NL1016029C2 - Method of eliminating disease causing agents by applying heating. - Google Patents

Description

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Brief indication: Method of switching off pathogens while applying heating.

Description.

The present invention relates to a method for switching off pathogens by heating for a certain time.

The causative agents of TSE, Transmissible Spongiform Encephalopathy, usually cause fatal neurodegenerative diseases. CJD comes to humans

(Creutzfeld-Jacob-Disease), GSS, FFI, Kuru and nvCJD (caused by transmission of the BSE agent to humans). TSEs have also been described in other animals: scrapie in sheep and goats, BSE in cattle, TME in mink, CWD in deer and FME in cats. Common to these diseases is that apparently no inflammatory or immune response occurs, and that the diseases cannot be detected at a pre-clinical, yet already infectious, stage. A possibility to prevent transmission of TSE pathogens therefore consists of inactivating or switching off the causative agent (the agent) involved. There is a need in the prior art for a method of switching off TSE pathogens in which no physical or chemical agents are used that leave traces in the treated material. This applies in general to all TSE agents and all materials and where TSE agents can occur, but in particular to material intended for human consumption. The method must of course be effective and reliable.

The method according to the present invention is characterized in that material containing TSE (Transmissible Spongiform Encephalopathy) agents is heated for 2-20 seconds at a temperature of 100-170 ° C.

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Research by the applicant shows that using this method surprisingly results in an effective and reliable elimination of any TSE agents present in the material. Due to the nature of the method, no traces of the method remain after the treatment. Therefore, there is no problem whatsoever with regard to possible residues. Moreover, the useful substances present in the material to be treated do not appear to deteriorate by heating for a short period of a few seconds.

It is preferred that the process be carried out in such a way that the temperature of the material to be treated is brought to 100-170 ° C for less than 15 seconds, to 100-170 ° C for 2-20 seconds, and lowered to less than 75 ° C for less than 15 seconds. Research shows that it is most preferred that heating is carried out for 4-10 seconds, and that heating is carried out at 130-145 ° C. It is most preferred that the heating be carried out at 138-140 ° C.

The material is preferably heated in the form of a liquid. In practice, it is most preferred that it be an aqueous solution, dispersion or suspension. Regardless of the nature of the liquid, it is recommended that during heating, such a pressure is maintained that the liquid does not boil. This prevents TSE causative agents from drying on the inner walls of the device in which the method is applied, whereby they can remain infectious.

Research has shown that the method according to the present invention can be used for switching off different TSE agents. Moreover, the method according to the invention can be applied to a variety of materials. Of course, it is recommended that the present method be applied to material intended for human consumption. For example, the present invention relates to a process for the preparation of gelatin, characterized in that TSE (Transmissible Spongiform Encephalopathy) causative agents are switched off during the preparation by applying one of the methods described above. The present invention furthermore relates to methods for the preparation of calcium phosphate, meat and bone meal, so-called Gel co (illustrated in Example 4), blood and blood products and milk and dairy products, wherein during the preparation TSE pathogens are switched off by one of the above described methods.

It is further preferred that an agent of a condition selected from the group consisting of BSE, scrapie, CJD and nvCJD is switched off. These TSE forms are of particular interest to humans.

The invention will be discussed below with reference to examples.

In general, the method according to the present invention is applied in a device consisting of three main parts: (1) a part in which the material to be treated (for example a pure liquid, a solution, an emulsion or a dispersion, quickly reaches the desired (2) a portion in which the fluid is maintained at the desired temperature for the desired period of time, and (3) a portion in which the fluid is rapidly cooled When the boiling point of the fluid or of the solution dissolved in the fluid is , dispersed or emulsified substances is lower than the heating temperature, means must be added to maintain a pressure such that the liquid will not boil The heating step (1) can be carried out in various ways, such as using direct steam injection, condensation of steam on the liquid when it is in a steam with the desired temperature 1016029

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4 filled chamber is sprayed, mix with a warm liquid, contact with a warm, externally heated, surface. The step of maintaining the liquid at the desired temperature (2) can be carried out in an insulated tube or chamber, or in a tube or chamber that is heated to maintain the temperature in question. The step of cooling and of the liquid (3) can be carried out, for example, by expanding the liquid, contacting the liquid with a cold surface or mixing the liquid with a cold substance. Of course, different combinations of heating, maintenance and cooling can be used.

Regardless of the treated material, the method according to the present invention will preferably be applied at the end of a preparation process in order to avoid cross-contamination as much as possible. In some cases, however, it may be advantageous to use the method according to the present invention at the beginning of a preparation process.

Tests to investigate the efficacy of the method.

To investigate the efficacy and reliability of the method, the method was applied to some samples of material, for example a gelatin solution to which a TSE agent was added, and a gelatin solution prepared from artificially infected freshly ground bone. The treated samples were then subjected to a biological determination in test animals.

For carrying out the biological determination in experimental animals, a method was used that was generally based on Dickingson, A.G. and Meilke, V.M.H., Genet. Res., 13, 213 (1969); Dickingson, A.G., Meilke, V.M.H. and Fraser, H. J., Comp. Path., 78, 294 (1968); and Kimberlin, R.H. (1976) in "Scrapie in the Mouse", Meadowfield 30 Press Co., Durham (UK), pp. 7-10. Briefly, the (residual) infectivity of a material was examined by making a series of ten-fold dilutions of the material and injecting an amount of 0.02 ml of each of these dilutions into the brains of test animals. Of course, the choice of the test animals depended on the nature of the TSE causative agent involved. For example, the test animals were mice when the causative agent is mouse-adapted BSE causative agent. Of the group of test animals to which less than one effective unit per test animal was added, no test animal will show any signs of disease. Of test animals belonging to groups that exhibited about one infectious unit per test animal, only a part of the test animals received disease symptoms. Based on the number of test animals with disease symptoms per dilution group, it was possible to calculate which dilution corresponds to one infectious unit, on the basis of which the infectivity per gram of material could be calculated. The efficacy of the method in question could be assessed by comparing the infectivity of the starting material with the infectivity of the material subjected to the heating according to the present invention.

In general, using the method of the present invention, a reduction in infectivity of about 104 infectious units was observed. In this regard, account must be taken of the fact that the level of infectivity in the laboratory experiments was 100,000 times higher than the actual level of infectivity. In the laboratory experiments, the level of infectivity was increased to obtain numerical values for the loss of infectivity.

Example 1

The present example describes the preparation of gelatin from bovine bones. Because it could not be excluded that the bovines in question had BSE without showing clinical signs, at the end of the preparation process, a method according to the present invention was introduced to prevent the gelatin ultimately obtained from being able to pose a PSE risk. invention.

The total preparation process included the following 5 steps.

(1) Degreasing: the bones were reduced to pieces of less than 2 cm and were degreased with water of 70-90 ° C; soft tissue and small bone particles were removed, and the resulting bone chips ("bone chips") were dried with warm air.

(2) Demineralization: the bone discs were treated with dilute hydrochloric acid for several days; the resulting organic fraction is called ossein.

(3) Liming treatment: the ossein obtained was treated with saturated lime for a few weeks.

(4) Neutralization: the lime-treated ossein was treated with an acid and washed several times with water.

(5) Extracting: the gelatin was extracted with warm water; a diluted gelatin solution was obtained.

(6) Purification: the resulting crude gel atine solution was purified by filtration and ion exchange.

(7) Concentration by evaporation and adjustment of the pH value with sodium hydroxide.

(8) Elimination of TSE causative agents at the last point at which the gelatin was still in solution. The gelatin was heated to 138 ° C for 3 seconds, maintained at a temperature between 136 ° C and 142 ° C for 4 seconds, and then cooled to room temperature for 1 second. The above-described biological assay was performed on a sample of the treated gelatin; no infectivity could be established.

(9) Drying: the concentrated gelatin solution, after cooling and to a gel, was dried with warm air; the resulting solid gelatin was cut, packaged and stored.

When a sample of gelatin obtained as described above was previously artificially contaminated with TSE causative agents, a decrease in infectivity of more than 104 infectious units was found. Favorable results were also obtained when the temperature was raised for 1.9 seconds, when the heating temperature was 133 °, and when the heating temperature was lowered to room temperature for 0.1 seconds.

Example 2

In the present example, a method for preparing calcium phosphate from the bone chips described above is described.

The effluent obtained in step (2) of the preparation process described in Example 1 consists of a solution of monocalcium phosphate. In order to eliminate any TSE pathogens originating from the bone material, a method according to the invention was used during the preparation process.

(1) Precipitation: with stirring, calcium hydroxide was added to the monocalcium phosphate solution until the pH was about 5; the obtained emulsion of dicalcium phosphate was separated in a liquid phase and in a thick slurry of dicalcium phosphate.

(2) Inactivation: the slurry was heated to 145 ° C in 4 seconds, maintained at a temperature between 142 ° C and 149 ° C for 5 seconds, and cooled to 50 ° C for 1 second.

(3) Centrifugation, washing with water and drying with warm air.

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When TSE causative agents were added to the starting material, a more than satisfactory decline in infectivity was found using the present method.

Example 3

In the present example a method for preparing meat and bone meal is described. Meat and bone meal were prepared from the soft tissue and small bone particles described in step (1) of Example 1. According to the relevant European directives, this material must be treated to prevent contamination with TSE-10 pathogens. The applied preparation process included the following steps.

(1) The water stream with soft tissue and fine bone particles was thickened to an emulsion to which solid particles obtained from the degreasing emulsion were added.

15 (2) Inactivate. The resulting emulsion was heated to 133 ° C in 2 seconds, could be maintained at a temperature of 136-138 ° C for 4 seconds and then cooled to room temperature for 1 second.

(3) Drying and cutting.

When TSE agents were added to the starting material, the present method showed a more than satisfactory deterioration of infectivity.

Example 4

In the present example, a process for preparing "Gelco" is described.

From the emulsion of fat and solid particles in water obtained by treating ground bone during step (1) of Example 1, the fat material and the solid material were separated. The resulting liquid, called Gel co, contains a certain amount of dissolved protein and can be used for various purposes.

Because it could not be excluded that the bones were obtained from an infected bovine that did not show clinical signs, it could not be excluded that this fluid would contain a TSE causative agent. The preparation process used therefore included a process step according to the invention.

(1) Separating. The fat material and the solid material were separated for further processing, and the remaining liquid, with a certain dissolved protein content, was the so-called "Gelco".

(2) Inactivate.

The liquid (Gelco) was heated to 136 ° C for 1 second, maintained at a temperature between 133 ° C and 138 ° C for 4 seconds, and cooled to room temperature for 1 second.

15 (3) Storage.

When TSE causative agents were added to the starting material, a more than satisfactory decline in infectivity was found using the present method.

Example 5

In the present example, a method for preparing blood and blood products is described.

A number of products with different uses are prepared from the blood of animals for slaughter. Since it cannot be excluded that blood may lead to infections with TSE causative agents, the method according to the present invention was also applied to preparation amounts of blood and blood products. The temperature was brought to 138 ° C for 2 seconds, maintained at a value between 136 ° C and 139 ° C for 3 seconds, and then lowered to room temperature for 1 second.

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When TSE causative agents were added to the starting material, a more than satisfactory decline in infectivity was found using the present method.

Example 6

In the present example a method for preparing milk and dairy products is described.

Since it cannot be excluded that milk and dairy products can lead to infections with TSE agents, the method according to the present invention was also applied to preparation quantities of milk and dairy products. The temperature was brought to 132 ° C for 3 seconds, maintained at a value between 130 ° C and 134 ° C for 5 seconds, and then lowered to room temperature for 2 seconds.

When TSE agents were added to the starting material, the present method revealed a more than satisfactory deterioration of infectivity.

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Claims (15)

1. Method for switching off pathogens by heating for a certain time, characterized in that material containing TSE (Transmissible Spongiform Encephalopathy) agents is heated for 2-20 seconds at a temperature of 100-170 ° C . A method according to claim 1, characterized in that the temperature of the material to be treated is brought to 100-170 ° C for less than 10 seconds, maintained at 100-170 ° C for 2-20 seconds, and less than 15 seconds to less than 75 ° C. Method according to claim 1 or claim 2, characterized in that the heating is carried out for 4-10 seconds. Method according to any of claims 1-3, characterized in that the heating is carried out at 130-145 ° C. Method according to claim 4, characterized in that the heating is carried out at 138-140 ° C. 6. A method according to any one of claims 1-5, characterized in that the material is heated in the form of a liquid. Method according to claim 6, characterized in that the liquid is an aqueous solution, dispersion or emulsion. 8. Method as claimed in claim 6 or claim 7, characterized in that during heating such a pressure is maintained that the liquid does not boil. A method for preparing gelatin, characterized in that TSE (Transmissible Spongiform Encephalopathy) causative agents are switched off during the preparation by applying one of the methods according to any of claims 1-8. 1016029 *. A method for preparing calcium phosphate, characterized in that TSE (Transmissible Spongiform Encephalopathy) causative agents are switched off during the preparation by applying one of the methods according to any of claims 1-8. A method for preparing meat and bone meal, characterized in that TSE (Transmissible Spongiform Encephalopathy) causative agents are switched off during the preparation by applying one of the methods according to any of claims 1-8. 12. Method for the preparation of Gel co, characterized in that TSE (Transmissible Spongiform Encephalopathy) pathogens are switched off during the preparation by applying one of the methods according to any one of claims 1-8. 13. Method for the preparation of blood and blood products, characterized in that TSE (Transmissible Spongiform Encephalopathy agents are eliminated by applying one of the methods according to any of claims 1-8. 14. Method for preparing milk and dairy products, characterized in that TSE (Transmissible Spongiform Encephalopathy) causative agents are switched off during the preparation by applying one of the methods according to any one of claims 1-8. A method according to any one of claims 1-14, characterized in that an agent of a disorder selected from the group consisting of BSE, scrapie, CJD and nvCJD is switched off. 1016029 COOPERATION TREATY (PCT) REPORT ON NEWNESS RESEARCH OF INTERNATIONAL TYPE IDENTIFICATION OF THE NATIONAL APPLICATION CHARACTERISTICS OF THE APPLICANT OR OF THE AUTHORIZED 42395 / EP / pr Dutch application no. Date of request for an international type examination by International Institution (ISA) request for international type examination no. _SN 35779 GB_ I. CLASSIFICATION OF THE SUBJECT (if different classifications are applied, state all dassification symbols) ) According to the International Classification (IPC) lnt.CI.7: A6112 / 64 A23L3 / t6 II. TECHNICAL FIELDS EXAMINED Minimum Documentation Examined Classification System _ Class Symbols_ lnt.CI.7: A61L A23L Documentation other than the minimum documentation examined, to the extent that such documents are included in the investigated areas III. □ NO INVESTIGATION FOR CERTAIN CONCLUSIONS (comments on supplement sheet) IV. □ LACK OF UNIT OF INVENTION (comments on supplement sheet) IJ-Form PCT / ISA201 a (11/2000)