DE10357232B3 - Artifical fecal matter composition useful e.g. for testing cleansing compositions comprises Escherichia coli biomass, ballast materials from vegetable cell wall, glycerin or substance of similar viscosity and aqueous solution - Google Patents

Abstract

Artifical fecal matter composition (C1) comprises bacterial biomass (I), ballast material (II) from plant cell wall material, glycerin or a substance with similar viscosity and an aqueous solution. Independent claims are also included for the following: (1) preparation of (C1); (2) conservation of (C1) by freeze-drying; and (3) freeze dried (C1).

Description

The The present invention relates to an artificial feces replacement and its use for the production of artificial fecal contamination.

Natural feces are because of their unpleasant smell as well as due to hygienic and emotional reasons not suitable for cleaning performance tests For example, to be used by toilet cleaners.

It There is therefore a need for artificial Fäkalienersatz, if possible have the same physical properties as natural fecal matter.

In Fecal substitutes have already been described in this context.

So describes DE 2855062 a Fäkalienersatzstoff consisting of water, a pulp component, binder, dye and optionally an extender such. As polystyrene and / or fungicidal additives.

JP 2000352101 describes a Fäkalienersatzstoff consisting of fatty acids, fatty acid esters and salts and also dyes and salts (carbonates, sulfates) as viscosity regulators.

Further will be published in the SÖFW-Journal (1998) 124, p 1029 an IKW ballast dirt from food ingredients (starch, whole egg, Tomato ketchup, etc.), which is actually as ballast dirt for the Dishwashing has been developed.

It is also known that the large intestine contains about 10 ¹¹ to 10 ¹² bacteria per gram of intestinal contents (Journal of Nutritional Medicine, Vol. 2, pp 6-9, 2000) and that insoluble fiber mechanically loosen the intestinal contents and excreted unchanged ( www.apoverlag.at/DieApotheke/2003_04/seiten/haupt02_laxasoft_0403.html)

Surprisingly has now been found that easily a feces replacement can be produced reproducibly, the physical properties of natural feces reflects very well.

• a) bakterielle Biomasse,
• b) Ballaststoffe aus pflanzlichem Zellwandmaterial,
• c) Glycerin oder eine Substanz mit ähnlicher Viskosität und
• d) eine wässrige Lösung.

This faecal substitute includes:

• a) bacterial biomass,
• b) fiber from plant cell wall material,
• c) glycerol or a substance of similar viscosity and
• d) an aqueous solution.

In a preferred embodiment consists of fecal substitute essentially and / or exclusively from the components mentioned.

Of the Content of bacterial biomass according to the invention is preferably from 10-90 Wt .-%, in particular from 20-70 Wt .-%. As bacterial biomass, basically all bacteria come into question, preferably those that are naturally occur in the human intestine such as Enterobacteriaceae such as Enterobacter, Citrobacter, Klebsiella, Proteus, Providentia, Morganella, Hafnia, Edwardsiella, Enterococcus, Pseudomonas, Staphylococcus, Streptococcus or Serratia and anaerobes such as Bacteroides, Clostridium, Bifidobacterium, Lactobacillus, Fusobacterium or Eubacterium or Mixtures thereof. In particular, derivatives of the natural Intestinal flora, which no longer have any potential for infection, for the use according to the invention suitable. By mixtures of different species can be possibly the natural one Depict occurrences even better. As bacterial biomass come in particular E. coli bacteria, especially those of strain K12 or derivatives thereof, in question.

The bacterial biomass can be obtained by a method of culturing known to the person skilled in the art, for example by cultivation in LB medium, but preferably by cultivation in a medium, contains the material of harder consistency, such as soybean meal, wheat flour, beef protein or pulp or mixtures thereof, and / or permits the production of high cell densities, in particular greater than 1 × 10 ⁹ ml ^-1 . The cultivation is preferably carried out in laboratory shaking flasks, but for larger quantities is about the cultivation under controlled conditions in the fermenter possible.

Of the Content of fiber is preferably from 10-40, especially from 15-25 Wt .-%. Being fiber from plant cell wall material preferably used cell wall polysaccharides, in particular those which are not or only partially cleaved in the digestive tract. in this connection it may be, for example, xylan, cellulose, xyloglucan, pectins or other hemicelluloses or mixtures of said components act.

Of the Moisture content is preferably from 50-90 Wt .-%, particularly preferably from 60-80 wt .-%. To adjust the moisture content is preferably an aqueous solution, especially preferably a buffered aqueous solution uses. This may be, for example, a phosphate buffer, in particular PBS, a Tris buffer or a citrate buffer act.

Of the pH of the aqueous solution and also the substitution of faeces is preferably between 5.8 and 8.5, especially between 6.8 and 8.0.

When Alternatives to glycerin are all substances that are similar viscosity such as glycerol, especially those with a dynamic Viscosity η of 1000-2000, in particular of about 1500 mPa · s at 20 ° C. As an example, polyethylene glycols are mentioned in particular.

Of the Content of the agent according to the invention to glycerol or substances with similar viscosity in a preferred embodiment between 3-30 Wt .-%, in particular in the range of 5-20 wt .-%.

• a) Mikroorganismen der Species E. coli werden in einem Nährmedium kultiviert und vermehrt, das insbesondere Sojamehl enthalten kann, bis die Keimzahl mehr als 10⁹, insbesondere etwa 4·10⁹ Keime pro ml beträgt,
• b) die Biomasse wird durch Zentrifugation geerntet und gegebenenfalls durch Autoklavieren inaktiviert,
• c) die Abwesenheit vermehrungsfähiger E. coli-Keime wird gegebenenfalls durch Ausplattieren auf Tetracyclin-haltigem Medium überprüft,
• d) die Biomasse (bspw. ca. 100 Teile; „Teile" bezieht sich im folgenden jeweils auf das Gewicht) wird mit pflanzlichem Zellwandmaterial (bspw. ca. 49,5 Teilen Xylan), Glycerin (bspw. ca. 33 Teilen) und gepufferter wässriger Lösung (bspw. ca. 61,5 Teilen PBS) portionsweise vermischt, so dass sich eine homogene Masse mit einer leicht viskos tropfenden bis teigigen, gerade noch streichfähigen Konsistenz ergibt.

The fecal substitute can be produced, for example, in the following way:

• a) microorganisms of the species E. coli are cultured and propagated in a nutrient medium, which may in particular contain soybean flour, until the bacterial count is more than 10 ⁹ , in particular about 4 × 10 ⁹ germs per ml,
• b) the biomass is harvested by centrifugation and optionally inactivated by autoclaving,
• c) the absence of replicable E. coli bacteria is optionally checked by plating on tetracycline-containing medium,
• d) the biomass (for example, about 100 parts, "parts" refers to the following in each case on the weight) with plant cell wall material (eg., About 49.5 parts xylan), glycerol (eg., About 33 parts) and buffered aqueous solution (eg. About 61.5 parts PBS) mixed in portions, so that a homogeneous mass with a slightly viscous dripping to doughy, just spreadable consistency results.

The consistency or the rheological behavior of Fäkalienersatzes is preferably from slightly viscous dripping to just spreadable, more preferably from viscous dripping to well spreadable and is adjusted depending on the requirements, in particular by varying the moisture content. In a preferred embodiment, the viscosity of the Fäkalienersatzes at 20 ° C at a shear rate of 10 s ^-1 between 10 and 400 Pa, at a shear rate of 30 s ^-1 between 5 and 120 Pa and at a shear rate of 100 s ^-1 between 1 and 50 Pa. Exemplary viscosities at different shear rates for a good spreadable and a viscous dripping Fäkalienersatz are shown in the following table.

Since the Fäkalienersatz corresponds in consistency and adhesion behavior largely natural fecal matter, it is used as a test substrate for the evaluation of the performance of detergents, especially WC-Rei niger, excellent, in particular to assess the removal of fecal matter or the prevention of the deposition of feces on toilet materials, which may consist in particular of ceramic, stainless steel or plastic. In addition, it can be used to assess the flow behavior in the development of toilet flushing systems, basins and outlets. But as an artificial soiling in the development of detergents, especially for hospital hygiene or diaper products, the substance is suitable.

The chemical composition of feces replacement corresponds largely to the natural feces (see Roche Lexicon Medicine, 4th Edition, Urban & Fischer Verlag Munich or Römpp Online, Georg Thieme Verlag) and accordingly are the physical Properties are largely similar. these can further by varying the amount of the different components be varied according to requirements without artificial, the rheological properties modulating additives required are.

In a preferred embodiment the Fäkalienersatzes invention is the bacterial biomass used hygienic non-pathogenic cells, in particular E. coli cells a derivative of the K12 strain, which may additionally be Autoclaving can be disabled.

One Infection risk due to biomass accidentally not completely killed exists here in particular for E. coli K12 derivatives are not, since these derivatives in the biological Security level S1 (without risk potential) are classified. An intensive anaerobic fermentation and decomposition the nutrients, as it normally occurs in the intestine, found in the production The substance does not take place, so even the usually contained odor causing sulfur compounds and biogenic amines are not excessively strong are included.

Opposite natural feces is therefore the Fäkalienersatz advantageously harmless to health and has inventive handling no unpleasant odor development and no germs capable of reproduction more on. Therefore he can without additional Protective and security measures, z. B. according to the BioStoffVO, for a product development can be used. He is also in Its composition is defined and constant.

One Another advantage of the invention is that pollution it can be preserved by freeze-drying, by Addition of the amount of water removed by freeze drying the properties of feces replacement can be restored. That's why the samples stored very well and virtually unlimited and transported as well become. The present invention is therefore also freeze-dried Faeces replacement, which is obtainable by freeze-drying the Fäkalienersatzes invention.

Of another leaves the viscosity of feces replacement adjust as required by adding or removing water.

object The present invention further contemplates the use of the artificial one Fäkalienersatzes to Production of an artificial Fecal soiling.

The Application of the faeces substitute This can, for example, depending on the set Consistency of feces replacement done with the help of a hole template, the z. B. consist of sheet metal and may have a diameter of 10 mm and a sheet thickness of 0.1 mm or the job can be done with the help of a pipette.

object The present invention is in particular the use of the artificial faeces substitute to carry out from tests to review the Cleaning performance of toilet cleaners, detergents, diaper products and / or toilet flushing systems. The method can be carried out here, for example, as in the embodiments specified.

pictures

In 1 soiling was carried out with natural feces and fecal substitute according to the invention and then subjected to a standardized rinsing process (see Example 3). Fecal replacement and natural feces were applied in alternating columns, beginning with Fäka lien substitute material.

embodiments

The Abbr. "Upm" is below for the Unit rpm.

Example 1: Preparation bacterial biomass

For cultivation a medium is used consisting of: soy flour 30 g / l PUG 10 g / l K ₂ HPO ₄ 10 g / l glucose 30 g / l MgSO ₄ .7H ₂ O 1 g / l MnSO ₄ 50 mg / l FeSO ₄ .7H ₂ O 50 mg / l pH = 7.4

For cultivation, a preculture of the strain Escherichia coli XL-1 (Fa. Stratagene, # 200268) is grown for about 16 h in complete medium (eg Luria-Bertani-Broth) and 1% (based on the total culture volume) of the previously sterilized above Medium added. The incubation is carried out in 500 ml culture filled 2 l glass shake flasks with 4 baffles each for 48 hours at 37 ° C and 130 rpm. The bacterial count is determined by plating and is 4 x 10 ⁹ nuclei per ml in a typical experiment. The biomass is separated by centrifugation at 12,000 rpm (eg Sorvall RC 5B centrifuge, GSA rotor) and subsequently inactivated by autoclaving. The absence of replicable E. coli can be checked by plating on tetracycline containing medium. The yield of biomass (wet weight) is about 60 to 70 g per liter of culture volume.

Example 2: Preparation of feces replacement

From the biomass of Example 1, a Fäkalienersatzstoff is then prepared. 100 g of biomass with 49.5 g of xylan (Oat spelts, Sigma No. X0627), 33 g of glycerol and 61.5 g of phosphate-buffered saline (PBS per liter 0.21 g of KH ₂ PO ₄ , 9.00 g of NaCl, 0.726 g of Na ₂ HPO ₄ .7H ₂ O) are mixed in portions, so that a homogeneous mass with a doughy, spreadable consistency results. Since the moisture content of the biomass can vary, it may be necessary to adjust the liquid content by adding PBS.

Example 3: Comparison of stains with natural Faeces and feces replacement

In 1 A comparison of the model soiling with faeces of human origin can be seen. In each case 3 different amounts of 50 .mu.l to 250 .mu.l were applied to a ceramic tile and subjected to 5 successive rinsing cycles. Good to see depending on the amount applied a different degrees of detachment through the rinsing.

Especially for larger quantities is a largely the same detachment behavior between natural Faeces and faeces substitute to observe.

Example 4: Preparation a freeze-dried feces replacement

One Faeces substitute with the composition of Example 2 is by lyophilization dewatered. The water loss is 54%. You get a free-flowing Powder without sticky properties. By adding the previously withdrawn Quantity of water will get a stain that is not from the soiling from Example 2 differs. In addition, through Variation of water content to adjust the viscosity accurately. The implementation the soiling tests from Example 3 with a soiling from Example 2 and 4 gives identical results.

Claims (17)

Faeces substitute including: a) bacterial biomass, b) fiber from plant cell wall material, c) glycerol or a substance more similar viscosity and d) an aqueous Solution. Faeces substitute according to claim 1, characterized in that the bacterial biomass Contains E. coli. Faeces substitute according to claim 2, characterized in that it is at the E. coli bacteria is a non-pathogenic E. coli strain. Faeces substitute according to one of the claims 1 to 4, characterized in that it is in the dietary fiber from plant cell wall material to cell wall polysaccharides. Faeces substitute according to claim 4, characterized in that it is in the cell wall polysaccharide is about xylan. Faeces substitute according to one of the claims 1 to 5, characterized in that it is in the aqueous solution is a buffer. Faeces substitute according to one of the claims 1 to 6, characterized in that the content of bacterial Biomass of 10-90 Wt .-% is. Faeces substitute according to one of the claims 1 to 7, characterized in that the content of fiber from plant cell wall material of 10-40 wt .-% is. Faeces substitute according to one of the claims 1 to 8, characterized in that the moisture content of 50-90 Wt .-% is. Faeces substitute according to one of the claims 1 to 9, characterized in that the glycerol content of 3-30 wt .-% is. Faeces substitute according to one of the claims 1 to 10, characterized in that the pH is between 5.8 and 8.5 is. Faeces substitute according to one of the claims 1 to 11, characterized in that the consistency of light viscose dripping until just spreadable. Use of a Fäkalienersatzstoffes after a the claims 1 to 12 for making an artificial Fecal soiling. Use of a Fäkalienersatzstoffes after a the claims 1 to 12 to review the Cleaning performance of toilet cleaners, detergents, diaper products and / or toilet flushing systems. A method of producing a faeces substitute according to any one of claims 1 to 12, comprising the following steps: a) microorganisms of the species E. coli are cultured in a nutrient medium and propagated until the number of microorganisms is more than 10 ⁹ germs per ml, b) the biomass becomes harvested by centrifugation, c) the biomass is mixed in portions with fiber from plant cell wall material, glycerol or a substance of similar viscosity and with an aqueous solution, so that a homogeneous mass results with a slightly viscose dripping to doughy, just spreadable consistency. Method of preserving a faecal substitute according to one of the claims 1 to 12, characterized in that the Fäkalienersatzstoff freeze-drying is subjected. Freeze-dried faeces substitute, obtainable by freeze-drying a faecal substitute according to one of claims 1 to 12.