WO2001053010A1 - Enzymatic process for fluidizing or detaching biofilms from different interfaces - Google Patents

Enzymatic process for fluidizing or detaching biofilms from different interfaces Download PDF

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Publication number
WO2001053010A1
WO2001053010A1 PCT/ES2000/000360 ES0000360W WO0153010A1 WO 2001053010 A1 WO2001053010 A1 WO 2001053010A1 ES 0000360 W ES0000360 W ES 0000360W WO 0153010 A1 WO0153010 A1 WO 0153010A1
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biofilm
solid
liquid
elimination
facilitate
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PCT/ES2000/000360
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Spanish (es)
French (fr)
Inventor
Carmen San Jose Serran
Almudena Jaspe Rodriguez
Begoña RUIZ PEREZ
Tomas Maira Litran
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Universidad Complutense De Madrid Rectorado
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Priority to AU75258/00A priority Critical patent/AU7525800A/en
Publication of WO2001053010A1 publication Critical patent/WO2001053010A1/en

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned

Definitions

  • TITLE Enzymatic procedure to fluidize or detach biofilms from different interfaces
  • the present invention falls within the technical field of cleaning and disinfection of surfaces. More specifically, the invention relates to a method for removing biofilms, also called biofilms, biological films, bio-layers or microbial films, formed by different microorganisms at the solid-liquid-air and solid-liquid interfaces of aqueous systems (systems that contain water, fluid systems); and to the use of at least one enzyme, belonging to the group of carbohydrases or proteases, in this procedure
  • microorganisms to surfaces are a frequent occurrence in aqueous systems.
  • the growth of microorganisms associated with surfaces or other interfaces involves the formation of biofilms, that is, a consortium of cells embedded in an exopolymeric matrix.
  • biofilms The presence of biofilms in different industrial processes is related to hygienic and technological problems. Among the former, its role as a reservoir of pathogenic and altering microorganisms, pollutants of food and biotechnology industry facilities stands out. On the other hand, the formation of biofilms is associated with the deterioration of materials, such as the corrosion of metallic substrates and the coating of other substrates, causing increases in energy expenditure, such as in heat exchangers and fluid distribution systems, decreased efficiency of processes, such as in water treatment or papermaking, and even the uselessness of pipes. The development of biofilms may also have place on surfaces of clinical interest, such as contact lenses or implants. and forming the basis of dental plaque formation.
  • the microbial population present in the biofilm depends on the initially adhered microorganisms, their proliferation in the biofilm and their detachment and passage to the liquid phase.
  • Gram-negative bacteria are particularly frequent, especially species of the genus Pseudomonas, Acinetobacter, Flcn'obacteriiim, Desulfovibrio, Escherichia and Enterobacter, and to a lesser extent Gram positive bacteria, molds, yeasts and algae.
  • a large part of these microorganisms are strict aerobes, so their growth in the solid-liquid-air interface is usually greater than in the solid-liquid (submerged zone), where oxygen availability is lower.
  • the exopolymeric matrix or glycocalix in which the adherent cells are trapped, is responsible for the structure and organization of the biofilm, and consists mainly of polysaccharides and proteins. Its production and composition, in addition to being related to the type of microorganism and surface (Ruiz et al., 1999 in Biofilms: The Good, the Bad and the Ugly. Edited by J. Wimpenny, P. Gilbert, J. Walker, M. Brading and R. Bayston.
  • Oxygen is one of the growth limiting factors of the main microorganisms involved in the formation of biofilms; the accumulation of cells and exopolymeric material can become greater in the solid-liquid-air interface than in the solid-liquid.
  • the elimination of industrial biofilms is usually done by mechanical cleaning procedures combined with the use of biocides.
  • the microorganisms accumulated in the biofilm are characterized by having a high resistance to the disinfection agents usually used in the industrial processes. Part of this effect is attributed to glycocalix, which constitutes a barrier to the penetration of the compounds present in the liquid phase. Although most of the viable biofilm cells are removed, residual exopolymeric material can support the growth of new microorganisms. Therefore, an effective elimination of the matrix will allow the biofilm to detach, also limiting the problems associated with microbial recolonization.
  • the enzymatic degradation of glycocalix has the advantage of being a cleaning procedure, respectful of the environment and the integrity of the surfaces.
  • the object of this invention is the development of a procedure that respects the environment and the surfaces of aqueous systems, effective to detach totally or partially biofilm accumulated in both the solid-liquid-air interface and the solid-liquid interface.
  • Another object of this invention is the development of a method capable of modifying the rheological properties of the exopolymeric matrix of the biofilm accumulated both in the solid-liquid-air interface and in the solid-liquid interface of the aqueous systems, so as to facilitate the elimination Biofilm after applying other physical or chemical cleaning and disinfection procedures.
  • This invention also aims to define the composition of a cleaning product consisting essentially of carbohydrases and proteases. to eliminate accumulated biofilm both in the solid-liquid-air interface, and in the solid-liquid of aqueous systems, compatible with the independent or combined use of other cleaning agents such as corrosion inhibitors, surfactants, biocides, among others.
  • the enzymatic process for fluidizing or releasing biofilms from different interfaces object of the invention, consists of a system to prevent, detach and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface (in this invention called biofilm in interface) as in the solid-liquid (in this invention called submerged biofilm) of aqueous systems, by exposure to a cleaning preparation that includes at least one enzyme of the carbohydrase or protease type, in an amount sufficient for the exopolymeric matrix the partial or total detachment of the biofilm is degraded and its rheological properties are modified, facilitating the subsequent elimination of the biofilm by other cleaning and disinfection procedures.
  • a cleaning preparation that includes at least one enzyme of the carbohydrase or protease type
  • Membrane, Cellubrix L, and Sigma Chemical Co products cellulase, alpha-amylase, pectinase, pectinase, pectolyase and dextranase.
  • Proteases useful for this invention include serine proteases such as trypsin and pepsin, both from Sigma Chemical Co.
  • the cleaning preparation of this invention is added to aqueous systems in the appropriate amount to release both the biofilm at the interface and the submerged and to modify the rheological properties of both types of biofilm.
  • the dose of each of the enzymes is not critical to the invention and is adjustable to the nature of the aqueous system, type of microorganism and environmental conditions.
  • the concentration of carbohydrase can vary from 0.0001% to 10% and that of protease between 0.001 and 100 U / mL.
  • the exposure of the biofilm at the interface and submerged to the cleaning preparation is preferably carried out in the range of pH and temperatures at which the enzymes applied are active, generally pH between 4 and 1 1, preferably between 4.5 and 8 and temperature in a range of 10 to 60 ° C, preferably between 25 and 50 ° C.
  • the exposure time is not critical to the invention, and may vary from 30 min to 24 h, preferably 1 h.
  • this procedure that allows to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid of aqueous systems, can include the exposure of biofilm to carbohydrases, or the exposure of biofilm to proteases. or the exposure of biofilm to carbohydrases and proteases.
  • the biofilm exposure is carried out in two sequential stages 1) exposure of the biofilm to a cleaning preparation consisting essentially of carbohydrases, 2) exposure of the biofilm to a cleaning preparation consisting essentially of proteases.
  • Figure 1 Represents digital images (2 magnifications) of biofilm biomass on glass (1 1) not exposed to enzymes and (1 2) exposed for 60 min first to Citrozym UltraL (1%) and then to trypsin (10 4 U / L ), of the interface (A) solid-liquid-air and (B) solid-liquid
  • Figure 2 Shows photographs taken by scanning electron microscopy at 2000 magnification of biofilm cells (2 1) not exposed to enzymes and (2 2) exposed for 60 min first to Citrozym UltraL (1%) and then to trypsin (10 4 U / L)
  • Figure 3 Shows the apparent viscosity, dependent on the strain rate of biofilm not exposed to enzymes ( ⁇ ) and exposed to CitrozymUltraL (1%) ( ⁇ ) and subsequently to CitrozymUltraL (1%) plus trypsin (10 4 U / L) ( ⁇ )
  • the log of the apparent viscosity is represented in Passes x second (Pa s) and on the ordinate axis the log of the deformation velocity in
  • the present invention is illustrated by the following examples, using Pseudomonas standardized biofilm blank. as this is one of the genera whose adhesion has been studied more frequently and is most commonly found in natural biofilms formed in very diverse habitats
  • Example 1 Obtaining and quantifying biofilm at interphase and submerged
  • the biofilms are formed on surfaces (coupons of 22 x 22 mm) of glass and stainless steel, arranged vertically, partially covered with the culture medium, so that part of the biofilm was formed at the solid interface. liquid-air and part in the solid-liquid interface (submerged zone), as described below.
  • the glass coupons were immersed in nitric acid (2 h) and subsequently rinsed with abundant double-distilled water.
  • Type 316 stainless steel coupons were left immersed overnight in neutral detergent; They were then rinsed with distilled water and kept in absolute ethanol (2 h), being finally rinsed with double distilled water.
  • a horizontal support with 16 radial grooves was used for the settlement of the coupons, these being perfectly subject.
  • the supports, loaded with the coupons, were introduced into beakers and the whole was sterilized by dry heat, subsequently adding the bacterial culture, so that it covered only the lower half of the coupons.
  • Pseudomonas fluorescens strain B52 was used, grown in a liquid mineral medium, pH 7, consisting of: 10.70 g L of N, N-bis- (2-hydroxyethyl) -2-aminoethane sulfonic acid ( BES);
  • biofilm staining was performed for 1.5 min, with a 1 g / L solution of Coomassie blue, 250 mL / L of methanol and 100 mL / L of acetic acid.
  • the biofilm obtained as described has a greater amount of biomass accumulated in the solid-liquid-air interface than in the solid-liquid interface. Specifically, the biofilm biomass at the interface is 30% and 50% more abundant per cm 2 than that of the submerged, in stainless steel coupons and glass coupons, respectively.
  • Figure 1 shows digital images of the biofilm on glass not exposed to enzymes (1.1), and exposed to Citrozym UltraL and trypsin (1.2), in which the solid-liquid-air (A) interface and the solid- liquid (B).
  • Example 4 Reduction of the number of adhered cells and effect on their viability by sequential exposure of biofilm to carbohydrases and proteases
  • the biofilms were treated for observation by confocal microscopy. as follows
  • the coupons were covered with a solution of the total cell indicator fluorochrome (SYTO 13 at 0.01%), after 5 min, they were washed with the phosphate buffer and then covered with the dead cell indicator fluorochrome (iodide) of 0.005% propidium). Finally, for 10 min, the coupons were sufficiently clarified with the same buffer. In each coupon the fluorescence was measured in a total of 16 observation fields, half corresponding to the interphase biofilm and the other half to the submerged biofilm.
  • the population present in the biofilm before exposure to enzymes was approximately 5.9 x 10 6 cfu / cm 2 both at the interface and in the submerged biofilm.
  • the percentage of non-viable biofilm cells at the interphase reached
  • Table 3 shows the percentage of cells eliminated by the various enzyme preparations, distinguishing the effect on biofilm at interphase and submerged biofilm. All enzymes cause cell shedding in both areas of the biofilm. Sequential exposure to carbohydrase and trypsin was more effective than that carried out with carbohydrases alone. The use of the C + P sequence caused a degree of cell shedding of 75-100%. The percentage of non-viable cells in the biomass that remains adhered after the various enzyme treatments does not exceed 15% at the interface and 10% in the submerged biofilm. None of the treatments affect the viability of the cells in the residual biofilms.
  • the samples were dehydrated by immersion for 15 min in a series of acetone solutions of increasing concentration (30, 40, 50, 70, 80, 90, 95 and 100%), and then treated with supercritical CO in a Balzen equipment and they were metallized with gold in a team of the same brand.
  • the preparations thus treated were examined in a scanning electron microscope.
  • Example 5 Modification of the rheological properties of biofilm by sequential exposure to carbohydrases and proteases
  • the weakly adhered biomass was removed by vertical immersion of the plates in a saline solution.
  • the biofilms of the 5 plates were collected manually by scratching with a plastic spatula and joined in an Eppendorf tube.
  • the excess water included was removed by centrifugation at 114,926 g for 5 min.
  • the rheological characterization of the biomass thus obtained was determined in a Bohlin rheometer, equipped with a concentric cylinder head. Incorporated two aliquots of 2 mL of biofilm, one with Citrozym TM Ultral to the
  • Figure 3 shows the rheological behavior of each of the samples.

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Abstract

The invention concerns an enzymatic process for detaching or facilitating removal of biofilm that has accumulated during solid-liquid-air interfaces and solid-liquid interfaces of aqueous systems. This is achieved by exposing the biofilm to a cleaning preparation containing at least one enzyme pertaining to the carbohydrase and protease group. Sequential treatment of the biofilm initially using a cleaning preparation essentially containing carbohydrase and then using a cleaning preparation essentially containing protease makes it possible to eliminate up to 85 % of accumulated biomass and up to 100 % of adhered cells.

Description

TITULO: Procedimiento enzimático para fluidificar o desprender biofilms de distintas interfasesTITLE: Enzymatic procedure to fluidize or detach biofilms from different interfaces
OBJETO DE LA INVENCIÓNOBJECT OF THE INVENTION
La presente invención se encuadra dentro del campo técnico de limpieza y desinfección de superficies. De forma más concreta, la invención se refiere a un procedimiento para eliminar biofilms, también denominados biopelículas, películas biológicas, biocapas o películas microbianas, formados por distintos microorganismos en las interfases sólido-líquido-aire y sólido-líquido de sistemas acuosos (sistemas que contienen agua, sistemas fluidos); y a la utilización de al menos una enzima, perteneciente al grupo de las carbohidrasas o proteasas, en este procedimientoThe present invention falls within the technical field of cleaning and disinfection of surfaces. More specifically, the invention relates to a method for removing biofilms, also called biofilms, biological films, bio-layers or microbial films, formed by different microorganisms at the solid-liquid-air and solid-liquid interfaces of aqueous systems (systems that contain water, fluid systems); and to the use of at least one enzyme, belonging to the group of carbohydrases or proteases, in this procedure
ANTECEDENTESBACKGROUND
La adhesión de microorganismos a superficies es un hecho frecuente en los sistemas acuosos. El crecimiento de los microorganismos asociados a superficies u otras interfases conlleva la formación de biofilms, esto es, un consorcio de células embebidas en una matriz exopoliméricaThe adhesion of microorganisms to surfaces is a frequent occurrence in aqueous systems. The growth of microorganisms associated with surfaces or other interfaces involves the formation of biofilms, that is, a consortium of cells embedded in an exopolymeric matrix.
La presencia de biofilms en distintos procesos industriales se relaciona con problemas higiénicos y tecnológicos. Dentro de los primeros, destaca su papel como reservorio de microorganismos patógenos y alterantes, contaminantes de instalaciones de la industria alimentaria y biotecnológica. Por otra parte, la formación de biopelículas se asocia con el deterioro de materiales, como la corrosión de sustratos metálicos y el recubrimiento de otros sustratos, provocando incrementos en el gasto energético, como en los intercambiadores de calor y los sistemas de distribución de fluidos, disminución de la eficacia de los procesos, como en el tratamiento de aguas o la fabricación de papel, e incluso la inutilización de las conducciones. El desarrollo de biofilms puede también tener lugar en superficies de interés clínico, como las lentes de contacto o los implantes. y constituyendo la base de la formación de placa dental.The presence of biofilms in different industrial processes is related to hygienic and technological problems. Among the former, its role as a reservoir of pathogenic and altering microorganisms, pollutants of food and biotechnology industry facilities stands out. On the other hand, the formation of biofilms is associated with the deterioration of materials, such as the corrosion of metallic substrates and the coating of other substrates, causing increases in energy expenditure, such as in heat exchangers and fluid distribution systems, decreased efficiency of processes, such as in water treatment or papermaking, and even the uselessness of pipes. The development of biofilms may also have place on surfaces of clinical interest, such as contact lenses or implants. and forming the basis of dental plaque formation.
La población microbiana presente en el biofilm depende de los microorganismos adheridos inicialmente, de su proliferación en el biofilm y de su desprendimiento y paso a la fase líquida. Entre los microorganismos encontrados en biofilms son particularmente frecuentes las bacterias Gram negativas, sobre todo, especies de los géneros Pseudomonas, Acinetobacter, Flcn'obacteriiim, Desulfovibrio, Escherichia y Enterobacter, y en menor proporción bacterias Gram positivas, mohos, levaduras y algas. Gran parte de estos microorganismos son aerobios estrictos, por lo que su crecimiento en la interfase sólido-líquido-aire, suele ser mayor que en la sólido-líquido (zona sumergida), donde la disponibilidad de oxígeno es menor.The microbial population present in the biofilm depends on the initially adhered microorganisms, their proliferation in the biofilm and their detachment and passage to the liquid phase. Among the microorganisms found in biofilms, Gram-negative bacteria are particularly frequent, especially species of the genus Pseudomonas, Acinetobacter, Flcn'obacteriiim, Desulfovibrio, Escherichia and Enterobacter, and to a lesser extent Gram positive bacteria, molds, yeasts and algae. A large part of these microorganisms are strict aerobes, so their growth in the solid-liquid-air interface is usually greater than in the solid-liquid (submerged zone), where oxygen availability is lower.
La matriz exopolimérica o glicocalix, en la que las células adheridas se encuentran atrapadas, es la responsable de la estructura y organización del biofilm, y está constituida principalmente por polisacáridos y proteínas. Su producción y composición, además de relacionarse con el tipo de microorganismo y superficie (Ruiz y col., 1999 en Biofilms: The Good, the Bad and the Ugly. Editado por J. Wimpenny, P. Gilbert, J. Walker, M. Brading y R. Bayston.The exopolymeric matrix or glycocalix, in which the adherent cells are trapped, is responsible for the structure and organization of the biofilm, and consists mainly of polysaccharides and proteins. Its production and composition, in addition to being related to the type of microorganism and surface (Ruiz et al., 1999 in Biofilms: The Good, the Bad and the Ugly. Edited by J. Wimpenny, P. Gilbert, J. Walker, M. Brading and R. Bayston.
Bioline), dependen de las condiciones nutritivas y ambientales (Sutherland, 1997 en Biofilms: Community Interactions and Control. Editado por J. Wimpenny, P. Handley, P. Gilbert, H. Lappin-Scott y M. Jones. Bioline). El oxígeno es uno de los factores limitantes del crecimiento de los principales microorganismos implicados en la formación de biofilms; la acumulación de células y material exopolimérico puede llegar a ser mayor en la interfase sólido-líquido-aire que en la sólido-líquido.Bioline), depend on nutritional and environmental conditions (Sutherland, 1997 in Biofilms: Community Interactions and Control. Edited by J. Wimpenny, P. Handley, P. Gilbert, H. Lappin-Scott and M. Jones. Bioline). Oxygen is one of the growth limiting factors of the main microorganisms involved in the formation of biofilms; the accumulation of cells and exopolymeric material can become greater in the solid-liquid-air interface than in the solid-liquid.
La eliminación de biofilms industriales suele realizarse mediante procedimientos de limpieza mecánicos combinados con el uso de biocidas. Los microorganismos acumulados en el biofilm se caracterizan por presentar una elevada resistencia a los agentes de desinfección habitualmente empleados en los procesos industriales. Parte de este efecto se atribuye al glicocalix, que constituye una barrera a la penetración de los compuestos presentes en la fase líquida Aunque se elimine la mayor parte de las células viables del biofilm, el material exopolimérico residual puede sustentar el crecimiento de nuevos microorganismos. Por ello, una eliminación eficaz de la matriz permitirá el desprendimiento del biofilm limitando además los problemas asociados a la recolonización microbiana. La degradación enzimática del glicocalix, tiene por otra parte la ventaja de ser un procedimiento de limpieza, respetuoso con el medio ambiente y con la integridad de las superficies.The elimination of industrial biofilms is usually done by mechanical cleaning procedures combined with the use of biocides. The microorganisms accumulated in the biofilm are characterized by having a high resistance to the disinfection agents usually used in the industrial processes. Part of this effect is attributed to glycocalix, which constitutes a barrier to the penetration of the compounds present in the liquid phase. Although most of the viable biofilm cells are removed, residual exopolymeric material can support the growth of new microorganisms. Therefore, an effective elimination of the matrix will allow the biofilm to detach, also limiting the problems associated with microbial recolonization. The enzymatic degradation of glycocalix, on the other hand, has the advantage of being a cleaning procedure, respectful of the environment and the integrity of the surfaces.
En la bibliografía científica se han descrito una amplia variedad de métodos para prevenir y eliminar biofilms formados en sistemas acuosos industriales, mediante la utilización de una o más enzimas que degradan la matriz exopolimérica, solas o combinadas con otros compuestos como biocidas, agentes surfactantes o quelantes (WO 9617632, WO 9826807, EP 0590746, WO 9631610,A wide variety of methods to prevent and eliminate biofilms formed in industrial aqueous systems have been described in the scientific literature, by using one or more enzymes that degrade the exopolymeric matrix, alone or in combination with other compounds such as biocides, surfactants or chelators (WO 9617632, WO 9826807, EP 0590746, WO 9631610,
WO 9213807, US 5071765, WO 9914312). Estos métodos han sido desarrollados fundamentalmente para prevenir y eliminar biofilms sumergidos. Su eficacia puede ser significativamente menor cuando se aplican a biofilms formados en la interfase sólido-líquido-aire que son bastante más gruesos, particularmente en los cultivos en continuo prolongados y en los discontinuos, al final de la fase exponencial.WO 9213807, US 5071765, WO 9914312). These methods have been developed primarily to prevent and eliminate submerged biofilms. Its effectiveness can be significantly lower when applied to biofilms formed at the solid-liquid-air interface that are much thicker, particularly in prolonged and discontinuous continuous cultures, at the end of the exponential phase.
EXPLICACIÓN DE LA INVENCIÓNEXPLANATION OF THE INVENTION
En líneas generales y para conseguir los objetivos preconizados, a la vez que eliminar los problemas citados en el apartado anterior, el objeto de esta invención es el desarrollo de un procedimiento respetuoso con el medio ambiente y las superficies de los sistemas acuosos, eficaz para desprender total o parcialmente biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido. Otro objeto de esta invención es el desarrollo de un procedimiento capaz de modificar las propiedades reológicas de la matriz exopolimérica del biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido-líquido de los sistemas acuosos, de forma que facilite la eliminación posterior del biofilm mediante la aplicación de otros procedimientos físicos o químicos de limpieza y desinfección.In general and to achieve the objectives recommended, while eliminating the problems mentioned in the previous section, the object of this invention is the development of a procedure that respects the environment and the surfaces of aqueous systems, effective to detach totally or partially biofilm accumulated in both the solid-liquid-air interface and the solid-liquid interface. Another object of this invention is the development of a method capable of modifying the rheological properties of the exopolymeric matrix of the biofilm accumulated both in the solid-liquid-air interface and in the solid-liquid interface of the aqueous systems, so as to facilitate the elimination Biofilm after applying other physical or chemical cleaning and disinfection procedures.
Esta invención tiene también por objeto definir la composición de un producto de limpieza que consiste esencialmente en carbohidrasas y proteasas. para eliminar biofilm acumulado tanto en la interfase sólido-líquido-aire, como en la sólido-líquido de sistemas acuosos, compatible con la utilización independiente o combinada de otros agentes de limpieza como inhibidores de la corrosión, surfactantes, biocidas, entre otros.This invention also aims to define the composition of a cleaning product consisting essentially of carbohydrases and proteases. to eliminate accumulated biofilm both in the solid-liquid-air interface, and in the solid-liquid of aqueous systems, compatible with the independent or combined use of other cleaning agents such as corrosion inhibitors, surfactants, biocides, among others.
Exactamente, el procedimiento enzimático para fluidificar o desprender biofilms de distintas interfases, objeto de la invención, consiste en un sistema para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire (en esta invención denominado biofilm en interfase) como en la sólido-líquido (en esta invención denominado biofilm sumergido) de sistemas acuosos, mediante la exposición a un preparado de limpieza que incluye al menos un enzima del tipo de carbohidrasas o proteasas, en una cantidad suficiente para que la matriz exopolimérica se degrade y permita el desprendimiento total o parcial del biofilm y se modifiquen sus propiedades reológicas, facilitando la posterior eliminación del biofilm mediante otros procedimientos de limpieza y desinfección.Exactly, the enzymatic process for fluidizing or releasing biofilms from different interfaces, object of the invention, consists of a system to prevent, detach and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface (in this invention called biofilm in interface) as in the solid-liquid (in this invention called submerged biofilm) of aqueous systems, by exposure to a cleaning preparation that includes at least one enzyme of the carbohydrase or protease type, in an amount sufficient for the exopolymeric matrix the partial or total detachment of the biofilm is degraded and its rheological properties are modified, facilitating the subsequent elimination of the biofilm by other cleaning and disinfection procedures.
Entre las carbohidrasas que resultan útiles para esta invención, pero no constituyen un límite, se incluyen los siguientes productos de Novo Nordisk AS; Citrozym Low GA, Citrozym Cloudy L, Citrozym Cloudy 100L, Citrozym LS, Citrozym Ultra L, Citrozym Ceo, Pectinex Ultra SP-L, Pectinex Smash, Bio-CipAmong the carbohydrases that are useful for this invention, but do not constitute a limit, the following Novo Nordisk AS products are included; Citrozym Low GA, Citrozym Cloudy L, Citrozym Cloudy 100L, Citrozym LS, Citrozym Ultra L, Citrozym Ceo, Pectinex Ultra SP-L, Pectinex Smash, Bio-Cip
Membrane, Cellubrix L, y los productos de Sigma Chemical Co: celulasa, alfa- amilasa, pectinasa, pectínesterasa, pectoliasa y dextranasa. Proteasas útiles para esta invención, pero que no constituyen un límite, incluyen serín proteasas como tripsina y pepsina, ambas de Sigma Chemical Co.Membrane, Cellubrix L, and Sigma Chemical Co products: cellulase, alpha-amylase, pectinase, pectinase, pectolyase and dextranase. Proteases useful for this invention, but not a limit, include serine proteases such as trypsin and pepsin, both from Sigma Chemical Co.
El preparado de limpieza de esta invención se añade a sistemas acuosos en la cantidad adecuada para desprender tanto el biofilm en interfase como el sumergido y para modificar las propiedades reológicas de ambos tipos de biofilm. La dosis de cada una de las enzimas no es crítica para la invención y es ajustable a la naturaleza del sistema acuoso, tipo de microorganismo y condiciones ambientales. La concentración de carbohidrasa puede variar de 0.0001% a 10% y la de proteasa entre 0.001 y 100 U/mL.The cleaning preparation of this invention is added to aqueous systems in the appropriate amount to release both the biofilm at the interface and the submerged and to modify the rheological properties of both types of biofilm. The dose of each of the enzymes is not critical to the invention and is adjustable to the nature of the aqueous system, type of microorganism and environmental conditions. The concentration of carbohydrase can vary from 0.0001% to 10% and that of protease between 0.001 and 100 U / mL.
La exposición del biofilm en interfase y sumergido al preparado de limpieza se lleva a cabo preferentemente en el rango de pH y temperaturas a los cuales las enzimas aplicadas son activas, generalmente pH entre 4 y 1 1, preferentemente entre 4,5 y 8 y temperatura en un intervalo de 10 a 60°C, preferentemente entre 25 y 50°C. El tiempo de exposición no es crítico para la invención, pudiendo variar desde 30 min a 24 h, preferentemente 1 h.The exposure of the biofilm at the interface and submerged to the cleaning preparation is preferably carried out in the range of pH and temperatures at which the enzymes applied are active, generally pH between 4 and 1 1, preferably between 4.5 and 8 and temperature in a range of 10 to 60 ° C, preferably between 25 and 50 ° C. The exposure time is not critical to the invention, and may vary from 30 min to 24 h, preferably 1 h.
Finalmente este procedimiento que permite prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido-líquido de sistemas acuosos, puede comprender la exposición del biofilm a carbohidrasas, o la exposición del biofilm a proteasas o la exposición del biofilm a carbohidrasas y proteasas. En este último caso, la exposición de biofilm se realiza en dos etapas secuenciales 1) exposición del biofilm a una preparación de limpieza que consiste esencialmente en carbohidrasas, 2) exposición del biofilm a una preparación de limpieza que consiste esencialmente en proteasas.Finally, this procedure that allows to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid of aqueous systems, can include the exposure of biofilm to carbohydrases, or the exposure of biofilm to proteases. or the exposure of biofilm to carbohydrases and proteases. In the latter case, the biofilm exposure is carried out in two sequential stages 1) exposure of the biofilm to a cleaning preparation consisting essentially of carbohydrases, 2) exposure of the biofilm to a cleaning preparation consisting essentially of proteases.
EXPLICACIÓN DE LOS DIBUJOS Para facilitar la comprensión de las características de la invención y formando parte de esta memoria descriptiva, se adjuntan una serie de figuras que representan lo siguiente: Figura 1 Representa imágenes digitales (2 aumentos) de biomasa de biofilm sobre vidrio (1 1) no expuesto a enzimas y (1 2) expuesto durante 60 min primero a Citrozym UltraL (1%) y luego a tripsina (104U/L), de la interfase (A) sólido- líquido-aire y (B) sólido-liquidoEXPLANATION OF THE DRAWINGS To facilitate the understanding of the features of the invention and as part of this specification, a series of figures are attached that represent the following: Figure 1 Represents digital images (2 magnifications) of biofilm biomass on glass (1 1) not exposed to enzymes and (1 2) exposed for 60 min first to Citrozym UltraL (1%) and then to trypsin (10 4 U / L ), of the interface (A) solid-liquid-air and (B) solid-liquid
Figura 2 Muestra fotografías tomadas por microscopía electrónica de barrido a 2000 aumentos de células de biofilm (2 1) no expuesto a enzimas y (2 2) expuesto durante 60 min primero a Citrozym UltraL (1%) y luego a tripsina (104U/L) Figura 3 Muestra la viscosidad aparente, dependiente de la velocidad de deformación de biofílm no expuesto a enzimas (μ) y expuesto a CitrozymUltraL (1%) (λ) y posteriormente a CitrozymUltraL (1%) más tripsina (104 U/L)(σ) En el eje de abscisas se representa el log de la viscosidad aparente en Paséales x segundo (Pa s) y en el eje de ordenadas el log de la velocidad de deformación enFigure 2 Shows photographs taken by scanning electron microscopy at 2000 magnification of biofilm cells (2 1) not exposed to enzymes and (2 2) exposed for 60 min first to Citrozym UltraL (1%) and then to trypsin (10 4 U / L) Figure 3 Shows the apparent viscosity, dependent on the strain rate of biofilm not exposed to enzymes (μ) and exposed to CitrozymUltraL (1%) (λ) and subsequently to CitrozymUltraL (1%) plus trypsin (10 4 U / L) (σ) On the abscissa axis, the log of the apparent viscosity is represented in Passes x second (Pa s) and on the ordinate axis the log of the deformation velocity in
MODO DE REALIZACIÓN DE LA INVENCIÓNEMBODIMENT OF THE INVENTION
La presente invención se ilustra mediante los ejemplos que siguen, empleando como blanco biofilm estandarizado de Pseudomonas. por ser éste uno de los géneros cuya adhesión se ha estudiado con más frecuencia y que se encuentra más habitualmente en biofilms naturales formados en muy diversos hábitatsThe present invention is illustrated by the following examples, using Pseudomonas standardized biofilm blank. as this is one of the genera whose adhesion has been studied more frequently and is most commonly found in natural biofilms formed in very diverse habitats
Estos ejemplos son sólo ilustrativos y no establecen los límites en cuanto a condiciones, eficacia o aplicaciones de la invención, que se definen en las correspondientes reivindicacionesThese examples are illustrative only and do not set limits on the conditions, efficacy or applications of the invention, which are defined in the corresponding claims.
Ejemplo 1: Obtención y cuantificación de biofilm en interfase y sumergidoExample 1: Obtaining and quantifying biofilm at interphase and submerged
Los biofilms se forman sobre superficies (cupones de 22 x 22 mm) de vidrio y acero inoxidable, dispuestas en posición vertical, parcialmente cubiertas con el medio de cultivo, de modo que parte del biofilm se formó en la interfase sólido- líquido-aire y parte en la interfase sólido-líquido (zona sumergida), según se describe a continuación.The biofilms are formed on surfaces (coupons of 22 x 22 mm) of glass and stainless steel, arranged vertically, partially covered with the culture medium, so that part of the biofilm was formed at the solid interface. liquid-air and part in the solid-liquid interface (submerged zone), as described below.
Antes de su uso, los cupones de vidrio se sumergieron en ácido nítrico (2 h) y se aclararon posteriormente con abundante agua bidestilada. Los cupones de acero inoxidable tipo 316, se dejaron sumergidos durante una noche en detergente neutro; a continuación se aclararon con agua destilada y se mantuvieron en etanol absoluto (2 h), siendo finalmente aclarados con agua bidestilada. Se empleó un soporte horizontal con 16 hendiduras radiales para el asentamiento de los cupones, quedando estos perfectamente sujetos. Los soportes, cargados con los cupones, se introdujeron en vasos de precipitados y el conjunto se esterilizó por calor seco, adicionándose posteriormente el cultivo bacteriano, de modo que cubriese tan solo la mitad inferior de los cupones.Before use, the glass coupons were immersed in nitric acid (2 h) and subsequently rinsed with abundant double-distilled water. Type 316 stainless steel coupons were left immersed overnight in neutral detergent; They were then rinsed with distilled water and kept in absolute ethanol (2 h), being finally rinsed with double distilled water. A horizontal support with 16 radial grooves was used for the settlement of the coupons, these being perfectly subject. The supports, loaded with the coupons, were introduced into beakers and the whole was sterilized by dry heat, subsequently adding the bacterial culture, so that it covered only the lower half of the coupons.
Como microorganismo formador de biofilms se empleó Pseudomonas fluorescens cepa B52, cultivado en un medio mineral líquido, de pH 7, compuesto por: 10,70 g L de ácido N,N-bis-(2-hidroxietil)-2-aminoetano sulfónico (BES);As a biofilm-forming microorganism, Pseudomonas fluorescens strain B52 was used, grown in a liquid mineral medium, pH 7, consisting of: 10.70 g L of N, N-bis- (2-hydroxyethyl) -2-aminoethane sulfonic acid ( BES);
11,00 g/L de piruvato sódico; 0,86 g/L de KH2PO4; 0,65 g/L de NH4CI; 0,20 g/L de MgSO y 0,11 g/L de CaCl . Se requirieron tan solo 20 h de cultivo para la formación de un biofilm visible.11.00 g / L sodium pyruvate; 0.86 g / L of KH 2 PO 4 ; 0.65 g / L of NH 4 CI; 0.20 g / L of MgSO and 0.11 g / L of CaCl. Only 20 h of culture were required for the formation of a visible biofilm.
Para cuantificar la biomasa adherida a los cupones de vidrio y acero inoxidable, se realizó un análisis de imágenes por densitometría. Tras la incubación se retiraron los cupones, y las células débilmente adheridas fueron eliminadas sumergiendo verticalmente los cupones en solución salina.To quantify the biomass attached to the glass and stainless steel coupons, an image analysis was performed by densitometry. After incubation the coupons were removed, and the weakly adhered cells were removed by vertically immersing the coupons in saline.
Manteniendo los cupones en posición vertical, los biofilms se secaron a temperatura ambiente, y a continuación se fijaron por inmersión en una solución de cloruro de cetilpiridinio 10 mM, 2 min y posterior secado a temperatura ambiente. La tinción del biofilm se realizó durante 1,5 min, con una solución 1 g/L de azul de Coomassie, 250 mL/L de metanol y 100 mL/L de ácido acético.Keeping the coupons in an upright position, the biofilms were dried at room temperature, and then fixed by immersion in a solution of 10 mM cetylpyridinium chloride, 2 min and subsequent drying at room temperature. Biofilm staining was performed for 1.5 min, with a 1 g / L solution of Coomassie blue, 250 mL / L of methanol and 100 mL / L of acetic acid.
Para eliminar el exceso de tinte, los cupones se lavaron con abundante agua destilada, dejándolos secar finalmente a temperatura ambiente. Los biofilms teñidos se analizaron en un densitómetro (BioRad GS-690) con detector de radiación visible, acoplado a un ordenador con un programa de análisis de imágenes (Molecular Analyst, BioRad). La cuantificación se baso en las medidas de densidad óptica (D O.) de miles de puntos de cada imagen, calculándose a continuación, la D O. media. La calibración de la cantidad de biomasa por medida de la densidad óptica se llevó a cabo con mucina porcina (Sigma Chemical Co), con cuyas soluciones se realizaron frotis sobre los cupones, que se fijaron y tiñeron siguiendo el procedimiento anterior.To remove excess dye, the coupons were washed with plenty of distilled water, finally allowing them to dry at room temperature. Biofilms stains were analyzed in a densitometer (BioRad GS-690) with visible radiation detector, coupled to a computer with an image analysis program (Molecular Analyst, BioRad). The quantification was based on the measurements of optical density (D O.) of thousands of points of each image, then calculating the average D O. The calibration of the amount of biomass by measurement of the optical density was carried out with porcine mucin (Sigma Chemical Co), with whose solutions smears were made on the coupons, which were fixed and stained following the above procedure.
El biofilm obtenido según se ha descrito, presenta mayor cantidad de biomasa acumulada en la interfase sόlido-líquido-aire que en la interfase sólido-líquido. Concretamente, la biomasa del biofilm en interfase es 30% y 50% más abundante por cm2 que la del sumergido, en cupones de acero inoxidable y cupones de vidrio, respectivamente.The biofilm obtained as described, has a greater amount of biomass accumulated in the solid-liquid-air interface than in the solid-liquid interface. Specifically, the biofilm biomass at the interface is 30% and 50% more abundant per cm 2 than that of the submerged, in stainless steel coupons and glass coupons, respectively.
Ejemplo 2: Desprendimiento de biomasa de biofilm por exposición a carbohidrasas o proteasasExample 2: Biofilm biomass release by exposure to carbohydrases or proteases
Los cupones de vidrio y acero en los que se había desarrollado biofilm, tal como se describe en el Ejemplo 1, se sumergieron en tampón fosfatoThe glass and steel coupons on which biofilm had been developed, as described in Example 1, were immersed in phosphate buffer
(NaH2PO /NaOH) 0,067M de pH 7, durante 1 min en condiciones estáticas, para eliminar las células débilmente adheridas. A continuación cada cupón se sumergió durante 60 min, sin agitación, en el tampón (controles) o en la solución enzimática correspondiente, de modo que los cupones quedaban en posición vertical y totalmente cubiertos por la preparación. Tras la incubación, cada cupón se sometió a un lavado con el mismo tampón, para eliminar los restos de biofilm desprendido y de enzimas. La cantidad de biomasa desprendida se estimó por diferencia, mediante la técnica de análisis de imágenes por densitometría descrita en el Ejemplo 1.(NaH 2 PO / NaOH) 0.067M pH 7, for 1 min under static conditions, to remove weakly adhered cells. Each coupon was then immersed for 60 min, without stirring, in the buffer (controls) or in the corresponding enzymatic solution, so that the coupons were upright and fully covered by the preparation. After incubation, each coupon was subjected to a wash with the same buffer, to remove the remains of detached biofilm and enzymes. The amount of biomass released was estimated by difference, using the densitometry image analysis technique described in Example 1.
Se ensayaron un total de 16 carbohidrasas puras o combinadas y 2 proteasas, a diferentes concentraciones. En la Tabla 1 se muestra el grado de desprendimiento de biomasa obtenido con algunas de las enzimas. El porcentaje de biomasa eliminada alcanzó el 24-29% en biofilms expuestos a carbohidrasas puras, el 57-58% en los expuestos a carbohidrasas combinadas y el 35-39% en los expuestos a tripsina.A total of 16 pure or combined carbohydrases and 2 proteases were tested at different concentrations. Table 1 shows the degree of biomass shedding obtained with some of the enzymes. The percentage of biomass removed reached 24-29% in biofilms exposed to pure carbohydrases, 57-58% in those exposed to combined carbohydrases and 35-39% in those exposed to trypsin.
Tabla 1.Table 1.
Nombre comercial de la enzima Porcentaje de biomasa desprendida (concentración)Commercial name of the enzyme Percentage of biomass released (concentration)
Biofilm en vidrio Biofilm en aceroBiofilm in glass Biofilm in steel
Citrozym Cloudy L ( 10%) 23 1 1Citrozym Cloudy L (10%) 23 1 1
Citrozym Ultra L ( 10%) 44 58Citrozym Ultra L (10%) 44 58
Citrozym Ceo (10%) 45 20Citrozym Ceo (10%) 45 20
Pectinex Ultra SP-L ( 10%) 34 31Pectinex Ultra SP-L (10%) 34 31
Pectinex Smash (10%) 57 31Pectinex Smash (10%) 57 31
Bio-Cip Membrane (10%) 50 26Bio-Cip Membrane (10%) 50 26
Cellubrix L (10%) 52 7 Cellubrix L (10%) 52 7
Celulasa (45 U/ml) 24Cellulase (45 U / ml) 24
Pectinasa (5.94 U/ml) - 29Pectinase (5.94 U / ml) - 29
Pectinesterasa (10 U/ml) - 21Pectinesterase (10 U / ml) - 21
Pectoliasa (0.32 U/ml) 18 22Pectoliase (0.32 U / ml) 18 22
Dextranasa (12.9U/ml) 23Dextranase (12.9U / ml) 23
Tripsina (105 U/L) 39 35Trypsin (10 5 U / L) 39 35
Ejemplo 3: Desprendimiento de biomasa de biofilm por exposición secuencial a carbohidrasas y proteasasExample 3: Biofilm biomass release by sequential exposure to carbohydrases and proteases
Los biofilms desarrollados sobre cupones de vidrio y acero, como se describe en el Ejemplo 1, se expusieron, según lo descrito en el Ejemplo 2, a carbohidrasas y proteasas en las dos etapas secuenciales posibles: 1) exposición del biofilm a carbohidrasas, seguida de exposición del biofilm a proteasas (en esta invención denominada secuencia C+P) y 2) exposición del biofilm a proteasas, seguida de exposición del biofilm a carbohidrasas (en esta invención denominada secuenciaThe biofilms developed on glass and steel coupons, as described in Example 1, were exposed, as described in Example 2, to carbohydrases and proteases in the two possible sequential stages: 1) exposure of the biofilm to carbohydrases, followed by exposure of biofilm to proteases (in this invention called the C + P sequence) and 2) exposure of biofilm to proteases, followed by exposure of biofilm to carbohydrases (in this invention called sequence
P+C). La exposición a cada una de las enzimas fue de 60 min, terminados con un lavado con el tampón fosfato antes descrito, para eliminar los restos de enzima y de biofilm desprendido. Como carbohidrasas se seleccionaron las que habían producido mayor desprendimiento (Tabla 1), a concentración de 10% y como proteasa, tripsina a concentración 104 U/L. Los resultados (Tabla 2), muestran que el empleo de ambos tipos de enzima de forma sucesiva produce mucho mayor desprendimiento que el obtenido con cada una de las enzimas aisladas, alcanzándose con la secuencia de tratamiento C+P, 84% de desprendimiento de biomasa de biofilm sobre acero y 75% de la de biofilm sobre vidrio. Tabla 2.P + C). The exposure to each of the enzymes was 60 min, terminated with a wash with the phosphate buffer described above, to remove the enzyme residues and detached biofilm. As carbohydrases, those that had produced the greatest detachment were selected (Table 1), at a concentration of 10% and as a protease, trypsin at a concentration of 10 4 U / L. The results (Table 2) show that the use of both types of enzyme successively produces much greater detachment than that obtained with each of the isolated enzymes, reaching with the C + P treatment sequence, 84% biomass shedding of biofilm on steel and 75% of that of biofilm on glass. Table 2.
Nombre comercial Porcentaje de biomasa desprendida según la de las carbohidrasas secuencia de aplicación de las enzimasCommercial name Percentage of biomass released according to the carbohydrase sequence of enzymes
P+C C+P C-PP + C C + P C-P
Vidrio Vidrio AceroGlass Glass Steel
Citrozym Cloudy L - - 71Citrozym Cloudy L - - 71
Citrozym Ultra L 55 75 84Citrozym Ultra L 55 75 84
Citrozym Ceo 58 34 -Citrozym Ceo 58 34 -
Pectinex Ultra SP-L 42 15 -Pectinex Ultra SP-L 42 15 -
Pectinex Smash 29 68 68Pectinex Smash 29 68 68
Biocip Membrane 40 62 70Biocip Membrane 40 62 70
Cellubrix L 51 47Cellubrix L 51 47
En la figura 1 se muestran imágenes digitales del biofilm sobre vidrio no expuesto a enzimas (1.1), y expuesto a Citrozym UltraL y tripsina (1.2), en las que se distingue la interfase sólido-líquido-aire (A) y la sólido-líquido (B).Figure 1 shows digital images of the biofilm on glass not exposed to enzymes (1.1), and exposed to Citrozym UltraL and trypsin (1.2), in which the solid-liquid-air (A) interface and the solid- liquid (B).
Ejemplo 4: Reducción del número de células adheridas y efecto en su viabilidad por exposición secuencial de biofilm a carbohidrasas y proteasasExample 4: Reduction of the number of adhered cells and effect on their viability by sequential exposure of biofilm to carbohydrases and proteases
Tras observar que la exposición del biofilm a carbohidrasas o proteasas, produce una considerable reducción en la biomasa adherida (mostrada en los Ejemplos 2 y 3), se estudió si esta se debe tan solo al desprendimiento de la matriz del biofilm o por el contrario también provoca la disminución del numero de células adheridasAfter observing that the exposure of biofilm to carbohydrases or proteases, produces a considerable reduction in the adhered biomass (shown in Examples 2 and 3), it was studied whether this is due only to the detachment of the matrix of the biofilm or on the contrary it also causes the decrease of the number of adhered cells
Biofilms desarrollados sobre cupones de vidπo, como se muestra en el Ejemplo 1, fueron expuestos a carbohidrasas y proteasas en las dos secuencias posibles (C+P y P+C). como se indica en el Ejemplo 2 A continuación, con el fin de determinar la proporción de células que permanecían adheπdas, asi como su viabilidad después del tratamiento enzimatico. los biofilms fueron tratados para su observación por microscopía confocal. como se indica a continuación Los cupones se cubrieron con una solución del fluorocromo indicador de células totales (SYTO 13 al 0.01%), tras 5 min, se lavaron con el tampon fosfato y a continuación, se cubπeron con el fluorocromo indicador de células muertas (yoduro de propidio al 0,005%). durante 10 min, finalmente, los cupones se aclararon suficientemente con el mismo tampon En cada cupón se midió la fluorescencia en un total de 16 campos de observación, la mitad correspondientes al biofilm en interfase y la otra mitad al biofilm sumergidoBiofilms developed on voucher coupons, as shown in Example 1, were exposed to carbohydrases and proteases in the two possible sequences (C + P and P + C). as indicated in Example 2 Next, in order to determine the proportion of cells that remained adhered, as well as their viability after enzymatic treatment. The biofilms were treated for observation by confocal microscopy. as follows The coupons were covered with a solution of the total cell indicator fluorochrome (SYTO 13 at 0.01%), after 5 min, they were washed with the phosphate buffer and then covered with the dead cell indicator fluorochrome (iodide) of 0.005% propidium). Finally, for 10 min, the coupons were sufficiently clarified with the same buffer. In each coupon the fluorescence was measured in a total of 16 observation fields, half corresponding to the interphase biofilm and the other half to the submerged biofilm.
Para relacionar los datos de intensidad de fluorescencia con el numero de células, se tomaron diversas diluciones de células en suspensión de un cultivo con 107 unidades formadoras de colonias (ufc)/ml de Pseudomonas. tiñendose como se describe anteriormente Seguidamente se prepararon con ellas frotis sobre cupones, en los que se determino la intensidad de fluorescencia La correspondencia entre el numero de células y la intensidad de fluorescencia fue linealTo relate the fluorescence intensity data to the number of cells, various dilutions of suspension cells were taken from a culture with 10 7 colony forming units (cfu) / ml of Pseudomonas. staining as described above Next, smears on coupons were prepared with them, in which the fluorescence intensity was determined. The correspondence between the number of cells and the fluorescence intensity was linear.
La población presente en el biofilm antes de la exposición a enzimas, fue de aproximadamente 5,9 x 106ufc/cm2 tanto en la interfase como en el biofilm sumergido El porcentaje de células no viables de biofílm en inteifase alcanzó elThe population present in the biofilm before exposure to enzymes was approximately 5.9 x 10 6 cfu / cm 2 both at the interface and in the submerged biofilm. The percentage of non-viable biofilm cells at the interphase reached
7-8% (aproximadamente 4 x 105 ufc/cm2), mientras que en biofilm sumergido no supero el 1% (alrededor de 6 x 104 ufc/cm2) En la Tabla 3 se muestra el porcentaje de células eliminadas por las diversas preparaciones enzimáticas, distinguiendo el efecto sobre biofilm en interfase y biofilm sumergido. Todas las enzimas provocan desprendimiento celular en ambas zonas del biofilm. La exposición secuencial a carbohidrasa y tripsina fue más efectiva que la realizada con carbohidrasas solas. El empleo de la secuencia C+P provocó un grado de desprendimiento celular de 75-100%. El porcentaje de células no viables en la biomasa que permanece adherida tras los diversos tratamientos enzimáticos, no supera el 15% en la interfase y el 10% en el biofilm sumergido. Ninguno de los tratamientos afecta a la viabilidad de las células en los biofilms residuales.7-8% (approximately 4 x 10 5 cfu / cm 2 ), while in submerged biofilm I do not exceed 1% (about 6 x 10 4 cfu / cm 2 ) Table 3 shows the percentage of cells eliminated by the various enzyme preparations, distinguishing the effect on biofilm at interphase and submerged biofilm. All enzymes cause cell shedding in both areas of the biofilm. Sequential exposure to carbohydrase and trypsin was more effective than that carried out with carbohydrases alone. The use of the C + P sequence caused a degree of cell shedding of 75-100%. The percentage of non-viable cells in the biomass that remains adhered after the various enzyme treatments does not exceed 15% at the interface and 10% in the submerged biofilm. None of the treatments affect the viability of the cells in the residual biofilms.
Tabla 3.Table 3.
Tratamiento enzimático aplicado Porcentaje de células desprendidasEnzymatic treatment applied Percentage of detached cells
iofilm en la Biofilmiofilm in the Biofilm
Interfase SumergidoSubmerged Interface
Citrozym Ultra L 47 76Citrozym Ultra L 47 76
Citrozym Ultra L+Tripsina (C+P) 60 86Citrozym Ultra L + Trypsin (C + P) 60 86
Tripsina+Citrozym Ultra L (P+C) 64 83Trypsin + Citrozym Ultra L (P + C) 64 83
Pectinex Smash 23 63Pectinex Smash 23 63
Pectinex Smash +Tripsina (C+P) 87 82Pectinex Smash + Trypsin (C + P) 87 82
Tripsina+ Pectinex Smash (P+C) 86 91Trypsin + Pectinex Smash (P + C) 86 91
Bio-Cip Membrane 27 32Bio-Cip Membrane 27 32
Bio-Cip Membrane+Tripsina (C+P) 100 100Bio-Cip Membrane + Trypsin (C + P) 100 100
Tripsina+ Bio-Cip Membrane (P+C) 84 87 Cellubrix 32Trypsin + Bio-Cip Membrane (P + C) 84 87 Cellubrix 32
Cellubriχ-1-Tripsina (C+P) 93 75 Tripsina+Cellubrix (P+C) 86 81Cellubriχ-1-Trypsin (C + P) 93 75 Trypsin + Cellubrix (P + C) 86 81
Otro método empleado para estudiar cualitativamente la biomasa residual presente en los cupones, tras la exposición del biofilm a las enzimas, fue la microscopía electrónica de barrido. Un cupón en el que se había desarrollado biofilm según el Ejemplo 1, se expuso secuencialmente a Citrozym UltraL y tripsina (C+P), tal como se muestra en el ejemplo 3. Este cupón junto con otro con biofílm no expuesto a enzimas, fueron recortados con un diamante, hasta un tamaño aproximado de 1 cm2, fijándose con una solución de glutaraldehído al 1.5% en tampón cacodilato 0, 1 M pH 7,4 durante 5 h a 4°C. Posteriormente, se lavaron por inmersión en tampón fosfato de pH 7 durante 1 h., renovando el tampón cada 15 min. Las muestras se deshidrataron por inmersión durante 15 min en una serie de disoluciones de acetona de concentración creciente (30, 40, 50, 70, 80, 90, 95 y 100%), y a continuación se trataron con CO supercrítico en un equipo Balzen y se metalizaron con oro en un equipo de la misma marca. Las preparaciones así tratadas se examinaron en un microscopio electrónico de barridoAnother method used to qualitatively study the residual biomass present in the coupons, after exposure of biofilm to enzymes, was scanning electron microscopy. A coupon in which biofilm had been developed according to Example 1, was sequentially exposed to Citrozym UltraL and trypsin (C + P), as shown in example 3. This coupon together with another with biofilm not exposed to enzymes, were trimmed with a diamond, up to an approximate size of 1 cm 2 , fixing with a solution of 1.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 5 h at 4 ° C. Subsequently, they were washed by immersion in pH 7 phosphate buffer for 1 h., Renewing the buffer every 15 min. The samples were dehydrated by immersion for 15 min in a series of acetone solutions of increasing concentration (30, 40, 50, 70, 80, 90, 95 and 100%), and then treated with supercritical CO in a Balzen equipment and they were metallized with gold in a team of the same brand. The preparations thus treated were examined in a scanning electron microscope.
Jeol, mod. JSM.6400 tomándose fotografías a 2000 aumentos que permitieron visualizar los cambios provocados por la exposición secuencial a carbohidrasas y proteasas. En la Figura 2 se muestran las fotografías del biofilm antes de su exposición a enzimas y tras su exposición secuencial a Citrozym UltraL y tripsina, con lo que se observa una dramática disminución en el número de células adheridas.Jeol, mod. JSM.6400 taking photographs at 2000 magnifications that allowed to visualize the changes caused by the sequential exposure to carbohydrases and proteases. Figure 2 shows the photographs of the biofilm before exposure to enzymes and after sequential exposure to Citrozym UltraL and trypsin, which shows a dramatic decrease in the number of adhered cells.
Ejemplo 5: Modificación de las propiedades reológicas del biofilm por exposición secuencial a carbohidrasas y proteasasExample 5: Modification of the rheological properties of biofilm by sequential exposure to carbohydrases and proteases
Para determinar los cambios provocados por la exposición a enzimas en las propiedades reológicas del biofilm, fue necesario en primer lugar la obtención de suficiente biomasa adherida, empleando como substrato placas grandes inmersas en cubetas de cromatografía, como se describe a continuación. Las placas de vidrio de 20 x 20 x 0,5 cm, antes de ser utilizadas, se limpiaron tal y como se ha descrito en el Ejemplo 1 para los cupones de vidrio. En una cubeta de las empleadas en cromatografía de capa fina, se introdujeron 5 placas en posición vertical, sujetas por las hendiduras laterales presentes en la cubeta. El conjunto se esterilizó por calor seco. Seguidamente, y en condiciones estériles, se incorporó el cultivo de Pseudomonas en el medio mineral descrito en el Ejemplo 1. Transcurridas 24 h de cultivo, se retiraron las placas de la cubeta. La biomasa débilmente adherida se eliminó por inmersión vertical de las placas en una solución salina. Los biofilms de las 5 placas se recogieron manualmente por rascado con una espátula de plástico y se juntaron en un tubo Eppendorf. El exceso de agua incluido se eliminó por centrifugación a 114.926 g durante 5 min.In order to determine the changes caused by exposure to enzymes in the rheological properties of the biofilm, it was first necessary to obtain enough adhered biomass, using as a substrate large immersed plates in chromatography cuvettes, as described below. The 20 x 20 x 0.5 cm glass plates, before being used, were cleaned as described in Example 1 for glass coupons. In a cuvette used in thin layer chromatography, 5 plates were inserted in a vertical position, held by the lateral grooves present in the cuvette. The whole was sterilized by dry heat. Then, and under sterile conditions, the culture of Pseudomonas was incorporated into the mineral medium described in Example 1. After 24 hours of culture, the plates were removed from the cuvette. The weakly adhered biomass was removed by vertical immersion of the plates in a saline solution. The biofilms of the 5 plates were collected manually by scratching with a plastic spatula and joined in an Eppendorf tube. The excess water included was removed by centrifugation at 114,926 g for 5 min.
La caracterización reológica de la biomasa así obtenida, se determinó en un reómetro Bohlin, equipado con un cabezal de cilindros concéntricos. Dos alícuotas de 2 mL de biofilm, una de ellas con CitrozymTMUltraL incorporado alThe rheological characterization of the biomass thus obtained was determined in a Bohlin rheometer, equipped with a concentric cylinder head. Incorporated two aliquots of 2 mL of biofilm, one with Citrozym TM Ultral to the
1%, se incubaron separadamente durante 60 min, a 40°C y a presión constante de1%, incubated separately for 60 min, at 40 ° C and at constant pressure of
0,1401 Pascales, en la propia cubeta del reómetro. Durante este tiempo, se registraron los cambios de viscosidad correspondientes. A continuación, cada una de las muestras se sometió a una serie de presiones crecientes, desde 0,1404 Pa, incrementando la presión aplicada un 25% cada 80 seg, lo que permitió la caracterización del comportamiento reológico de la muestra. La muestra que ya había sido incubada con la carbohidrasa y sometida a presiones altas, se incubó con tripsina a concentración de 10 U/L, en las condiciones previamente señaladas, antes de ser sometida a la misma serie de presiones indicadas, para medir su viscosidad aparente.0,1401 Pascals, in the rheometer cuvette itself. During this time, the corresponding viscosity changes were recorded. Then, each of the samples was subjected to a series of increasing pressures, from 0,1404 Pa, increasing the applied pressure by 25% every 80 sec, which allowed the characterization of the rheological behavior of the sample. The sample that had already been incubated with the carbohydrase and subjected to high pressures, was incubated with trypsin at a concentration of 10 U / L, under the conditions previously indicated, before being subjected to the same set of pressures indicated, to measure its viscosity apparent.
En la Figura 3 se observa el comportamiento reológico de cada una de las muestras. El biofilm no expuesto a enzimas, con viscosidad inicial del orden de 1 Pa s, presentó un comportamiento pseudoplástico, y la viscosidad disminuyó progresiva e indefinidamente al incrementarse la velocidad de deformación.Figure 3 shows the rheological behavior of each of the samples. The biofilm not exposed to enzymes, with initial viscosity of the order of 1 Pa s, showed a pseudoplastic behavior, and the viscosity decreased progressively and indefinitely as the strain rate increased.
Durante la incubación del biofilm con Citrozym™UltraL, a presión constante de 0.1404 Pascales, se observó un comportamiento marcadamente reopexo y la viscosidad aumentó hasta más de 500 Pascales x seg. La aplicación de presiones crecientes provocó sin embargo una rápida e intensa disminución de la viscosidad en un rango de velocidades de deformación muy bajas. Cuando la velocidad de deformación alcanzó aproximadamente 48 s" (1.6 en la escala logarítmica de laDuring the incubation of the biofilm with Citrozym ™ UltraL, at constant pressure of 0.1404 Pascals, a markedly reoperative behavior was observed and the viscosity increased to more than 500 Pascals x sec. The application of increasing pressures caused, however, a rapid and intense decrease in viscosity in a range of very low deformation rates. When the strain rate reached approximately 48 s " (1.6 on the logarithmic scale of the
Figura 3), provocada por la aplicación de presiones de aproximadamente 0.7 Pa. se produjo una transición de comportamiento pseudoplástico a Newtoniano. alcanzándose una viscosidad mínima y constante de 0,01 1 Pa s (-1,9 en la escala logarítmica de la Figura 3). Cuando este biofilm fue expuesto posteriormente a tripsina mostró comportamiento pseudoplástico solo a presiones muy bajas, que se convirtió en Newtoniano a velocidades de deformación semejantes a las ya mencionadas para el biofilm tratado con carbohidras.Figure 3), caused by the application of pressures of approximately 0.7 Pa. There was a transition from pseudoplastic to Newtonian behavior. reaching a minimum and constant viscosity of 0.01 1 Pa s (-1.9 on the logarithmic scale of Figure 3). When this biofilm was subsequently exposed to trypsin, it showed pseudoplastic behavior only at very low pressures, which became Newtonian at deformation rates similar to those already mentioned for the carbohydrate-treated biofilm.
En conclusión, a pesar de que la viscosidad de la muestra incubada con carbohidrasa es mucho mayor que la del biofilm control, la aplicación de muy ligeras presiones consigue convertir el biofilm tratado en un fluido Newtoniano de viscosidad comparable a la del agua. La acción de las enzimas facilita en gran medida el arrastre del material adherido mediante métodos mecánicos y disminuye también su capacidad de retención de las células. In conclusion, although the viscosity of the sample incubated with carbohydrase is much higher than that of the control biofilm, the application of very light pressures manages to convert the treated biofilm into a Newtonian fluid of comparable viscosity to that of water. The action of enzymes greatly facilitates the entrainment of adhered material by mechanical methods and also decreases its capacity to retain cells.

Claims

REIVINDICACIONES
1- Procedimiento enzimatico para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, caracterizado por la exposición (por inmersión, baño, aspersión, etc) del biofilm a un preparado de limpieza que incluye al menos una enzima perteneciente al grupo de las carbohidrasas o proteasas, en una cantidad suficiente para que la matriz exopolimérica se degrade y permita el desprendimiento total o parcial del biofilm y se modifiquen sus propiedades reológicas, para facilitar la posterior eliminación del biofilm mediante otros procedimientos de limpieza y desinfección.1- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, characterized by exposure (by immersion, bathing, spraying, etc.) of the biofilm to a cleaning preparation that includes at least one enzyme belonging to the group of carbohydrases or proteases, in an amount sufficient for the exopolymeric matrix to degrade and allow the partial or total detachment of the biofilm and its rheological properties to be modified, to facilitate the subsequent elimination of biofilm by other cleaning and disinfection procedures.
2- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicación anterior, caracterizado por la utilización de una carbohidrasa perteneciente al grupo de celulasas, hemicelulasas, pectinasas, amilasas o mezclas de las anteriores.2- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to the preceding claim, characterized by the use of a carbohydrase belonging to the group of cellulases , hemicellulases, pectinases, amylases or mixtures of the above.
3- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicación 2, caracterizado por la utilización de los siguientes productos de Novo Nordisk AS: Citrozym Cloudy L, Citrozym LS, Citrozym Ultra L, Citrozym Ceo, Pectinex Ultra SP-L, Pectinex Smash, Bio-Cip Membrane, Cellubrix L.3- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid of aqueous systems, according to claim 2, characterized by the use of the following Novo Nordisk AS products : Citrozym Cloudy L, Citrozym LS, Citrozym Ultra L, Citrozym Ceo, Pectinex Ultra SP-L, Pectinex Smash, Bio-Cip Membrane, Cellubrix L.
4- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicaciones 2 y 3, caracterizado porque la preparación de limpieza incluye carbohidrasas a una concentración comprendida entre 0,0001% y 100%, preferentemente entre 0, 1 y 10%, y posee un pH entre 3 y 1 1, preferentemente entre 4,5 y 8, y una temperatura entre 10 y 60° C. preferentemente entre 25 y 50° C.4- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to claims 2 and 3, characterized in that the cleaning preparation includes carbohydrases at a concentration between 0.0001% and 100%, preferably between 0.1 and 10%, and has a pH between 3 and 1 1, preferably between 4.5 and 8, and a temperature between 10 and 60 ° C. preferably between 25 and 50 ° C.
5- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sόlido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicación 1, caracterizado por la utilización de una proteasa perteneciente al grupo de serín proteasas, metalo proteasas, cistein proteasas, aspártico proteasas o mezclas de las anteriores.5- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to claim 1, characterized by the use of a protease belonging to the serine group proteases, metallo proteases, cysteine proteases, aspartic proteases or mixtures of the above.
6- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicación 5, caracterizado por la utilización de tripsina como proteasa perteneciente al grupo de las serín proteasas.6- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to claim 5, characterized by the use of trypsin as a protease belonging to the group of The serine proteases.
7- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicaciones 1, 5 y 6, caracterizado porque la preparación de limpieza incluye proteasas a una concentración comprendida entre 0,001 y 100 U/mL, preferentemente entre 1 y 10 U/mL, y posee un pH entre 3 y 11, preferentemente entre 6 y 8, y una temperatura entre 10 y 60° C, preferentemente entre 30 y 45° C.7- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to claims 1, 5 and 6, characterized in that the cleaning preparation includes proteases at a concentration between 0.001 and 100 U / mL, preferably between 1 and 10 U / mL, and has a pH between 3 and 11, preferably between 6 and 8, and a temperature between 10 and 60 ° C, preferably between 30 and 45 ° C.
8- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicaciones anteriores, caracterizado por comprender las siguientes etapas secuenciales 1 ) exposición (por inmersión, baño, aspersión, etc) del biofilm a una preparación de limpieza que consiste esencialmente en carbohidrasas, 2) exposición (por inmersión, baño, aspersión, etc) del biofilm a una preparación de limpieza que consiste esencialmente en proteasas. 9- Procedimiento enzimático para prevenir, desprender y facilitar la eliminación de biofilm acumulado tanto en la interfase sólido-líquido-aire como en la sólido- líquido de sistemas acuosos, según reivindicaciones anteriores, caracterizado porque las preparaciones enzimáticas de limpieza son compatibles con la utilización independiente o combinada de uno o más agentes pertenecientes al grupo de biocidas, surfactantes, quelantes, dispersantes, encapsulantes de baja densidad, inhibidores de la corrosión, agentes poliméricos, fosfatos, polifosfatos. silicatos, tampones, carboxilatos, sulfonatos. 8- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to previous claims, characterized by comprising the following sequential steps 1) exposure (by immersion, bathing, spraying, etc.) of the biofilm to a cleaning preparation consisting essentially of carbohydrases, 2) exposure (by immersion, bathing, spraying, etc.) of the biofilm to a cleaning preparation consisting essentially of proteases. 9- Enzymatic procedure to prevent, release and facilitate the elimination of accumulated biofilm both in the solid-liquid-air interface and in the solid-liquid interface of aqueous systems, according to previous claims, characterized in that the enzymatic cleaning preparations are compatible with the use independent or combined of one or more agents belonging to the group of biocides, surfactants, chelants, dispersants, low density encapsulants, corrosion inhibitors, polymeric agents, phosphates, polyphosphates. silicates, buffers, carboxylates, sulfonates.
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