WO2002017974A1

WO2002017974A1 - Method for deactivating pathogens using heat

Info

Publication number: WO2002017974A1
Application number: PCT/NL2001/000625
Authority: WO
Inventors: Adrianus Hendrikus Grobben; Thomas Boudewijn Versterre
Original assignee: Gelatine Smits Beheer B.V.
Priority date: 2000-08-28
Filing date: 2001-08-27
Publication date: 2002-03-07
Also published as: AU2001294364A1; NL1016029C2

Abstract

The present invention relates to a process for inactivating disease causing agents by heating during a certain period, characterised in that material containing TSE (Transmissible Spongiform Encephalopathy) causing agents is heated during 2-20 second at a temperature of 100-170 °C. Furthermore, the invention relates to a process for preparing gelatine, calcium phosphate, meat and bone meal, gelco, blood and blood products and milk and dairy products.

Description

METHOD FOR DEACTIVATING PATHOGENS USING HEAT

Description. The present invention relates to a process for inactivating disease causing agents by heating during a certain period.

The causative agents of TSE, Transmissible Spongiform Encephalopathy, cause neurodegenerative diseases which are usually fatal. CJD (Creutzfeld-Jacob-Disease), GSS, FFI, Kuru and nvCJD (caused by the transmission of the BSE-agent to humans) occur. TSE's have also been reported in other animals: scrapie in sheep and goats, BSE in bovines, TME in mink, CWD in deer and FME in cats. Common to all these diseases is that, apparently, there is no inflammatory reaction or immune reaction, and that the diseases in a preclinical, but already infective stage, can not be detected. Therefore, a possibility to prevent transmission of TSE causing agents consist of inactivating or eliminating the particular disease causing agent (the agent).

There is a need in the art for a process for inactivating TSE causing agents, wherein no physical or chemical means which leave residues in the treated material are used. This applies for all TSE causing agents and all materials in which TSE causing agents can occur in general, but in particular for material which is destined for human consumption. As a matter of course, the process has to be effective and reliable. It is generally known that disease causing agents can be inactivated by heating the material in which the disease causing agents are present. Thus, a process for processing food stuffs and the like, wherein microorganisms are inactivated by, for instance, heating milk during 20 minutes at 120 °C or during 15 seconds at 135 °C, is known from GB 1143405. Inactivating TSE (Transmissible Spongiform Encephalopathy) causing agents is not known therefrom. The process according to the present invention is characterised in that the material containing TSE (Transmissible Spongiform Encephalopathy) causing agents is heated during 2-20 seconds at a temperature of 100-170 ^°C. From research carried out by applicant, it appears that, surprisingly, an effective and reliable inactivation of TSE causing agents which may be present in the material is obtained by using said process. Due to the character of the process, no traces of the process are left behind following the treatment. Therefore, there is no problem whatsoever concerning possible residues. Furthermore, the useful substances which are present in the material to be treated are not deteriorated by heating during a short period of several seconds.

GB 409290 relates to treating milk and similar products by heating during less than 1 minute at more than 120 "C. EP 0943243 relates to treating a product which can flow, in particular milk, by heating at 120-160 "C. EP 1027835 relates to a method and apparatus for continually sterilising a fluid, by heating it at 90 "C-160 °C during several seconds. US 5097018 relates to treating a lyophilised material containing blood-clotting factors, by heating during up to 10 minutes at 100 °C- 130 °C. EP 0571771 relates to a process for inactivating viruses in preparations of proteins, by heating at 45 °C to 95 °C during several seconds. None of these references relates to a process for inactivating TSE (Transmissible Spongiform Encephalophaty) causing agents.

Preferably, the process is carried out in such a way that the temperature of the material to be treated is raised to 100-170 °C during less than 15 seconds, is maintained at 100-170 °C during 2-20 seconds, and is decreased to 75 °C during less than 15 seconds. It appears from experiments that it is more preferable that the heating is carried out during 4-10 seconds, and also that the heating is carried out at 130-145 ^°C. It is most preferable that the heating is carried out at 138-140 °C. The material is preferably heated in the form of a fluid. In practice, it is most preferable that the fluid is an aqueous solution, dispersion or emulsion. Independent of the character of the fluid, it is preferred that during the heating, the pressure to prevent the fluid from boiling is maintained. In this way, drying of TSE causing agents on the inner surfaces of the apparatus in which the process is applied, while remaining infective, is avoided.

Research shows that the process according to the present invention can be used for inactivating different TSE causing agents. Furthermore, the process according to the present invention can be applied on varying materials. Obviously, the present invention is preferably applied on material destined for human consumption. Thus, the present invention relates to a process for the preparation of gelatine, characterised in that during the preparation TSE (Transmissible Spongiform Encephalophaty) agents are inactivated by applying one of the above described processes. Furthermore, the present invention relates to processes for the preparation of calcium phosphate, meat and bone meal, so-called Gelco (illustrated in Example 4), blood and blood products and milk and dairy products, wherein, during the course of the preparation, TSE causing agents are inactivated by applying one of the above- described processes.

Furthermore, it is preferable that a causative agent of a disease chosen from the group consisting of BSE, scrapie, CJD and nvCJD is inactivated. These TSE forms are particularly important for humans. Hereinafter, the invention will be illustrated by way of examples.

In general, the process according to the present invention is applied in an apparatus comprising three main parts: (1) a part in which the material to be treated (for instance a pure liquid, a solution, an emulsion or a dispersion), is rapidly heated to the desired temperature; (2) a part in which the fluid is maintained at the desired temperature during the desired period of time; and (3) a part in which the fluid is rapidly cooled. If the boiling point of the fluid or of the dissolved, dispersed or emulsified substances is lower than the heating temperature, means must be added to maintain such a pressure that the fluid will not boil. The heating step (1) can be carried out in several ways, such as by using direct steam injection, condensation of steam on the liquid when it is sprayed in a chamber filled with steam having the required temperature, mixing with the heated liquid, contacting with a hot, externally heated, surface. The step of maintaining the fluid at the desired temperature (2) can be carried out in an insulated tube or chamber, or in a tube or chamber heated to maintain the particular temperature. The step of cooling the liquid (3) can, for instance, be carried out by expanding the liquid, contacting the liquid with a cold surface, or mixing the liquid with a cold substance. As a matter of course, several combinations of heating, maintaining and cooling can be applied.

Independent of the treated material, the process according to the present invention is preferably applied at the end of a preparation process, in order to prevent cross-contamination as much as possible. However, in some cases it may be advantageous to carry out the process according to the present invention at the beginning of a

( preparation process.

Tests to determine the effectiveness of the process.

In order to determine the effectiveness and the reliability of the process, the process was applied to several samples of material, for instance a gelatine solution to which a TSE causing agent had been added, and a gelatine solution prepared from artificially infected, freshly ground bone. The treated samples were then subjected to a biological determination in experimental animals. For carrying out the biological determination in experimental animals, use was made of a process generally based on Dickingson, A.G. and eilke, V.M.H., Genet. Res., 13, 213 (1969); Dickingson, A.G., Meilke, V.M.H. and Fraser, H.J., Co p. Path., 78, 294 (1968); and Ki berl n, R.H. (1976) in "Scrapie in the Mouse", Meadowfield Press Co., Durham (UK), pp. 7-10. Briefly, the (residual) infectivity of a material was analysed by preparing a series of tenfold dilutions of the material, and by injecting an amount of 0.02 ml of each of these dilutions in the brains of experimental animals. The choice of the experimental animals of course depended on the character of the particular TSE causing agent. Thus, the experimental animals were mice, when the agent is mouse-adapted BSE. Of the group of experimental animals to which less than one effective unit per experimental animal was administered, no experimental animal will show any signs of disease. Of the experimental animals belonging to groups which had received approximately one effective unit per experimental animal, only a part of the experimental animals showed any disease signs. Starting from the number of experimental animals showing disease signs per dilution group, it could be calculated which dilution corresponded to one infective unit, on the basis of which the infectivity per gram of material could be calculated. The effectiveness of the particular process could be evaluated by comparing the infectivity of the starting material with the infectivity of the material subjected to the heating process according to the present invention.

Using the process according to the present invention, a decrease of the infectivity by approximately 10⁴ infective units was observed in general. In this connection, the fact that the level of infectivity of the laboratory experiments was 100,000 times higher than the level of infectivity occurring in reality has to be taken into account. For the laboratory experiments, the level of infectivity was increased in order to obtain numerical values for the loss of infectivity.

Example 1. In the present example, the preparation of gelatine from cattle bones is described. As the possibility that the cattle did not have BSE without showing clinical signs, a process according to the present invention was carried out at the end of the process for the preparation, in order to prevent the possibility that the eventually obtained gelatine may form a BSE risk.

The total preparation process comprised the following steps.

(1) Degreasing step: the bones were crushed to pieces smaller than 2 cm, and were degreased with water at a temperature of 70-

90 °C; soft tissue and small bone particles were discarded and the resulting "bone chips" were dried with hot air.

(2) Demineralisation step: the bone chips were treated with diluted hydrochloric acid during several days; the organic fraction obtained thereby is called ossein.

(3) "Liming" step: the obtained ossein was treated with saturated lime during several weeks.

(4) Neutralisation step: the limed ossein was treated with an acid, and was washed with water several times. (5) Extraction step: the gelatine was extracted with warm water; a diluted gelatine solution was obtained thereby.

(6) Purification step: the obtained rude gelatine solution was purified using filtration and ion exchange.

(7) Concentrating by evaporation and adapting the pH value with sodium hydroxide.

(8) Inactivating TSE causing agents at the last point of the process at which gelatine was still in solution. The gelatine was heated to 138 °C during 3 seconds, was maintained at a temperature between 136 °C and 142 °C during 4 seconds, and was then cooled to chamber temperature during one second. A sample of the thus treated gelatine was subjected to the above described biological determination; no infectivity could be detected.

(9) Drying step: following cooling down to a gel, the concentrated gelatine solution was dried with warm air; the resulting solid gelatine was cut, packed and stored. When a sample of gelatine obtained in the above described way had been artificially infected with TSE causing agents beforehand, a decrease of the infectivity of more than 10⁴ infective units was observed. Favourable results were also obtained when the temperature was increased during 1,9 seconds, when the heating temperature was 133 °C, and when the heating temperature was decreased to chamber temperature during 0,1 seconds.

Example 2.

The present example describes a process for preparing calcium phosphate starting from the above described "bone chips". The effluent obtained in step (2) of the above described

Example 1 consists of a solution of monocalcium phosphate. In order to inactivate TSE causing agents possibly deriving from the bone material, a process according to the present invention was carried out during the preparation process. (1) Precipitation step: while stirring, calcium hydroxide was added to the solution of monocalcium phosphate unit, the pH value was about 5; the obtained emulsion of di -calcium phosphate was separated in a liquid phase and a thick slurry of di -calcium phosphate.

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

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

When TSE causing agents had been added to the starting material beforehand, a more than satisfactory decrease of the infectivity was observed when using the present process. Example 3.

The present example describes a process for the preparation of meat and bone meal . Meat and bone meal were prepared started from the soft tissue and small bone parts obtained in step (1) of the above described Example 1. Based on the relevant European Directives, this material has to be treated in order to prevent infections with TSE causing agents. The process for the preparation which was carried out comprised the following steps.

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

(2) Inactivation step. The obtained emulsion was heated to 133 °C in 2 seconds, was maintained at a temperature of 136-138 °C during 4 seconds, and was then cooled to chamber temperature during 1 second. (3) Drying and cutting step.

When TSE causing agents had been added to the starting material, a more than satisfactory decrease of the infectivity was observed when using the present process. Example 4. The present example describes a process for the preparation of "Gel co".

The fat material and the solid material was separated from the emulsion of fat and solid particles in water, obtained by treating crushed bones during step (1) of Example 1. The resulting fluid, referred to as Gelco, contains a certain amount of dissolved protein, and can be used for several purposes.

As it could not be excluded that the bones had been obtained from infected cattle showing no clinical signs, it could also not be excluded that said fluid contained a TSE causing agent. Therefore, the used preparation process comprised a process according to the present invention. (1) Separation. The fat material and the solid material were separated for further processing, and the remaining fluid, with a certain content of dissolved protein, was the so-called "Gelco".

(2) Inactivation. The fluid (Gelco) was heated to 136 °C during 1 second, was maintained at a temperature of between 133 °C and

138 °C during 4 seconds, and was cooled to chamber temperature during 1 second.

(3) Storage. When TSE causing agents had been added to the starting material, a more than satisfactory decrease of the infectivity was observed when using the present process.

Example 5.

The present example describes a process for the preparation of blood and blood products.

Starting from the blood of slaughtered animals, several products with different uses were prepared. As it cannot be excluded that blood leads to infections with TSE causing agents, the process according to the present invention was also carried out on prepared amounts of blood and blood products. The temperature was raised to 138 °C during 2 seconds, was maintained at a value of between 136 °C and 139 ^°C during 3 seconds, and was then decreased to chamber temperature during 1 second.

If TSE causing agents had been added to the starting material, a more than satisfactory decrease of the infectivity was observed when applying the process according to the present invention. Example 6. The present example describes a process for the preparation of milk and dairy products.

As it cannot be excluded that milk leads to infections with

TSE causing agents, the process according to the present invention was also carried out on prepared amounts of milk and dairy products. The temperature was raised to 132 °C during 3 seconds, was maintained at a value of between 130 °C and 134 °C during 5 seconds, and was then decreased to chamber temperature during 2 seconds.

If TSE causing agents had been added to the starting material, a more than satisfactory decrease of the infectivity was observed when applying the process according to the present invention.

Claims

1. A process for inactivating disease causing agents by heating during a certain period, characterised in that material containing TSE (Transmissible Spongiform Encephalopathy) causing agents is heated during 2-20 seconds at a temperature of 100-170 °C.

2. The process according to claim 1, characterised in that the temperature of the material to be treated is raised to 100-170 °C during less than 15 seconds, is maintained at 100-170 °C during 2-20 seconds, and is decreased to 75 °C during less than 15 seconds.

3. The process according to claim 1 or claim 2, characterised in that the heating is carried out during 4-10 seconds.

4. The process according to any one of claims 1-3, characterised in that the heating is carried out at 130-145 °C.

5. The process according to claim 4, characterised in that the heating is. carried out at 138-140 °C.

6. The process according to any one of claims 1-5, characterised in that the material is heated in the form of a fluid.

7. The process according to claim 6, characterised in that the fluid is an aqueous solution, dispersion of emulsion.

8. The process according to claim 6 or claim 7, characterised in that, during the heating, a pressure to prevent the fluid from boiling is maintained.

9. A process for preparing gelatine, characterised in that during the preparation TSE (Transmissible Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

10. A process for preparing calcium phosphate, characterised in that during the preparation TSE (Transmissible Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

11. A process for preparing meat and bone meal, characterised in that during the preparation TSE (Transmissible Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

12. A process for preparing Gelco, characterised in that during the preparation TSE (Transmissible Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

13. A process for preparing blood and blood products, characterised in that during the preparation TSE (Transmissible

Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

14. A process for preparing milk and dairy products, characterised in that during the preparation TSE (Transmissible Spongiform Encephalopathy) agents are inactivated by applying one of the processes according to any one of claims 1-8.

15. The process according to any one of claims 1-14, characterised in that an agent causing a disease selected from the group consisting of BSE, scrapie, CJD and nvCJD is inactivated.