DE102009025542A1 - Detecting intact capsule-forming or non-capsule-forming parasites, e.g. Trichinella papuae, in a meat comprises macerating the meat with a basic digestive solution containing a digestive enzyme, which is active in basic medium - Google Patents

Abstract

Detecting intact capsule-forming or non-capsule-forming parasites in a meat comprises macerating the meat with a basic digestive solution containing a digestive enzyme, which is active in a basic medium. Independent claims are included for: (1) use of the digestive enzyme, preferably serine endopeptidase of subtilisin group, such as Alcalase(RTM: Protease subtilisin Carlsberg) and Alcalase 2.5 L(RTM: Protease) in a diagnosing method for detecting an infection with the intact capsule-forming or non-capsule-forming parasites; and (2) a kit comprising the digestive enzymes and basic digestive solutions.

Description

The The present invention relates to a method for detecting capsule-forming ends or non-capsule-forming parasites in meat that cause maceration of the meat with a digestive solution comprising a Serine endopeptidase contains. The further concerns the present Invention a method for detection of capsule-forming or non-capsule-forming ends Parasites in meat, which is the maceration of the meat with a Digestive solution includes a serine endopeptidase optionally comprising the steps of (a) mechanical Crushing the meat to be tested; (b) the maceration the meat to be tested by adding a digestive solution; (c) Maceration with simultaneous movement of the digestive approach and / or simultaneous ultrasound treatment; (d) inactivation the maceration; (e) filtration of the macerate; and (f) control, Detection, diagnosis and / or typing for parasite infestation. Furthermore, uses of a serine endopeptidase are in one Method for detecting capsule-forming or non-capsule-forming ends Parasites described in meat. The present invention provides also serine endopeptidases for use in a diagnostic procedure for detection of capsule-forming or non-capsule-forming parasites in meat. Finally, a kit is also revealed that the enzymes and digestive solutions of the present Invention.

Trichinella (Trichinella) are a genus of tiny roundworms of the Tribal Nematoda with parasitic lifestyle. mammals, Birds and humans serve as intermediate and final hosts. Principal vector on humans are pigs or their raw or insufficient cooked meat. By cooking or big cold can Trichina be killed.

The Trichinae are spread worldwide by several species. There is Eight species, the most important occurring in Europe are Trichinalla spiralis, Trichinella britovi, Trichinella pseudospiralis, and Trichinella nativa. The parasitic nematode Trichinella spp. is able to, many different species, including humans, pigs, Rats, bears, horses and birds. In western In Europe, trichinae occur predominantly in the "silvatic Cycle ", where foxes and rodents eat by eating infected animals spread the worms. In the north Areas can also be bears, sled dogs and Robben serve as an intermediate host. An "urban cycle" is also pronounced; in this the pathogens become predominant spread by rats and pigs.

The adult animals reach a length of up to 4 mm (females) or 1.5 mm (male). You can clearly the thickened Recognizing the rear end, which houses the intestine. The larvae encyst in the muscle tissue and form a "nurse cell complex" there, a capsule that provides plenty of blood vessels and thereby keeps the larva alive. She reaches a size of about a millimeter and is tall infectious. However, there are also variants that no Form capsule, such. B. Trichinella pseudospiralis.

recorded Muscle cysts are dissolved in the small intestine and so the larvae released. The larvae drill into the small intestinal epithelium and develop within 30 hours to the adult, then the mating takes place. In the small intestine, the females bring viviparous up to 1500 larvae to the world. The larvae then drill through the small intestine and so reach the lymph or the bloodstream. They drift through the Body and especially good in striated good hemorrhaged muscle tissue. Diaphragm, Eyes and tongue. There begins the formation of the "nurse complex", the infectious remains as long as the parasite lives. From the fifth month, calcification occurs in human tissue instead, with the encysted muscle trichinae probably still 5 remain viable for up to 10 years at an incubation period from 8 to 15 days develop from the first in the Small intestine larvae adult worms. there In addition to often asymptomatic disease processes also occur general weakness, abdominal pain, nausea, Vomiting and diarrhea; after 1 to 3 weeks then fever, muscle aches and edema in the eye area. These symptoms usually last up to a year and then disappear without lasting consequences. As a complication, the heart muscle can be affected, causing the Worm infection can take a deadly course. The trichinae caused disease is called trichinellosis designated. The infections are subject to European Union of registration and are under the Infection Protection Act Notifiable in Germany.

The most important preventive measure is the statutory Trichinenschau, which is also called Trichinenuntersuchung. This refers to a microscopic examination of meat for trichinae after slaughter, whereby the capsules of the larvae are specifically recognized. Meat of domestic swine, solipeds, wild boars, bears, foxes, beaver rats, nutrias and badgers, as well as all other animals that carry trichinae are subject to human consumption , the obligation to examine (slaughtered animals requiring examination).

Essentially, two methods are used to detect Trichinella spp. in animals. The first method is called trichinoscopy or trichinoscopic examination. In this direct detection method, the tissue of the trichina samples taken from the pylons and forelimb musculature for examination is compressed in a so-called compressorium (ie squeeze-glass consisting of two glass plates) and then examined microscopically. This method is less sensitive and labor intensive. In addition, no capsule-free (non-capsule-forming) trichinae can be identified by this method. The second method is a mechanically assisted method of artificial digestion. In the process, the muscle tissue surrounding the larvae is artificially digested by imitation of natural digestion with the addition of pepsin in an acidic environment in order to release the larvae. Subsequently, the larvae are examined microscopically. The Trichinenschau is by the " Commission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules for official post-mortem inspection for trichinae "Regulated. The process used in the prior art, in which the natural digestion is mimicked with the addition of the enzyme pepsin, an aspartate endopeptidase, and the digestion is carried out in an acidic environment, uses the commercially available pepsin form obtained from the stomach of the pig ,

newer ELISA-based methods are more sensitive, but the risk of wrong positive results is correspondingly high, which is routine that leads to a much higher probability entire batches of carcasses were confiscated and reexamined would have to be. However, ELISA-based methods are not yet approved.

Around to obtain a license for routine examination, must have a similar sensitivity and comparability to common examination methods of already validated and approved procedures. New Approved Methods while respecting equality of rights, they need an alternative to already approved methods.

According to regulations The EU should no longer accept trichinoscopy as the standard method of investigation be. The above-mentioned prescribed methods for Examination of Trichinella are: (i) the magnetic stirrer method for the artificial digestion of bulk samples; (ii) the mechanically assisted method (Stomacher Lab Blender) the artificial digestion of bulk samples under application the sedimentation technique; (iii) the mechanically assisted Method (Stomacher Lab-blender) of artificial digestion of bulk samples using the on-filter-isolation technique; and (iv) the automatic process (with the trichomatome Trichomatic-35) for bulk samples up to 35 g.

The currently used testing methods for the detection of Trichinella have a number of disadvantages. All procedures based on artificial Based on digestion, are relatively time and labor intensive. Especially the material costs used for the investigation have to be, are quite high. This is especially true attributed the high price of the enzyme pepsin, that in high quantities to the above-mentioned artificial Digestion must be added. Due to the worldwide scarcity of pepsin, prices for this enzyme have risen sharply. Whether the need for pepsin can be met in the long term is uncertain. Supply shortages could lead to that the legally required Trichinenkontrol no longer can be carried out.

task Therefore, the present invention is alternative methods and means for isolating Trichinella or other parasites from organic tissue.

The technical problem is solved by providing a method for detecting capsule-forming or non-capsule-forming parasites in meat, which method comprises macerating the meat with a digestion solution containing serine endopeptidase. It has surprisingly been shown that a serine endopeptidase, preferably Alcalase ^® (subtilisin Carlsberg), obtained with no obvious disadvantages compared to that used in the prior art method, the maceration of the to be tested meat to detect capsule-forming or non-capsule forming parasites in the meat.

Consequently The present invention relates to a method for the detection of Capsule-forming or non-capsule-forming parasites in meat, that the maceration of the meat with a digestive solution comprising a serine endopeptidase.

The term "meat" includes meat after slaughter of slaughter animals. As described herein The term "meat" preferably refers to the meat of slaughtered animals subject to inspection, the term "slaughtered animals requiring examination" being generally known to the person skilled in the art. Thus, the meat to be examined may preferably come from domestic pigs, wild boars, solipeds, bears, foxes, beaver rats (Nutria) and badgers. However, the meat may also be derived from any other animal that may be infected by capsule-forming or non-capsule-forming parasites. Preferably, therefore, the meat may also originate from other animals which are carriers of parasites of the strain of nematoda (roundworms), in particular carriers of trichinae, such as Trichinella, or are infected by them.

In accordance With the foregoing, the present invention includes a method for detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution which contains a serine endopeptidase, optionally the following steps include: (a) mechanically crushing the to examining meat; (b) the maceration of the substance under investigation Meat by adding a digestive solution; (c) maceration with simultaneous movement of the digestive approach and / or simultaneous Ultrasound treatment; (d) inactivation of maceration; (e) filtration the macerate; and (f) control, detection, diagnosis and / or typing on infestation with parasites.

Of the Term "maceration" as described herein circumscribes the process of dissolution of organic Tissue. The term does not describe a chemical, but one purely physical process, since no chemical reaction takes place. "Maceration" refers focus on a physical process in which a body or subject some time to the action of a liquid such as being exposed to water, oil or alcohol, which as a solvent for certain ingredients this product is used, the product is called macerate. In this case, the object or body as such is not dissolved, but only certain components of it go in the liquid over, acting as a solvent serves. Preferably, the body or object is the to understand as the meat described above.

Of the Term "maceration" as described herein is equivalent to the digestion reaction described below.

As illustrated in the examples, the present invention provides Method for detecting capsule-forming or non-capsule-forming ends Parasites in meat, which is the maceration of the meat with a Digestive solution includes a serine endopeptidase contains, optionally, the following steps: (a) the mechanical Crushing the meat to be tested; (b) the maceration the meat to be tested by adding a digestive solution; (c) inactivation of maceration; (d) filtration of the macerate; and (e) control, detection, diagnosis and / or typing for infestation prepared with parasites, taking the meat / digestive solution preferably 1:10. In a further embodiment the ratio may differ and, for example 1: 5 or 1:20.

In accordance with the methods set forth above, is preferably a Serine endopeptidase, wherein the enzyme is an enzyme of the enzyme group the subtilisin is.

Stronger Preferably, the subtilisins used are a Subtilisin produced recombinantly or non-recombinantly.

In a highly preferred embodiment, wherein the subtilisin is selected from the group consisting of Alcalase ^® (subtilisin Carlsberg) and Alcalase 2.5 L ^®.

In accordance With the foregoing, the present invention includes a method for detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution wherein the digestive solution is preferably basic is.

Stronger it is preferably a digestive solution, wherein the digestive solution preferably has a pH between pH 7 and 10 has. In a highly preferred embodiment the digestive solution has a pH between pH 7 and 9. The invention is not limited only to the mentioned pH ranges. In a further preferred embodiment, depending after the enzyme has been used, a pH range is chosen in which the pH optimum of each enzyme used is.

In a preferred embodiment comprises the above described method, a digestion solution, wherein the Digestion solution contains NaCl.

In particular, the digestive solution may optionally include further ingredients, these being preferably contains fat emulsifiers, surfactants and / or other auxiliaries. In a further embodiment, the digestion solution may contain dyes or coloring agents with which the parasites to be detected can be stained. In one embodiment, the parasites stained in this way facilitate the visual-optical detection of the parasites in the samples and / or the typing of the parasites.

In a highly preferred embodiment, the digestion solution comprises a fat emulsifier, wherein the fat emulsifier is preferably Supralan UF ^®.

In another preferred embodiment the digestion solution described herein further excipients, wherein the adjuvants are selected from the group consisting from: coenzymes, enzyme substrates, catalysts.

In accordance With the foregoing, the present invention includes a method for detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution comprising a serine endopeptidase, wherein the Maceration preferably with simultaneous ultrasound treatment he follows.

In accordance With the foregoing, the present invention includes a method for detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution comprising a serine endopeptidase, wherein the Maceration prefers simultaneous movement and / or simultaneous Stirring takes place. In a further embodiment the maceration takes place with simultaneous movement and / or simultaneous Stir with a magnetic stirrer.

In In another preferred embodiment, this includes herein described method a maceration, these preferably carried out at a temperature between 40 and 65 ° C. becomes. In a further preferred embodiment the maceration is carried out at a temperature between 55 and 65 ° C. In a highly preferred embodiment, the maceration carried out at a temperature of 55 ° C. The Invention is not limited to the mentioned temperature ranges limited. In a further preferred embodiment Depending on the enzyme used, a temperature range is selected. in which the temperature optimum of the particular enzyme used is.

Prefers For example, the method described herein consists of a step in the maceration described herein by filtration of the reaction or terminated by cooling the reaction. These Termination of the reaction as described herein is as inactivation to understand the reaction.

In In a preferred embodiment, the maceration is by Filtration terminated, wherein in the filtration of macerate the Mesh size of the filters used on the size the organisms to be detected is adjusted. In another Embodiment, the filtration for termination of the Reaction optionally with the addition of pressure or vacuum.

As Illustrated in the examples may be filtration, or forced filtration (Filtration of the solution, optionally with the addition of Pressure or vacuum). Like the rest illustrates, during the maceration sedimentation down and a floating up of material observed become. In addition, the digestive fluid during the sedimentation is flocculent (see examples). The precipitation Floats upwards and thus prevents (partially) a complete Sedimentation of the parasites to be detected. This process can by forced filtration, optionally with the addition of pressure or vacuum, counteracted.

In accordance with the foregoing, the present invention encompasses a method of detecting capsule-forming or non-capsule-forming parasites in meat which comprises macerating the meat with a digestion solution containing a serine endopeptidase, the control, detection, diagnosis and detection / or the typing for parasite infestation is visual or via the detection of nucleic acid molecules or proteins. In a particular embodiment, the detection of the nucleic acid molecules takes place via a PCR reaction. It is particularly noteworthy that the nucleic acid molecules in the above-described maceration as possible not or little degraded and should remain as intact as possible. In a further particular embodiment, the detection, the diagnosis and / or the typing for infestation with parasites should be based on serum-based methods or serological methods. These serological / immunological methods for the detection of capsule-forming or non-capsule-forming parasites are based in a preferred embodiment on the detection of corresponding antigens these capsule-forming or non-capsule-forming parasites. In the case where antibodies are used, an immune complex is formed, which can be detected by methods known to those skilled in the art. In another embodiment, other serological / immunological methods may be used, e.g. As immunoblot method, ELISA, RIA, SPR (surface plasmon resonance) or agglutination tests, only a few examples are listed here.

Prefers is the present invention an invention, which is a method of detecting capsule-forming or non-capsule-forming ends Parasites in meat, which is the maceration of the meat with a Digestive solution includes, wherein it is the capsule-forming or non-capsule-forming parasites around organisms of the strain of Nematoda (roundworms) acts. In a preferred embodiment The present invention is the capsule-forming ends Parasites around organisms belonging to the genus Trichinella (trichinae) belong. In another preferred embodiment The Trichinella species is selected from the group consisting from: Trichinella britovi, Trichinella murrelli, Trichinella nativa, Trichinella nelsoni, and Trichinella spiralis. In another Embodiment are the non-capsule-forming parasites Trichinella pseudospiralis.

As illustrated in the examples, the present invention provides Method for detecting capsule-forming or non-capsule-forming ends Parasites in meat, which is the maceration of the meat with a Digestive solution includes a serine endopeptidase contains, with the meat to be examined preferably meat of slaughter animals is. As used herein, the term "meat" preferably refers on meat from slaughtered animals subject to inspection, the The term "slaughter animals requiring examination" is understood by the person skilled in the art is well known. Thus, the meat to be examined preferably of domestic pigs, wild boars, solipeds, bears, foxes, Beaver rats (Nutria) and badgers come from. But the meat can also come from all other animals, those from Kapselbildenden or non-capsule-forming parasites. Preferably, therefore, the meat can also come from other animals, the carriers of parasites of the strain of Nematoda (roundworms), especially carriers of trichinae, such as Trichinella or are infected by these.

The The present invention also includes the use of a Endopeptidase in one of the methods described above. Especially Accordingly, the present invention includes a method for detection capsule-forming or non-capsule-forming parasites in meat, that the maceration of the meat with a digestive solution comprising a serine endopeptidase.

In a preferred embodiment, the present invention comprises the illustrated use wherein the endopeptidase is a serine endopeptidase. In another embodiment, the serine endopeptidase is preferably an enzyme of the subtilisin enzyme group. In a further preferred embodiment, the enzyme of the enzyme group of subtilisins is preferably selected from the group consisting of Alcalase ^® (subtilisin Carlsberg) and Alcalase 2.5 L ^®.

The The present invention also includes a serine endopeptidase in any of the diagnostic methods described above. In a special Embodiment of the invention includes this serine endopeptidase for use in a method for detection of capsule-forming ends or non-capsule-forming parasites in meat that cause maceration of the meat with a digestive solution that includes these Serine endopeptidase contains. In a further embodiment For example, the present invention includes an enzyme of the enzyme group of Subtilisins for use in a diagnostic method for detection capsule-forming or non-capsule-forming parasites in meat, that the maceration of the meat with a digestive solution includes.

The present invention further relates to preferably a Alcalase ^® (subtilisin Carlsberg) and Alcalase 2.5 L ^® for use in a method for the detection of capsular or non-capsular parasites in meat, comprising the maceration of the meat with a digestive solution.

Finally, the present invention relates to a kit comprising a digestion solution containing a serine endopeptidase as described above for detecting capsule-forming or non-capsule-forming parasites in meat comprising maceration of the meat containing a serine endopeptidase. In a preferred embodiment, the kit comprises an enzyme of the enzyme group of the subtilisins, a farther one preferred embodiment, the enzyme Alcalase ^® (subtilisin Carlsberg) and Alcalase 2.5 L ^®.

The Embodiments related to the method of the present invention apply mutatis mutandis also for the kit of the present invention.

advantageously, the kit of the present invention further comprises a digestive solution, optionally (a) NaCl, (b) fat emulsifiers, (c) surfactants and / or (d) contains adjuvants. Advantageously, this includes Kit also more reaction buffers, storage solutions, Wash solutions, and / or other reagents or Materials necessary for carrying out procedures, as described herein for detection of capsule-forming or non-capsule-forming ends Parasites in meat, which includes the maceration of the meat, with a digestive solution containing a serine endopeptidase contains.

In In a further embodiment, the kit comprises a digestive solution, which is basic, with the pH of this digestion solution in a preferred further embodiment, a value between pH 7 and 10. In a highly preferred embodiment the digestive solution has a pH between pH 7 and 9th

In a particularly preferred embodiment, the kit comprises a digestion solution, wherein the fat emulsifier described therein Supralan UF is preferably ^®. In further embodiments, the kit of the present invention may include a digestive solution, wherein the adjuvants described are selected from the group consisting of: coenzymes, enzyme substrates, and catalysts. In a further embodiment, this digestion solution may contain dyes or coloring agents with which the parasites to be detected can be stained. In one embodiment, the parasites stained in this way facilitate the visual-optical detection of the parasites in the samples and / or the typing of the parasites.

Of the Kit of the present invention may advantageously, inter alia be used to process as described herein Detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution comprising a serine endopeptidase. This kit can, in accordance with the above, in a method of detecting capsule-forming or non-capsule-forming ends Parasites are used in meat, which is the maceration of meat with a digestive solution containing a serine endopeptidase contains, optionally, the following steps: (a) the mechanical Crushing the meat to be tested; (b) the maceration the meat to be tested by adding a digestive solution; (c) Maceration with simultaneous movement of the digestive approach and / or simultaneous ultrasound treatment; (d) inactivation of maceration; (e) filtration of the macerate; and (f) control, evidence, Diagnosis and / or typing for parasite infestation.

The Preparation of the kits preferably follows standard procedures appropriate to the Those skilled in the art are aware of. The kit of the present invention is preferably useful in as provided herein Method.

With The method described herein makes it possible to 100 To digest samples within about a quarter of an hour (in the trichomatoes, Of the common method in the art, 35 samples processed per pass). With the method described herein can also do this with a relatively low level of laboratory equipment Carrying out the digestion will be carried out. The samples can after the digestion reaction with the in the state the technique known sedimentation method with the separating funnel to be examined.

A Automation for rational trichinine control in large companies, analogous to the automatic method recognized in the art with the trichomats, can be in accordance with the present Invention be carried out, with a new device has to be developed.

With The method described herein is material cost lower. Because a larger amount of sample in one go could be processed, is also the personnel expenses lower.

In the examples described below, an enzyme from the enzyme group of the subtilisins is used, and the invention described herein is not limited to this enzyme. Subtilisin is the name of a group of enzymes belonging to the other group of serine proteases / serine endopeptidases (enzyme classification EC category EC3.4.21) and whose catalysed reaction is therefore the cleavage of protein chains. The serine proteases / serine endopeptidases include, among others, the enzymes chymotrypsin (EC3.4.21.1), trypsin (EC3.4.21.4), elastase (EC3.4.21.11), plasmin (EC3.4.21.7), thrombin (EC3. 4.21.5), Savinase (EC3.4.21.14) and subtilisin (EC3.4.21.62). Subtilisins occur mainly in bacteria of the genus Bacillus and have been used industrially in detergents for decades. A wide variety of subtilisins and similar enzymes have been isolated from bacteria of the genus Bacillus, especially the species B. subtilis (subtilisin E), B. lentus, B. licheniformis (subtilisin Carlsberg, Alcalase ^®), B. amyloliquefa ciens, but also from the fungi Tritirachium album, Thermoactinomyces vulgaris. All of these microorganisms naturally occur in the soil and the subtilisins serve to break down proteins outside the cell. The enzyme is known under the name Subtilisin Carlsberg and registered under the name Alcalase ^® and patented. Alcalase ^® is a protease used to remove protein-based stains and is mainly used in detergents for textiles and dishwashing detergents. Alcalase ^® is produced in only one activity. It is produced in liquid form, as a powder and as granules. The use of the liquid form is preferred in the present invention because dusts in general and enzyme dusts in particular are considered hazardous to health. The type of Alcalase 2.5 L ^® used is derived from genetically modified bacteria. There are other types of Alcalas that are derived from non-genetically modified bacteria. These are significantly more expensive and are used in products that may only be made from non-genetically modified organisms. There are also so-called "Ultra" types, which are additionally pre-stabilized in order to be better mixed with other types of enzymes can.

As discussed above and illustrated in the examples below, In a preferred embodiment, the method for detection of capsule-forming or non-capsule-forming parasites in meat, which is the maceration of the meat with a digestive solution comprising a serine endopeptidase, wherein the Maceration preferably with simultaneous ultrasound treatment he follows. As illustrated below, the use of Ultrasound significantly increased the digestion rate. By the sonication of the fleet with ultrasound it is possible to digest the samples within about 15 minutes. So it is possible to process large quantities of samples in large companies.

As illustrated in the examples described below, can be found in an embodiment of the present invention normal Tap water can be used. In the ultrasonic bath it was found that a white "precipitate" takes place, which is lime. If the microscopic evaluation by the lime precipitation would be disturbed can in a preferred embodiment for the digestive liquor resorted to distillate-like water become.

In a further preferred embodiment, in the digestion reaction, a pH range of pH is preferably 7 to 9, a pH range in which the optimum of Alcalase ^® is located. The preferred temperature with the best activity is about 55 ° C. Since it comes with the introduction of the samples into the digestive liquor to a cooling, can be used in a further embodiment with an initial starting temperature of about 60 ° C.

Of the Term "liquor" as used herein the maceration solution including all additives. Under the term "fleet relationship", such as used herein is the weight ratio of the maceration solution understood to the amount of meat to be digested.

Of the Term, "liquor relationship", as used herein It also includes that by using more enzyme the digestive speed is accelerated. The ratio of meat (to digesting amount) to the enzyme crucial. At one too low Liquor ratio meat comes to the digestive fluid it at the sample addition (room temperature) to the digestion liquor (60 ° C) too much cooling of the fleet.

In In a further preferred embodiment, the herein described method under movement of the fleet during carried out the digestion process, there purulent Meat sedimented on the one hand and partly on the other Fleet floats.

As discussed above, in a preferred embodiment The present invention fat emulsifiers used since with the Meat samples also always fat tissue is introduced into the digestive liquor. The fat content is significantly higher in older animals as in young animals. Floats without the use of fat emulsifiers this fat after digestion in the form of grease eyes on the water surface. Since this fat hang in the filter during forced filtration remains, it may interfere with the examination under the microscope. As illustrated in the examples presented below, through the use of fat emulsifiers the existing fat in solution brought. It must be used emulsifiers, the compatible with the enzyme. The product Supralan UF of the company Zschimmer and Schwarz from Lahnstein / Germany comes from the tanning industry and has very good emulsifying abilities even at low levels Concentrations. The function as a wetting agent also seems important because the digestion takes place in aqueous solutions. To Note that when cooling the fleet less fat dissolved / emulsified remains.

As discussed earlier, includes the digestive solution in one preferred embodiment of the present invention NaCl. When preparing bones with enzymes is always used NaCl (salt). This prevents the formation of lime soap. NaCl also acts as an activator for most enzymes. It is recommended to use 10-20 g per liter depending on water hardness (Aus: Bone preparation, Handbook for practitioners of Niederklopfer / Troxler). Of the Term "lime soap" as used herein on calcium or magnesium salts which are difficult to dissolve in water, usually of fatty acids. They form during use of soaps in hard water. Through the formation of these lime soaps the washing effect is reduced as the amount of active soap increases reduces and the lime soaps as an impurity on the deposit cleaning surfaces. The formation of lime soaps Can with softening agents containing the calcium and magnesium ions bind, be counteracted. Because of the mentioned problems included the detergents used in the household today in practice no or a little soaps in the classical sense, but other washing active Substances (surfactants). Made by the manufacturers as a substitute for "real" soap chosen means form little to no lime soaps and so do not cause flocculation and bad washing effect. The formation of smaller amounts of lime soap is used in modern Detergents used to excessive Inhibit development of foam. The lime soap arises from the in the samples present animal fats. In a special embodiment The present invention is described in all of the following Examples 20 g of salt / per liter of liquor used, although others Salt concentrations can be used.

As discussed above, the method described herein consists of a step in which the maceration described herein by filtration the reaction or by cooling the reaction terminates becomes. This termination of the reaction as described herein is to understand as inactivation of the reaction. This is important because at too long residence time of the trichinae in the digestive liquor these are damaged. Therefore, according to the planned Digestion time the process stopped. This can be done in a preferred way Embodiment either by an immediate filtration or by cooling, z. B. Dilute with cold Water or a 0.1% NaCl solution.

Of the Term "fascia" as described herein the soft tissue components of the connective tissue that cover the whole body as an enveloping and connecting power network penetrate. These include all collagenous fibrous Connective tissue, in particular joint and organ capsules, tendon plates (aponeuroses), Muscle septa, ligaments, tendons, retinacula (so-called "fetters", for example on the feet), as well as the "actual Fasciae "in the form of flat solid connective tissue layers, like the plantar fascia on the sole of the foot. Preferably, like discussed previously and illustrated in the examples below It takes a long time for the fascia in the maceration fleet are dissolved. If the dissolution of the fascia occurs not completely, the maceration fleet can in one preferred embodiment of this invention by a Screened coarse filter.

The Pictures show:

.: Digestion solution in a beaker. When this solution is allowed to stand, a flocculent precipitate is observed which floats upwards (see Example 4, Experiment 7).

.: Digestion solution in a separatory funnel after 10 min. Sedimentation. A flocculent precipitate is observed. The precipitate floats upwards and thus prevents (partial) complete sedimentation of the parasites to be detected (see Example 4, Experiment 7).

The The present invention is not limited by the following Examples illustrated.

EXAMPLES

EXAMPLE 1: Material and Methods

Material:

• Meat grinder Alexanderwerk 5 for the coarse shredding of the sample material (Hole size 4 mm)
• Beaker 1200 ml
• Beaker 200 ml
• Sample tubes wide-mouth bottles PVC 100 ml
• Blender Philips Cucina HR 1350/1351/80 / BC with blade insert
• Ultrasound device type Bandelin Sonorex RK 1028H with thermostat controlled water bath
• Magnetic Stirrer Pyro-Mag Stir Cat. No. 34534-200
• Digital scale Mettler PP200
• Digital thermometer Oregon Scientific SA880SSX
• Stopwatch Sportcount Combination
• pH measuring strips Macherey - Nagel 92122
• Separating funnel conical, without division, with PTFE plug, 1000 ml Lenz laboratory glassware, Art. No: 4.0041.7 with holder
• Coarse filter type coffee strainer, mesh size estimated 0.8 mm
• 20 ml syringe Tap water Distilled water NaCl Enzyme Subtilisin Carlsberg (Alcalase ^® ) Emulsifier Fatty alcohol polyglycol ether (Supralan UF ^® ) Sodium carbonate (soda)

Method:

The The meat sample of 100 g to be examined becomes coarse in the meat grinder crushed. A 1200 ml beaker is heated to 62 ° C with 500 ml filled with preheated distillate-like water. 5 g of NaCl and 5 to 20 g of subtilisin Carlsberg are added. Then the meat sample is added. It follows a thorough mixing and further comminution with the blender while 4 times 2 seconds. The prepared sample is either in the preheated and switched to 62 ° C ultrasonic water bath placed or processed on the magnetic stirrer. By Addition of sodium carbonate will lower the pH under review adjusted to 8.5 +/- 0.2 using pH measuring strips. To the desired incubation time, the liquor is through a coarse sieve transferred to a separating funnel. The process is going through Addition of cold 0.1% NaCl solution up to a total volume stopped by 1000 ml. The NaCl solution was previously as Rinse solution for the reaction vessel and the coarse filter so as not to lose sample material. After a sedimentation time of 10 min, the sediment becomes 20% the liquid is collected in a beaker 200 ml. The contents of the beaker turn into a separating funnel transferred and with 400 ml of 0.1% cold NaCl solution refilled. After a second sedimentation of 10 min turn the sediment with 100 ml of the liquid in collected from a sample tube. After a third sedimentation another 10 min in the sample tube becomes the supernatant Aspirate liquid to 20 ml with a syringe. The remaining sediment was adjusted to 50 with NaCl solution ml and in the laboratory of the Institute of Veterinary Parasitology in Berne examined for the presence and abundance of Trichinella larvae.

In selected experiments has also coincided with the serine protease 4 g of Fettemulgators Supralan added UF ^®.

Results:

• I. In one experiment, with 20 g of subtilisin Carlsberg / l in an ultrasonic bath at 60 ° C, pH 8.5 and 10 g NaCl / l, when incubated for 30 min, trichinones be proved (digested). DNA of the trichinae was in the PCR still identifiable, which is indispensable for diagnostics and typing Requirement is.
• II. In a further experiment, a sample with a known number of parasites with diaphragm of mother pigs was made up to 100 g sample amount, then turned through a meat grinder and pureed in 500 ml of water. With addition of 5 g NaCl, 20 g subtilisin Carlsberg and 4 g Supralan UF ^®, the whole was brought to a reaction temperature of 62 ° C and adjusted to a pH of 8.5 with sodium carbonate. After 40 minutes on the magnetic stirrer, only a few fascia residues were visible.

EXAMPLE 2: Comparative Verdauversuche with trichinenverseuchten sample with the enzymes Alcalase ^®, Savinase and papain under the action of ultrasound or without movement of the fleet

• Sample material: trichina contaminated mouse, frosted.
• Alcalase ^® = Subtilisin Carlsberg (derived from Bacillus licheniformis)
• Savinase: (derived from Bacillus lentus)

The digested samples were sedimented in the beaker 3 times for at least 10 minutes and each approx. 4/5 of the liquor was carefully aspirated with a syringe. Subsequently, the container was again with a 0.1% each NaCl solution filled. The evaluation of the samples took place at the University of Bern, Institute of Parasitology. Trial 1 Origin of the sample material: Breast right Test conditions: Alcalase ^® , in a warming cabinet (= without movement of the Fleet); 60 ° C, pH 8.5, 10 g salt / liter, 20 g / liter Alcalase ^® Reaction time: 5.5 hours

The maceration was carried out in a 50 ml PVC container. Result: Trichinas detectable (still intact, partially still encapsulated), also muscle fibers present; DNA the trichina still detectable. Trial 2 Origin of the sample material Back left Test conditions: Alcalase ^®, ultrasound; 60 ° C, pH 8.5, 10 g salt / liter, 20 g / liter of Alcalase ^® Reaction time: 0.5 hours

The maceration was carried out in a 50 ml PVC container. Result: Trichinae detectable (digested), liquid clear; DNA of the trichina still detectable. Trial 3 Origin of the sample material middle left Test conditions: Savinase in the warming cabinet (= without movement of the Fleet); 60 ° C, pH 8.0, 10 g salt / liter, 20 g / liter Savinase Reaction time: 3.5 hours

The maceration was carried out in a 50 ml PVC container. Result: no trichinae detectable. Trial 4 Origin of the sample material: Breast left Test conditions: Savinase, on ultrasound; 60 ° C, pH 8.0, 10 g salt / liter, 20 g / liter of Savinase Reaction time: 0.5 hours

The maceration was carried out in a 50 ml PVC container. Result: no trichinae detectable. Trial 5 Origin of the sample material: middle right Test conditions: Papain, in the warming cabinet (= without movement of the fleet) Sample previously cooked; 40 ° C, pH 8, 10 g salt / liter, 5 g / liter papain Trial 6 Origin of the sample material: Back right Test conditions: Papain, in ultra-sound, tasting before cooked; 40 ° C, pH 8, 10 g salt / liter, 5 g / liter papain Reaction time: 0.5 hours

The maceration was carried out in a 50 ml PVC container. Result: no trichinae detectable.

Example 3: Comparative Verdauversuche without trichinosis with the enzyme Alcalase ^®

The The following experiments were, unless otherwise stated, as described under Example 1 performed.

In particular, experiments 1 to 3 and 6 to 8 were carried out under ultrasonic treatment. Experiment 4 was carried out with simultaneous movement with a magnetic stirrer. Trial 1 Sample material: 100 g Diaphragm, young pigs, pureed in 500 ml of water Verdauansatz: 2000 ml of water (total) 60 ° C (ratio of meat, water 1:20); 20 g salt; 40 g of Alcalase ^®; Soda approx. PH 7.5. Test conditions: Add soda and salt only after the meat fleet. Result: Meat dissolved within 15 min. Strong, grainy Precipitates, probably lime. Small grease eyes on the Surface. Conclusion: Pre-adjust the solution to pH, mix in the salt beforehand. Use distillate-like water. Small amount Use fat emulsifier. Use another blender, meat stays stuck in holes. Trial 2 Sample material and digestion batch as in Experiment 1, but with fat emulsifier and distillate-like water: 0.5 g Supralan UF, increased to 2 g, soda approx. PH 8.5. Result: pH had to be corrected again after addition of meat become. Meat dissolved within 15 minutes except for a few Remainders (not completely mixed, since this with the used Blender bar can not be reached). Strong, grainy Precipitates. Conclusion: The granular precipitations may be to bone remnants from sawing the pigs. Despite Supralan still grease eyes on the surface. Trial 3 Sample material and digestion as in experiment 2, same enzyme concentration, but half of the digestion liquor (liquor ratio instead of 1:20 only 1:10): 100 g diaphragm, young pigs, pureed in 1000 ml water, 1000 ml water (total) 60 ° C (ratio of meat, water 1:10), 10 g salt, 20 g Alcalase ^® , soda ca pH 8.5. Result: Same amount of meat, but half of the water and Amount of enzyme. Nevertheless, the meat is down to a few fascia also dissolved after 15 minutes. Clear grease eyes on the surface, at the end add 2 g Supralan UF, clear emulsification of the fat. Trial 4 Sample material and digestion as in Experiment 1, but instead of the ultrasonic bath intensive liquor movement by magnetic stirrer. (Note: in the case of the pepsin method, it is an approved variant to intensively agitate the samples using a magnetic stirrer): 50 g diaphragm, young pigs, pureed in 1000 ml of water; 1000 ml of distilled water (total) 60 ° C (ratio of meat, water 1:20), 10 g salt, 20 g Alcalase ^® , soda about pH 8.5.

Experimental procedure on the magnetic stirrer. Result: Significantly more un-digested fascia after 15 minutes. Temperature not quite reached with my magnetic stirrer. Result not quite as good as with ultrasound. Trial 5 Digestion as in experiment 3, but with diaphragms from old sows, during which a total of 8 g Supralan UF was added. Result: The meat was also after a few fascia also after 15 Minutes resolved (no difference to young animals). Significantly more grease eyes on the surface, over 2 × 2 g Supralan added, at the end again 4 g, then everything was Emulsified fat. Trial 6 Sample material and digestion as in experiment 3, but the enzyme concentration halved and 2 g of Supralan UF added. Result: Dissolution of the meat rather only after 20 minutes. "Line" from tendons and bone chips. Not all fat is emulsified. Trial 7 Sample material and digestion batch as experiment 6, but liquor ratio only 1: 5 instead of 1:10. In addition, the meat was minced before mashing in the mincer: 100 g diaphragm, young pigs, mashed in 500 ml of water, 500 ml of water (total) 62 ° C (ratio of meat, water 1: 5), 5 g Salt, 20 g Alcalase ^®, soda about pH 8.5, 4 g Supralan UF. Result: Material dissolved very quickly. (visually approx. 8 min.) after 10 min sieved, only a few fascia residues. Trial 8 Sample material and digestion as in experiment 7, but the enzyme concentration halved In addition, the meat was minced before mashing in the mincer: 100 g diaphragm, young pigs, mashed in 500 ml of water, 500 ml of water (total) 62 ° C (ratio of meat, water 1: 5), 5 g Salt, 10 g Alcalase ^®, soda about pH 8.5, 4 g Supralan UF. Result: Material dissolved very quickly. (visually approx. 8 min.) after 10 min sieved, only a few fascia residues.

EXAMPLE 4: Comparative Verdauversuche with trichinenverseuchten samples with the enzyme Alcalase ^®

Sample material: for each of the following experiments, approx. 10 g meat of horse or pig was added, each containing 40 live trichinae (trichina contaminated meat). The digested samples were sedimented in the separating funnel 3 times for at least 10 minutes and in each case about 1/5 of the liquor was discharged into a beaker. The samples were each refilled with a 0.1% NaCl solution. The evaluation of the samples took place at the University of Bern, Institute of Parasitology. Trial 1 Sample material: Trichinae contaminated pork, sample approx. 10 g, plus approx. 90 g diaphragm old pig, total 100 g, Crushing of the meat by meat grinder, then in Puree 500 ml of water. Verdauansatz: 500 ml of distillate-like water (total) 62 ° C (meat, Water 1: 5), 5 g salt, 20 g Alcalase ^®, soda about pH 8.5 (pH at the beginning about 10), 4 g Supralan UF Reaction time: 15 min with simultaneous ultrasound treatment Result: Some fascia remnants, these were with a coffee sieve sieved; no trichinae detectable. Conclusion: The combination of fat emulsifier and ultrasound too strong. in the corresponding experiment without addition of emulsifier could Trichinen be detected (see Example 2, Experiment 2). Trial 2 Sample material: like experiment 1 Verdauansatz: as experiment 1, but the enzyme concentration was halved. Reaction time: Approximately 16.5 min with simultaneous ultrasound treatment Result: Some fascia residues (more than in experiment 1 (example 4)), approx. 1.7 g); these were sieved with a coffee sieve; few Detectable trichinae. Conclusion: The combination of fat emulsifier and ultrasound too strong. in the corresponding experiment without addition of emulsifier could Trichinen be detected (see Example 2, Experiment 2). Trial 3 Sample material: Trichinae contaminated horse meat; Sample approx. 10 g, plus about 90 g diaphragm old pig, total 100 g, crushing of the meat by mincer, then in 500 ml of water mashed. Verdauansatz: as experiment 1: 500 ml demineralised water (total) 62 ° C (Ratio of meat, water 1: 5), 5 g salt, 20 g Alcalase ^® , soda approx. pH 8.5 (pH at the beginning approx. 10), 4 g Supralan UF.

Reaction conditions as in Experiment 1, but the incubation was not carried out in an ultrasonic bath but by a magnetic stirrer, an intensive liquor movement was achieved. (Note: in the case of the pepsin method, it is an approved option to intensively agitate the samples using a magnetic stirrer). Reaction time: 40 min on magnetic stirrer. Result: Few fascia residues, comparable to example 3, experiment 8; very reliable result: found 32 out of 40 trichinae. in the Unlike having a conventional digestion with pepsin the trichinae does not survive the digestion; this has the advantage that the samples are no longer infectious. Trial 4 Sample material: Trichinae contaminated horse meat; Sample approx. 10 g, plus about 90 g diaphragm old pig, total 100 g, crushing of the meat by mincer, then in 500 ml of water mashed. Verdauansatz: as experiment 2; 500 ml distilled water (total) 62 ° C (Ratio of meat, water 1: 5), 5 g salt, 10 g Alcalase ^® , soda approx. pH 8.5 (initial pH approx. 10), 4 g Supralan UF. Reaction conditions as in experiment 2, but longer incubated in an ultrasonic bath. Reaction time: 25 min in the ultrasound. Result: Few fascia residues, comparable to example 3, experiment 8; no trichinae detectable. Conclusion: The combination of fat emulsifier and ultrasound too strong. in the corresponding experiment without addition of emulsifier could Trichinen be detected (see Example 2, Experiment 2). Trial 5 Sample material: Trichinae contaminated horse meat; Sample approx. 10 g, plus about 90 g diaphragm old pig, total 100 g, crushing of the meat by mincer, then in 500 ml of water mashed. Verdauansatz: 500 ml of demineralised water (total) 62 ° C (ratio Meat, water 1: 5), 5 g of salt, 20 g Alcalase ^®, soda about pH 8.0. Reaction time: 10 min. under simultaneous ultrasound treatment Result: No trichina detectable. Trial 6 Sample material: Trichinae contaminated pork; Sample approx. 10 g, plus approx. 90 g diaphragm old pig, total 100 g, Crushing of the meat by meat grinder, then in Puree 500 ml of water. Verdauansatz: 500 ml of demineralised water (total) 62 ° C (ratio Meat, water 1: 5), 5 g salt, 10 g Alcalase ^®, soda about pH 8.0. Reaction time: 11 min. with simultaneous ultrasound treatment Result: Approximately 0.8 g fascia residues, these were with a coffee sieve sieved. No trichina detectable. Trial 7 Sample material: Trichinae contaminated pork; Sample approx. 10 g, plus approx. 90 g diaphragm old pig, total 100 g, Crushing of the meat by meat grinder, then in Puree 500 ml of water. Verdauansatz: 500 ml of demineralised water (total) 62 ° C (ratio Meat, water 1: 5), 5 g salt, 5 g Alcalase ^®, soda about pH 8.0. Reaction time: 11 min. with simultaneous ultrasound treatment Result: see Figure 1. No trichina detectable.

A sedimentation down and a floating up of material is observed. In It can be seen that the digestive fluid precipitates flaky during sedimentation. This explains why sedimentation is unsuccessful. The precipitate floats upwards and thus prevents (partial) complete sedimentation of the parasites to be detected. A forced filtration (filtration of the solution, optionally with the addition of pressure or vacuum) can counteract this problem. Trial 8 Sample material: Trichinae contaminated pork; Sample approx. 10 g, plus approx. 90 g diaphragm, old pig, total 100 g, Crushing of the meat by meat grinder, then in Puree 500 ml of water. Verdauansatz: 500 ml of demineralised water (total) 62 ° C (ratio Meat, water 1: 5), 5 g salt, 5 g Alcalase ^®, soda about pH 8.0. Reaction time: 30 min. with simultaneous ultrasound treatment Result: see Figure 2. 2 trichinas were detectable, these were intact and not amalgamated.

A sedimentation down and a floating up of material is observed. In It can be seen that the digestive fluid precipitates flaky during sedimentation. This explains why sedimentation is unsuccessful. The precipitate floats upwards and thus prevents (partial) complete sedimentation of the parasites to be detected. A forced filtration (filtration of the solution, optionally with the addition of pressure or vacuum) can counteract this problem.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• - Commission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules for the official inspection of meat for Trichinella [0007]

Claims (36)

Method for detecting capsule ends or non-capsule-forming parasites in meat that cause the maceration of the Meat with a digestive solution that includes a Serine endopeptidase contains. Method according to claim 1, optionally comprising Steps: (a) the mechanical crushing of the to be examined meat; (b) the maceration of the meat to be tested by adding a digestive solution; (c) maceration with simultaneous movement of the digestive approach and / or simultaneous Ultrasound treatment; (d) inactivation of maceration; (E) Filtration of macerate; and (f) control, detection, diagnosis and / or typing for parasite infestation. Method according to one of claims 1 or 2, the meat / digestion solution preferably 1: 5, 1:10 or 1:20. Method according to one of claims 1 to 3, wherein the serine endopeptidase is preferably an enzyme of the enzyme group the subtilisin is. Method according to one of claims 1 to 4, wherein the subtilisin produced recombinant or non-recombinant is. Method according to one of claims 1 to 5, wherein the subtilisin is selected from the group consisting from: Subtilisin Carlsberg and Subtilisin Carlsberg 2.5 L. Method according to one of claims 1 to 6, wherein the digestive solution is basic. Method according to one of claims 1 to 7, wherein the digestive solution preferably has a pH between pH 7 and 10 has. Method according to one of claims 1 to 8, wherein the digestion solution contains NaCl. Method according to one of claims 1 to 9, the digestive solution optionally fat emulsifiers, surfactants and / or auxiliaries. Method according to one of claims 1 to 10, wherein the fat emulsifier is preferably fatty alcohol polyglycol ether is. Method according to one of claims 1 to 11, wherein the excipients are selected from the group consisting from: coenzymes, enzyme substrates, catalysts. Method according to one of claims 1 to 12, with maceration accompanied by ultrasound treatment he follows. Method according to one of claims 1 to 13, wherein the maceration with simultaneous movement and / or simultaneously stirring with a magnetic stirrer. Method according to one of claims 1 to 14, wherein the maceration at a temperature between 55 ° C and 65 ° C is performed. Method according to one of claims 1 to 15, wherein the inactivation of the maceration by filtration of the Reaction or by cooling the reaction is terminated. Method according to one of claims 1 to 16, wherein in the filtration of the macerate the mesh size of used filter on the size of the detected Organisms is adjusted. Method according to one of claims 1 to 17, whereby the control, the detection, the diagnosis and / or the Typing on parasite infestation visually or via the detection of nucleic acids or proteins takes place. Method according to one of claims 1 to 18, wherein the capsule-forming or non-capsule-forming parasites belong to the strain of nematoda (roundworms). Method according to one of claims 1 to 19, wherein the capsule-forming or non-capsule-forming parasites belong to the genus Trichinella (trichinae). Method according to one of claims 1 to 20, wherein the Trichinella species is selected from the group consisting from: Trichinella britovi, Trichinella murrelli, Trichinella nativa, Trichinella nelsoni, and Trichinella spiralis. Method according to one of claims 1 to 21, the non-capsule-forming parasites being Trichinella pseudospiralis are. Method according to one of claims 1 to 22, wherein the meat to be examined is selected from slaughter animals from the group consisting of: domestic pig, wild boar, equidae, Bear, fox, beaver rat (Nutria) and badger stems. Use of an endopeptidase in one of the methods according to one of claims 1 to 23. Use according to claim 24, wherein the endopeptidase is a serine endopeptidase. Use according to one of claims 24 or 25, wherein the serine endopeptidase is an enzyme of the enzyme group the subtilisin is. Use according to one of claims 24 to 26, wherein the subtilisin is selected from the group consisting from: Subtilisin Carlsberg and Subtilisin Carlsberg 2.5 L. Serine endopeptidase for use in a diagnostic method according to one of claims 1 to 23. Enzyme of the subtilisin enzyme group for use in a diagnostic method according to any one of claims 1 to 23. Subtilisin Carlsberg or 2.5 L for use in A diagnostic method according to any one of claims 1 to 23rd Kit comprising a digestive solution, the the enzyme according to any one of claims 28 to 30. The kit of claim 31, wherein the digestive solution further contains NaCl, fat emulsifiers, surfactants and / or auxiliaries Kit according to one of claims 31 or 32, wherein the digestive solution is basic. Kit according to one of claims 31 to 33, wherein the digestive solution preferably has a pH between pH 7 and 9 has. Kit according to one of claims 31 to 34, wherein the fat emulsifier is preferably fatty alcohol polyglycol ether is. Kit according to one of the claims 30 to 34, wherein the auxiliaries are selected from the group consisting of: coenzymes, enzyme substrates, and catalysts.

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