible with commonly used mechanical pouring machines. The hemagglutinin activity is no lesser than 128 HU/100 mg, ; Hg, As, Pb, and Cd content is much lower than EU norms; microbiological purity is compliant, and has a shelf-life of 3 years from production. Example 2

Hemagglutination test for determining the activity of the preparation

Preparation of erythrocytes

1 ml of blood collected from a Wistar rat (with 1 drop of heparin) was diluted 1 :20 in physiological saline (0.9% NaCI) and centrifuged (2000 RPM, 15 min, 4°C). The supernatant was poured off along with the interphase. The erythrocytes were resuspended in physiological saline and washed as shown above 3 times. Next, a 2% erythrocyte solution was made (1 :50 dilution).

Preparation of the preparation

For the analysis, 200 mg of the powdered preparation were taken and dissolved in 8 ml of physiological saline. Final concentration - 25 mg/ml.

A series of twofold dilutions was prepared:

Dilutions: 1 x 2x 4x 8x 16x 32x 64x 128x 256x

Test

The test were performed simultaneously in Eppendorf tubes as well as on microscope slides (not degreased!).

The reaction mixture in the Eppendorf tubes contains: 100μΙ physiological saline, 100μΙ erythrocytes 2% and 100μΙ of the examined solution (appropriate solution). After thoroughly mixing the components, 50 μΙ is collected from each examined sample and put onto the slides and incubated at room temperature for about 30 minutes to an hour (care should be taken that the drops do not dry out). The remaining part of the mixture is incubated in closed Eppendorfs (no less than 1 hour, elongation does not affect the readout). After the incubation, the contents of the Eppendorf tubes are mixed with a pipette to free the precipitate off the walls, and then 50 μΙ are collected and placed on a microscope slide. The test is performed in the presence of a negative control: 200μΙ physiological saline and 10ΟμΙ of 2% erythrocytes. The test result is read out under a microscope at 250x magnification. The results are written using the marks: +, +/-, -, where ++ means the presence of hemagglutinated erythrocytes, and - is their absence. To simplify, one can omit the incubation on microscope slides, as sufficient results are obtained in the incubation in closed Eppendorf tubes.

Determination of the hemagglutinin units in the preparation

One hemagglutinin unit (1 HU) is 1 mg of lectin preparation, whose concentration of 1 mg/ml causes the hemagglutination of at least 50% of erythrocytes in accordance with the abovementioned procedure of determining the degree of hemagglutination. The activity of the above preparation is 1 HU/mg.

The number of hemagglutination units (HU) in 1 mg of the examined lectin preparation is denoted as the inverse of the concentration of the solution (shown in mg/ml) in the next dilution, wherein one still observes the hemagglutination of at least 50% of erythrocytes.

For a preparation according to the present invention, activity expressed in hemagglutination units is usually given calculated per 100 mg of preparation.

Example 3. PHA extraction from bean seeds and production of the ready product using the method according to the invention of patent PL 384706

Preparation of common bean seeds

Seeds of the common bean were milled in a mill with internal cooling and milling temperature control. The milling process temperature varied from +34°C to 40°C. The milled particle diameter was set to grains with a diameter of 1 mm, which in practice yields 98% of a fraction with grain diameters of 0.98 - 1 .02 mm.

Preparation of the extraction solution:

An acetic acid solution is prepared for the extraction using a slow agitator, where glacial acetic acid is mixed into water at a rate of 1 .1 g acetic acid in 1000 ml solution. This solution is supplemented with 0.1 g ascorbic acid per every litre of solution well as propionic acid at 0.1 g per litre of solution. The amount of extraction solution is made dependent on the mass of milled bean seeds. The recommended ratio is 10 : 1 .

For the extraction, a measured amount of ground common bean seeds are added to the extraction solution and mixing is initiated. After 10 minutes, the pH control of the extraction process begins. The recommended pH for the extraction process is in the range 5.0±0.07. Any possible pH corrections should be made after a minimum 30 min of extracting, because after that point one can start to speak of the pH stabilisation of the extract system. The extraction process does not require exact temperature regimes because it is not significantly exergonic. The extraction temperature should be +16°C - +25°C. The mixing should last 4 hours altogether, whereafter the mixture is set aside without mixing for another 4 hours. Centrifuqation of the extract:

The separation of the solids occurs on high-speed drum centrifuges designed for the separation of protein precipitates. Drum diameter is 200 mm, and rotational speed is 18 000RPM. The process is periodic, and the cycle is determined by drum volume. The separated post-extraction residue, and the protein-rich product, are processed separately in order to prepare it for use as an animal feed.

Further extract processing:

The clarified extract is concentrated using reverse osmosis. The initial parameters of the extract are: dry mass 1 .242 %, activity 64 HU/100 ml. The concentrated solution is analysed for hemagglutinin activity and dry mass content. Immediately prior to the spray drying, the concentrated extract is supplemented with an appropriate, calculated amount of maltodextrin and alternatively sodium N-propyl p-hydroxybenzoate or sodium N-ethyl p-hydroxybenzoate. The spray drying process is performed at an inlet temperature not higher than +180°C. The product is analysed for biological activity, and a portion of material is formulated, which is analysed according to EU requirements for feed additives. The resulting product is a pale (white to cream-coloured) powder, highly pourable, a tendency to clump and easily forming a suspension in water. The powder is fully compatible with commonly used mechanical pouring machines. The product was used in tests on two Polish swine farms. Powder activity was 128 HU/100 mg.

The extraction results according to the new and old methods as well as the activity of the product resulting from them, lectin powder for preparing a suspension ex tempore is shown in Table 1 below.

Table 1 PHA activity of the extract and final product according to prior art and the present invention