FI101809B - Ways of analyzing a sample from a carbohydrate matrix - Google Patents

Description

101809

METHOD OF ANALYSIS OF A SAMPLE FROM A CARBOHYDRATE MATRIX

The present invention relates to a method according to the preamble of claim 1 for treating a biological sample containing or collected in a carbohydrate-based matrix so that the sample can be analyzed more reliably.

According to such a method, the sample is determined in a manner known per se, e.g.

10 by cultivation or instrumental analysis, at least one property essential for the material to be examined.

The invention further relates to a method for analyzing a biological material according to the preamble of claim 17 and to a method for examining a biological material containing a carbohydrate-based matrix according to the preamble of claim 20. The method according to the invention can thus be applied to cases where the sample to be examined together with the sampler or the sample as such is treated with an enzyme preparation.

The invention also relates to a test kit according to the preamble of claim 24.

In infectious diseases, pathogenic microbes multiply, usually locally, in the human or animal body. For example, the bacterium that causes laryngitis, group A streptococcus, forms a bacterial growth on the pharyngeal mucosa. These streptococci 25 can be detected by sampling the mucosa with a cotton swab or other material sampling device and culturing the sample on a culture medium. Streptococci, like other microbes, can also be detected by antigen detection analysis, such as latex agglutination, EIA (enzyme immunoassay), immunofluorescence staining, chromatography, or RIA (radioimmunoassay).

In this case, the sample bound to the cotton matrix of the sampler is usually transferred to the liquid phase before analysis. Other biological materials can be examined by methods similar to the basic technique 2 101809 described above, i.e., by absorbing a portion of the material into the fiber matrix of the sampling device and culturing the matrix with the samples as is or by extracting the sample from the matrix into the liquid. According to the prior art, various solutions of acid and buffer-5, which may contain, for example, surfactants, are used for the extraction of cotton and viscose sticks and sampling pads. Such an extraction solution may adversely affect the analyte to be determined from the sample, for example, impair the growth of microbes isolated from it or alter the structure of the antigens to be tested.

The studies aim to take as representative and sufficient a sample of the material as possible 10 in order to obtain a reliable result from the analysis. In practice, much of the sample remains attached to the sampler. In addition, if the concentration of the analyte in the sample is low, the analyte may often go undetected. This is particularly detrimental when examining clinical specimens from which a false negative result can lead to untreated disease.

15

The object of the present invention is to eliminate the drawbacks associated with the prior art and to provide a completely new solution for the treatment of biological samples. In particular, it is an object of the invention to provide a method for improving the reliability of the analysis of biological samples.

20

The invention is based on the idea that a sample of biological material, which itself may contain a carbohydrate-based matrix or is collected or absorbed into a sampler containing a carbohydrate-based matrix, is pretreated with a preparation containing hydrolytic enzyme activity before further examination of the sample. It has surprisingly been found in connection with the invention that pretreatment with a hydrolase, in particular: an enzyme acting on plant cell wall components, such as cellulase, hemicellulase, pectinase, amylase or a mixture thereof, can significantly improve the sensitivity and reproducibility of assays for samples. Substantially more test material is released into the liquid phase from a sample containing carbohydrate matrix by enzymatic treatment than if the sample is treated with acid or buffer solutions according to the prior art. Although we do not know exactly the mechanism of this phenomenon 3 101809, the loosening of the matrix and the resulting effective release of the analyte under study may be one factor that improves the reliability of the analysis. As can be seen from the examples below, the effect is not due to complete degradation or dissolution of the carbohydrate matrix, as only minor amounts of soluble sugars have been detected in the analyzes.

More specifically, the method of pretreating a carbohydrate-based sample according to the invention is characterized by what is set forth in the characterizing part of claim 1.

10

The method according to the invention for examining biological material, in turn, is characterized by what is set forth in the characterizing part of claim 17.

The method for examining a biological material containing a carbohydrate-containing matrix 15 and the test kit according to the invention are characterized by what is set forth in the characterizing parts of claims 20 and 24, respectively.

As stated above, the invention can be applied to the treatment of a sample containing or collected from a carbohydrate-based matrix. Such 20 samples are also referred to herein as the term “carbohydrate-based (analytical) sample”.

"Biological material" means, in a broad sense, any material that contains or has contained living organisms, their constituents and / or degradation products (eg antigens) and / or substances secreted or produced by them and / or antibodies raised against them; and / or other analytes of biological origin. Thus, the concept of biological material includes, for example, body fluids and secretions commonly used in clinical trials, such as e.g. blood, serum, plasma, cerebrospinal fluid, ascites, pleural fluid, urine, feces, wet, pharyngeal specimen, saliva and sputum.

30 The method can also be applied to the examination of tissue samples.

4 101809

Biological materials also include materials that consist of or contain carbohydrates. These are generally plant materials and foods and feeds made from plant materials. A particularly interesting application in this case is faeces, which according to the diet contain at least some polysaccharides and organic fibers.

5

The method can also be used in environmental research. In this case, significant applications of the invention are hygiene research in the healthcare sector (hospitals, health centers) and in the pharmaceutical, food and feed industries. In addition to the microbiological quality control of the products themselves, these applications also cover the premises (e.g.

10 hygienic examinations of tables, shelves, floors, walls, ceilings, sanitary fixtures, sewers) and process equipment (including machinery, equipment, piping, air conditioning pipes). Such research is increasingly carried out in the pharmaceutical, food and feed industries (e.g. bakeries, dairies, food factories, slaughterhouses, feed mills) as well as in the forest industry, etc. The invention can also be used in indoor air research in various buildings.

Potential applications are also plant pathological examinations of e.g. cereals, vegetables and fruits. Once the carbohydrate structure of the sample has been enzymatically modified, the pathogen (virus, bacterium, fungus) can be more easily extracted and isolated from the test sample.

However, the method is not limited to the study of samples derived from or produced by living organisms, but in principle any material that can be absorbed into or contain a carbohydrate-based matrix is suitable for testing by the present method.

The term "carbohydrate-based matrix" in the present context includes various structures composed of sugar polymers, which may be e.g. fibrous, layered or reticulated. Typical carbohydrate matrices include various structures containing plant-based fibers, such as cellulosic or lignocellulosic fibers (including cotton, viscose, ramie, and flax fibers). Many biological materials also contain non-fibrous polysaccharide-containing deposits, which are typically produced by microbes. Such deposits include, for example, plaque covering teeth, mucus contained in sputum, pharyngeal, salivary, and cervical samples, and biofilm of microorganisms and the polysaccharide-containing structures secreted by them.

5

What different carbohydrate-based matrices have in common is that they include hydrolyzable bonds such as ester bonds or glycosidic bonds.

"Sample" means a representative portion of the test material from which the desired property (analyte) of the material can be determined. In the present invention, the sample may be in or on the carbohydrate-based matrix of the sampler, or the sample itself may contain carbohydrate-containing material. In the latter case, the sample is subjected as such to the pretreatment according to the invention.

The pretreatment of the invention involves contacting a sample with carbohydrate matrices with an enzyme preparation having hydrolase activity that affects the carbohydrate structure prior to determining the desired property to release the desired analyte from the carbohydrate matrix. Preferably, the sample to be tested is then contacted with an amount of hydrolase enzyme sufficient to loosen the structure of the matrix 20, but which does not substantially dissolve the matrix. The dissolution of the matrix and the resulting degradation products may affect the analysis.

Thus, when a sample contains cellulosic fibers, it is generally intended that less than 20% of the fibers degrade into mono- or oligosaccharides. This is accomplished by using an appropriate enzyme composition and processing conditions.

25: According to the method of the invention, at least one preselected property (analyte) is determined from the biological material. This property may be the presence of a microorganism, such as a pathogenic or non-pathogenic bacterium, virus, fungus, yeast, protozoan, or a part or product thereof (e.g. an enzyme), or it may be the presence of a molecule (e.g. an antibody, hormone, prion or other marker), compound or element (eg trace element). Of these, 6,101,809 microbes can be detected, for example, by culturing, antigen detection, or determining antibodies raised against them.

By "hydrolase" is meant an enzyme that cleaves S ester bonds and glycosidic bonds contained in a carbohydrate structure. Suitable cellulases include cellobiohydrolases and endoglucanases, while suitable hemicellulases include xylanases and mannanases. / or arabinoglucan components.

Thus, it contains one or more of the following activities: pectin esterase, IS polygalacturonase, exopolesalacturonase, pectin lyase, endoglucanase, endoxylanase and mannanase. The enzyme preparation may contain mixtures of the above, in particular mixtures of cellulases and hemicellulases, but the preparation may contain other enzymes in addition to hydrolases.

According to the invention, a sample containing a carbohydrate-based matrix or a biological sample collected or absorbed in a sampler containing a carbohydrate-based matrix is contacted with the enzyme, e.g. by immersing the sample in the enzyme solution for a desired time. The sample is shaken in solution and / or allowed to stand. When the sample as such already consists of a carbohydrate-based matrix, the enzyme treatment 25 is performed, e.g., by mixing the sample with the enzyme solution, after which the enzyme is allowed to act for the desired time before the analysis is performed.

The enzyme preparation is used in an amount of 5 mg to 50 mg per gram of sample (dry weight). The enzyme treatment is performed at 0-60 ° C, pH 3-10, preferably about 3-8.

30 Processing time can range from 1 minute to about 48 hours.

7 101809

The appropriate amount and composition of enzyme depends on the origin of the sample to be treated and the sample matrix itself. In those cases where the sample is collected with a cellulose-based sampler, the preferred enzyme dose is 0.02 to 5 mg / g of sample. If the sample itself contains a carbohydrate-based matrix (e.g. faeces, cereals, feed), a suitable enzyme dose 5 is 0.5 to 20 mg / g of sample.

The preferred enzyme composition is determined by the sample matrix: an enzyme preparation suitable for a sample collected in a fibrous and cellulose-based sampler contains in particular cellulase and pectinase. A preferred enzyme preparation for sample 10 containing a carbohydrate-based matrix includes cellulase, pectinase and hemicellulase. If the treatment is applied to a material rich in starch (eg cereals, feed), it is appropriate for the enzyme preparation to contain amylase in addition to the above.

In many cases, the hydrolase treatment is preferably performed at room temperature, in a buffer solution having a pH of about 4-6, with the duration of the enzyme treatment typically being about 10 to 60 minutes. As the buffer, for example, Na acetate solution can be used.

Especially for hygiene studies, an alternative in which the enzyme treatment is carried out at the storage temperature of the sample, i.e. at about 0-6 ° C, is suitable. Most preferably, a buffer solution is used as the medium in order to stabilize the pH at the optimum pH of the enzyme. The treatment time is 1 to 30 h. Said application option can be performed, for example, by placing the sample taken for hygiene examination in the enzyme solution immediately after sampling and storing it overnight or until the sample is transported to the laboratory for analysis.

25

The test kit of the invention for sampling biological material includes sampling means comprising a carbohydrate-based fibrous material sampler for collecting a sample of the biological material and a pretreatment agent for releasing the analyte in the fibrous material for examination. The test kit may further include analytical means by which the analyte of the sample can be determined, e.g., by the rapid diagnostic methods set forth below. The test kit may also contain the pretreatment agent alone.

The test kit pretreatment contains an enzyme preparation with hydrolysis activity that releases the test analyte from the carbohydrate matrix of the sampler.

In some situations where the sample itself contains a carbohydrate matrix, a sampler is not required but treatment is performed directly on the sample. In addition to the enzyme or enzymes, the enzyme preparation may contain a solution acid (pH) stabilizing buffer, antimicrobial components and enzyme stability enhancing components. The concentration of enzymes in the preparation is adjusted so that a sufficient dose of enzyme is achieved at the place of use of the preparation. In many cases, a suitable enzyme concentration in the preparation is 0.05 to 0.5% by weight.

According to a preferred embodiment, a sampling pad or swab consisting of a cellulose-15 matrix is used as the sampling means, optionally attached to a holder suitable for sampling. The cellulose matrix then consists of cotton, viscose or cellulose pulp or a product made from these by chemical modification, such as viscose. Absorbent paper strips are also suitable as sampling media.

20

After the pretreatment according to the invention, the sample is analyzed in a manner known per se. Thus, any microbes present in the sample can be detected by conventional, microbial culture-based methods or other diagnostic methods. Of the latter, mention may be made of the detection of antigens based on the identification of components which are typical of microbes by means of antibodies or other reagents which react sepsis with them. Common antigen detection methods include immunological methods such as latex agglomeration, EIA, RIA, FIA (fluorescence immuno assay), immunofluorescence staining, immunoelectrophoresis, etc. Microbial-specific nucleic acid sequences can also be detected using labeled nucleic acid as a probe (nucleic acid hybrid).

9 101809

Detection of microbes by conventional culture typically occurs in three steps: first, any microbes in the sample are cultured, then pure culture is performed, and finally the microbes are identified. In the culture step, the sample can be cultured on a plate by dilution, resulting in distinct colonies of individual bacteria that can be identified. Enrichment conditions in which only the microbe under study grows can also be used for culture. The above methods can be used in the identification step.

Particularly preferably, the method is used for the study of microorganisms belonging to the genera Pseudomonas, Bacillus, Pediococcus, Candida, Streptococcus, Staphylococcus, Escherichia, Salmonella, Campylobacter 10 or Chlamydia and some viruses, such as, for example, rat and adenoviruses. However, in principle, any microbial or virus that can be cultured or otherwise identified is suitable for testing by the method of the invention.

The invention provides considerable advantages. Thus, the sample treatment according to the invention, unlike the known extraction solutions, does not adversely affect the analyte to be determined from the sample. Because the action of the hydrolase on the carbohydrate matrix releases slightly soluble sugars, which can be utilized by, for example, microorganisms, making it easier to determine these by culturing. The method can further significantly improve the analyzability of the sample; as shown in the examples below, the sensitivity of the assay may even multiply.

The invention will now be examined in more detail with the aid of the accompanying figures and a few application examples.

25

Figure 1 shows a chromatogram run from a commercial cellulase preparation and Figure 2 shows the disintegration of the cellulose fiber-based sample pad as a function of time at different enzyme dilutions.

101809 ΙΟ

Example 1

Characterization of Enzymes This example describes some enzyme preparations suitable for use in the method of the invention.

Cellulase means a preparation containing one or more cellobiohydrolases (E.C. 3.2.1.91) and endoglucanase (E.C.3.2.1.4). Several different preparations are commercially available, which have been produced with e.g. Trichoderma or Humicola molds. Here 10, a product suitable for this purpose, the product name of which is Econase (Primalco Oy), is described in more detail. The manufacturer declares the following activities for the product: FPU (filter paper unit) 31 U / ml HEC (hydroxyethylcellulosa) 13,500 nkat / ml 15 β-glucosidase 1,030 nkat / ml

Figure 1 shows a chromatogram run from Econase (DEAE Sepharose, Pharmacia; phosphate buffer pH 7.2; elution with a salt gradient) showing that the product contains all major Trichoderma mold cellulases (CBH I, CBH Π, EG I and EG Π).

20

Pectinases are a large number of enzymes that are able to break down plant cell wall pectin material. The branched pectin material is composed of galacturonic acid, rhamnose, xylose, fucose, arabinose and galactose. In addition, the pectin material also includes xyloglucans and arabinoglucans, through which the pectin material is attached to the cellulose. Arabinogalactans form a bridge between the plant cell wall protein and pectin.

Several enzyme preparations are commercially available that contain enzymes that degrade or modify plant cell wall pectin. In this connection, 30 commercial preparations called Pectinex Ultra have been used, which contain at least the following activities: 101809 π pectin esterase E.C. 3.1.1.11 polygalacturonase E.C. 3.2.1.15 exopolyactacturonase E.C. 3.2.1.67 pectin lyase E.C. 4.2.2.2 E. endoglucanase E.C. 3.2.1.4 endoxylanase E.C. 3.2.1.8 Mannanase E.C. 3.2.1.78

Pectin esterase, polygalacturonase, exopolygalacturonase and pectin lyase are precursors to pectin-acting enzymes.

By hemicellulases is meant in this context an enzyme preparation containing xylanases, mannanases and enzymes acting on the substance pectin. Xylanases (E.C. 3.2.1.8) and mannanases (E.C.3.2.1.78) were produced with Trichoderma mold and purified as previously described (Tenkanen et al. 1992, Stälbrand et al. 1993).

These enzymes were added to a commercial pectinase preparation (Pectinex Ultra, Novo Nordisk, Denmark) to give a mixture of e.g. the following activities: xylanase 7 150 nkat / ml 20 mannanase 9 000 nkat / ml polygalacturonase 15 150 nkat / ml

Example 2 Dispersion of an Omni-SAL sampler with cellulase

An Omni-SAL sampling pad made of cellulose-based material for collecting saliva samples was used as the material to be examined, which was cut into small pieces for the experiment. Samples were incubated for 2-6 hours at room temperature (18-30 ° C) in buffer solution (100 mM Na acetate buffer, pH 5) to which various amounts of cellulase (Econase, Primalco Oy, Biotec, Rajamäki) were added. The enzyme dilutions were 12,101,809 1: 1,000, 1: 5,000, and 1: 10,000. A buffer solution without enzyme and the Omni-SAL solution supplied with the product were used as controls. After incubation, the absorbance of the solutions was measured at 600 nm (A? NJ, reflecting the dispersion of the sample pad in buffer).

5

Figure 2 shows the disintegration of the sample pad structure (A # »„ J as a function of time with different enzyme dilutions, as well as with buffer and Omni-SAL solution.

It can be seen from Figure 2 that the structure of the sample pad is significantly released at all enzyme-10 dilutions, but hardly with buffer or Omni-SAL solution alone. When soluble sugars were determined from the sample solution (enzyme dilution 1: 1,000) by liquid chromatography after 6 hours of incubation, it was surprisingly found that only about 10% of the cellulose was cleaved by enzymes into glucose. Thus, the disintegration of the sampler structure, i.e. the dispersion of the swab, took place by the action of the enzymes apparently before the actual hydrolysis of the cellulose. For example, after one (1) hour, which is a practical incubation time, the amount of soluble sugars is very small, but the opening of the cellulosic structure is already clearly noticeable.

20 Example 3

Effect of enzyme treatment on the culture result of a biofilm sample collected with a cotton swab

A stainless steel plate (AISI 304 / 2B) was used as the biofilm growth medium. The steel plates were washed with warm (50 ° C) 2% RBS 35 detergent (Chemical Products SPRL, .. 25 Belgium), rinsed five times with warm water and sterilized by autoclaving. The following test organisms were grown on the surface of the steel plate:

Pseudomonas fragi (Gram-negative bacterium)

Bacillus licheniformis (Gram-positive bacterium 30 Pediococcus inopinatus (Gram-positive bacterium)

Candida utilis (yeast) 13 101809

The composition of the culture medium was as follows: 2.4 g of LAB-Lemco powder (Oxoid), 8 g of Nutrient Broth (Difco), 50 g of sucrose, 10 g of glucose and 10 g of fructose in 1000 ml of distilled water.

5 Plates in broth (200 ml) were inoculated with 2 ml of a microbial suspension with a cell concentration of 108-109 cfu / ml (du, Colony forming units). The steel plates were shaken (60 rpm) in growth tanks at +25 * C for 5 days. The culture broth was changed every other day by pouring off the old medium and adding about 200 ml of new medium. After culturing, the sample plates were rinsed twice in the culture tanks with sterile water.

10

Bacterial mass was collected from sample plates with cotton sticks dipped in distilled water. After sample collection, cotton swabs were treated with the following enzyme solutions: cellulase (1: 100 and 1: 500 dilutions) 15 pectinase (1: 100 and 1: 500 dilutions) mixture of cellulase and pectinase (1: 1, diluted 1: 100 and 1: 500)

Enzyme treatments (three cotton sticks / treatment) were performed in buffer (50 mM Na-acetate buffer, pH 5) at room temperature (18-23 ° C) for one (1) hour. For comparison, the corresponding treatment (buffer) without enzyme was used. Sample tubes were mixed at the beginning and end (30 s). After treatment, the sample was diluted in peptone-containing physiological saline, and two parallel nutrient agar plates were inoculated from the appropriate dilutions and grown at + 30 ° C for 2 days.

25 The results are shown in Table 1: 14 101809

Table 1. Effect of enzyme treatment on the culturability of a biofilm sample 5 Organism__Red colony count, log cfu / cm2 when the sample was treated with cellulase pectinase and a mixture of pectinase ___ buffer__ P. fragi_5.49 ± 0.32 5.45 ± 0.44 5.43 5.33 5.33 .0.9 ± 0.44_ B. licheniformis 4.44 ± 0.32 4.73 ± 0.41 4.94 ± 0.42 4.35 ± 0.29_ P. inopinatus 3.46 ± 0.42 3.04 ± 0.15 2.98 ± 0.31 2.72 ± 0.24_ 10 C, utilis 3.97 ± 0.44 3.61 ± 0.29 3.74 ± 0.29 3.69 ± 0.37

Enzyme treatment improved microbial culture from a biofilm sample taken with a cotton swab. Depending on the composition of the biofilm, either cellulase alone or a mixture of cellulase and pectinase gave the best results.

Example 4

Effect of Enzyme Treatment on the Performance of the Strep A Rapid Test 20 The test was performed using the Tandem ICON Strep A Rapid Test, with a cotton swab and the dacron stick provided with the kit as samplers. Artificial samples were prepared by taking a throat swab (healthy throat) with a cotton or dacron stick as instructed and then adding 50 μΐ of the overnight Streptococcus pyogenes strain grown to the sticks at a concentration! 106 or 105 bacteria / ml. Sampling sticks were immersed in the enzyme solution for 5 seconds each and placed in a sterile tube for 30 minutes or 1 hour. Enzyme solutions contained cellulase, hemicellulase, or a mixture of cellulase and hemicellulase. Prior to treatment, the enzymes were diluted 1:10 in physiological NaCl. After enzyme treatment, a rapid test was performed according to the instructions. Color intensity was interpreted visually.

30 The results are shown in Table 2.

15 101809

Table 2. Effect of enzyme treatment on the performance of the Strep A rapid test Bacterial- Sampling Test result1 when the sampler it & dteHH concentration 5 with a mixture of hemicellulase cellulase and buffer hemisululase _ 30 min 60 min 30 min 60 min 30 min 60 min 30 min 60 min

Even cotton »++ + + + ++ ++ m + + + ++ + + 10t / ml DacroräSkin ^" + + + + "

Cotton swab -M- ++ + + + + ++ + + 10 lOj / ml "" "^ äcröimkki ^” 1 + -f- +, blue; + +, light blue; +, light blue; no color 2 ET, not tested 15 In conclusion, when a cotton swab is used as a sampler, enzyme treatment enhances the release of bacteria from the sampler, which increases the sensitivity of the antigen detection test.At a lower bacterial concentration, a cotton swab treated with enzyme gave a better result than in a commercial test.

20

Example 5

Effect of Enzyme Treatment on Klamydia Antigen Detection A Wellcozyme Chlamydia Specimen Collection kit (female) 25 cotton swab was used as a sampler. Chlamydia antigen was detected by EIA (Wellcozyme Chlamydia Murex). As an antigen ("sample"), a crude preparation of Chlamydia containing tax cells (Orion Diagnostica, Espoo) diluted in PBS was used. The antigen solution was absorbed into a sample stick, which was then placed in a sterile tube and allowed to stand overnight at room temperature (= transport to the laboratory).

For pretreatment of the cotton swab, the enzyme solution (cellulase) was diluted (1/100 and 1/500) in dilute HCl pH 5.0. Enzyme buffer (dilute HCl) without enzyme was used as a negative control. Cotton swabs were shaken in enzyme solutions in test tubes with a level mixer, at room temperature, for 1 hour, after which the enzyme solutions were subjected to an EIA assay according to the instructions of the Wellcozyme Chlamydia kit. There were two sticks in each of 16,101,809 readings and two parallel EIA assays were performed on each stick. The results were compared with the EIA result obtained without pretreatment (cotton stick for direct extraction according to the kit).

5 The results are shown in Table 3:

Table 3. Effect of cellulase treatment on Chlamydia antigen detection

Pretreatment_EIA absorbance x 1000

Cellulase 1 / 100__1.694_ 10 Cellulase 1 / 500__2.512_

Entsyymipuskuri__1.078_

No processing 1.779

Cellulase treatment (1: 500) clearly increased the sensitivity of the test.

15

Example 6

Effect of enzyme treatment on faecal dispersion

Enzyme treatments were performed with the following enzyme preparations: cellulase, hemicellulase 20 (xylanase + mannanase + pectinase) and a mixture of cellulase and hemicellulase.

. Enzyme solutions were diluted 1:10 in physiological NaCl. A stool sample (1 g) was mixed (by vortexing for 1 min) with 9.0 ml of enzyme solution. The samples were then allowed to stand at room temperature for various times (30, 60, 120 and 480 min), after which the absorbances of the samples at 600 nm were measured. Prior to each measurement, device 25 was first reset with a control sample (1 g of feces without enzyme) and then with enzyme solution (colored). The results are shown in Table 4.

17 101809

Table 4. Effect of enzyme treatment on faecal dispersion psssssBESBaBssssaBsess = asBaBasBmBS = sesBSSsasBBBsa

Enzyme _Absorbance (A ^ „J_ 5 30 min. 60 min. 120 min. 480 min.

Cellulase__0.020 0.060 0.120__0.260

Hemisellulaasi__0,040__0,110__0,220__0,345

Cellulase + hemicellulase 0.010 0.040 0.080 0.160 10

In addition, the enzyme solutions were made into bacterial cultures on nutrient agar plates, which were allowed to grow for 2 days at 36 ° C. This gave the total number of endogenous, aerobically growing bacteria present in the samples. The results are reported in Table 5.

15

Table 5. Effect of enzyme treatment on the number of bacteria whistling in the faeces Treatment _Bacterial concentration (x 10s) _ __60 min .__ 480 min._ 20 No enzyme ___ 0.50__0.25_

Cellulase K25__1.60_

Hemisellulaasi__K25__L60_

JeHu ^ smjahemsellul ^ smjeo ^ __ 3.00 25

As shown in Table 4, all enzyme treatments significantly contributed to fecal dispersion. Enzyme-induced fecal dispersion clearly increased the number of bacteria that could be cultured from the feces (Table 5).

Claims (27)

1. Process for pretreating a carbohydrate-based assay, characterized by 5. The sample is contacted with an enzyme preparation which affects the structure of the carbohydrate matrix and contains cellulase, hemicellulase, amylase and / or pectinase activity, in an amount sufficient for unlocking the matrix structure without substantial dissolution of the matrix. 10 The method of claim 1, wherein a sample containing cellulosic fibers is processed, characterized in that at most about one-fifth of the cellulosic fibers are broken down into mono- or oligosaccharides. 3. Process according to claim 1, characterized in that an enzyme preparation containing cellobiohydrolase and endoglucanase activity is used. 4. A process according to claim 1, characterized in that an enzyme preparation containing xylanase and / or mannanase activity is used. 20 5. A process according to claim 3 or 4, characterized in that an enzyme preparation containing cellulase and hemicellulase activity is used, in particular cellulase and xylanase and / or mannanase activity. 6. A process according to claim 1, characterized in that a pectinase composition is used which acts on the galacturonic acid, ramnose, xylose, fructose, arabinose, galactose, xyloglucan and / or arabinoglucan components in the cell wall of a plant fiber. Process according to claim 6, characterized in that a pectinase preparation containing one or more of the following activities is used: pectin esterase, polygalacturonase, exopolygalacturonase, pectin lyase, endoglucanase, endoxylanase and mannanase. 101809 Method according to any of the preceding claims, characterized in that it is used 0.02 mg - 50 mg of enzyme preparation per sample gram (dry weight). Method according to any of the preceding claims, characterized in that the sample 5 is immersed in or mixed with an enzyme solution. Process according to any of the preceding claims, characterized in that the enzyme treatment is carried out at a temperature of 0 - 60 ° C for a period of 1 minute. 48 hours and with a pH of 3 - 10, preferably about 3 - 8. 10 A method according to any of the preceding claims, wherein, after the pretreatment, at least the pre-selection characteristic of the pre-selection property sample is characterized by the determination of the microorganisms in the sample. 15 Method according to claim 11, characterized in that the bacterial, fungal, yeast, protozoal and / or viral contents of the sample are determined. Method according to claim 11 or 12, characterized in that a microorganism or a root or adenovirus is determined in the sample, belonging to the species Pseudomonas, Method according to any of claims 11-13, characterized in that the microorganisms are determined by culture or by detection of their antigens or antibody pairs. Method according to any of claims 11-13, characterized in that the microorganisms are determined with antibodies that react with them. 30 The method according to any of claims 1-10, wherein, after the pre-treatment, at least one pre-selected characteristic of the sample is determined, characterized in that the hormones, markers, prions and / or spare substances of the sample are determined. 17. A method for analyzing a biological material, according to which the method - the taking of the material, a sample for which a predetermined characteristic is characterized, characterized by - the sample is taken by means of a sampling device consisting of plant fibers and - the sampling means is processed after the sampling, prior to determining the selected property, a hydrolase activity containing enzyme preparation in an amount sufficient to dissolve the matrix structure without substantial dissolution of plant fibers is required. 18. A method according to claim 17, characterized in that as a sampling unit a cellulose-based matrix is used, which is possibly attached to a holder suitable for sampling. 19. A process according to claim 18, characterized in that cotton, viscose or cellulose pulp or a product made by chemical modification is used as a cellulose-based matrix. 20 20. A method for analyzing a biological material containing a carbohydrate matrix, according to which the method - one of the material takes a sample, which determines a predetermined property of, characterized by - the sample is processed after sampling, before determining it selected property, by a hydrolase activity containing enzyme preparations in an amount sufficient for the digestion of the matrix structure without substantial dissolution of the carbohydrate matrix. 30 Bacillus, Pediococcus, Candida, Streptococcus, Staphylococcus, Escherichia, Salmonella, Campylobacter or Chlamydia. Method according to claim 20, characterized in that the sample consists of an excrement. 101809 22. A method according to claim 20, characterized in that the sample consists of plaque, of mucus that is included in expectoration or pharyngeal, salivary or cervical samples. 5 Method according to claim 20, characterized in that the sample consists of plant material. A test package for sampling a biological material and for analysis by the method of claim 17, which comprises • a sampling means comprising a portion consisting of fibrous material on which the biological material may be collected or absorbed, and - a pretreatment agent, characterized in that - the pretreatment agent contains an enzyme preparation with hydrolase activity which substantially unlocks the structure of the fibrous material without dissolving the fibrous material. Test package according to claim 24, characterized in that it also contains analyzers for analysis of the sample material. 26. A test package according to claim 24 or 25, characterized in that the pre-treating substance contains an enzyme preparation which acts on hydrolase activity and a standardizing buffer and protective agent which act on the carbohydrate matrix. Use of an enzyme preparation which affects the structure of a carbohydrate matrix and contains cellulase, hemicellulase, amylase and / or pectinase activity, for pretreatment of assay samples in such a way that the structure of the carbohydrate matrix is digested by said enzyme. 30