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WO2001053010A1 - Procede enzymatique servant a fluidifier ou a detacher des biofilms de differentes interfaces - Google Patents

Procede enzymatique servant a fluidifier ou a detacher des biofilms de differentes 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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WO
WIPO (PCT)
Prior art keywords
biofilm
solid
liquid
elimination
facilitate
Prior art date
Application number
PCT/ES2000/000360
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English (en)
Spanish (es)
Inventor
Carmen San Jose Serran
Almudena Jaspe Rodriguez
Begoña RUIZ PEREZ
Tomas Maira Litran
Original Assignee
Universidad Complutense De Madrid Rectorado
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad Complutense De Madrid Rectorado filed Critical Universidad Complutense De Madrid Rectorado
Priority to AU75258/00A priority Critical patent/AU7525800A/en
Publication of WO2001053010A1 publication Critical patent/WO2001053010A1/fr

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Classifications

    • 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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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Detergent Compositions (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un procédé enzymatique servant à détacher et à faciliter l'élimination de biofilms accumulés dans les interfaces solide-liquide-air et solide-liquide de systèmes aqueux. Le procédé consiste à exposer le biofilm à une préparation de nettoyage comprenant au moins une enzyme appartenant au groupe des carbohydrases et des protéases. Le traitement séquentiel du biofilm, d'abord avec une préparation de nettoyage essentiellement composée de carbohydrases, puis avec une préparation comprenant essentiellement des protéases, permet d'éliminer jusqu'à 85 % de la biomasse accumulée et jusqu'à 100 % des cellules collées.
PCT/ES2000/000360 2000-01-20 2000-09-25 Procede enzymatique servant a fluidifier ou a detacher des biofilms de differentes interfaces WO2001053010A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75258/00A AU7525800A (en) 2000-01-20 2000-09-25 Enzymatic process for fluidizing or detaching biofilms from different interfaces

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200000113A ES2162593B1 (es) 2000-01-20 2000-01-20 Procedimiento enzimatico para fluidificar o desprender biofilms de distintas interfases.
ESP200000113 2000-01-20

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Publication Number Publication Date
WO2001053010A1 true WO2001053010A1 (fr) 2001-07-26

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ES (1) ES2162593B1 (fr)
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2846665A1 (fr) * 2002-10-31 2004-05-07 Karine Marion Procede d'elimination du biofilm
EP1423337A4 (fr) * 2001-09-07 2005-08-24 Advanced Biocatalytics Corp Reduction du film biologique dans les systemes de filtration a flux tangentiel
EP2099466A4 (fr) * 2006-12-01 2012-04-11 Laclede Inc Utilisation d'enzymes hydrolytiques et oxydatives pour dissoudre un biofilm dans les oreilles
US20120301946A1 (en) * 2010-06-21 2012-11-29 Toyota Motor Corporation Thermolysin for easy-cleaning of insect body stains
US8597927B2 (en) 2007-12-20 2013-12-03 Danisco Us Inc. Enzymatic prevention and control of biofilm
DE10304331B4 (de) * 2003-02-04 2017-12-28 Henkel Ag & Co. Kgaa Enzymatische Entfernung von Biofilmen auf Haushaltsoberflächen
EP3505609A1 (fr) 2017-12-29 2019-07-03 Itram Higiene, S.L. Composition détergente pour le contrôle et l'élimination de biofilms
US10563094B2 (en) 2011-09-09 2020-02-18 Toyota Motor Engineering & Manufacturing North America, Inc. Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
US10683529B2 (en) * 2011-06-24 2020-06-16 Realco Kit for detecting biofilms
US10781438B2 (en) 2006-11-22 2020-09-22 Toyota Motor Engineering & Manufacturing North America, Inc. Biofunctional materials
US10988714B2 (en) 2010-06-21 2021-04-27 Regents Of The University Of Minnesota Methods of facilitating removal of a fingerprint from a substrate or a coating
US11015149B2 (en) 2010-06-21 2021-05-25 Toyota Motor Corporation Methods of facilitating removal of a fingerprint
WO2021207687A1 (fr) 2020-04-10 2021-10-14 Liberty Biosecurity Llc Compositions de polypeptides et utilisations associées
WO2021207679A1 (fr) 2020-04-10 2021-10-14 Liberty Biosecurity, Llc Compositions de polypeptides et utilisations associées
WO2022171120A1 (fr) * 2021-02-10 2022-08-18 Novozymes A/S Produit de nettoyage enzymatique amélioré pour membranes et son procédé de nettoyage
US11624044B2 (en) 2010-06-21 2023-04-11 Toyota Motor Corporation Compositions for facilitating biological stain removal

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2397333B1 (es) 2011-08-31 2014-01-16 Betelgeux, S.L. Composición marcadora de biofilms y método de detección de los mismos en superficies.

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WO1992013807A1 (fr) * 1991-02-12 1992-08-20 Buckman Laboratories International, Inc. Compositon et procedes de suppression ou de prevention de la formation de couches d'organismes biologiques
EP0590746A1 (fr) * 1992-09-28 1994-04-06 W.R. Grace & Co.-Conn. Protéases pour inhiber et éliminer la formation d'un film biologique
WO1998026807A1 (fr) * 1996-12-18 1998-06-25 Novo Nordisk A/S Procede de traitement enzymatique d'un film biologique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013807A1 (fr) * 1991-02-12 1992-08-20 Buckman Laboratories International, Inc. Compositon et procedes de suppression ou de prevention de la formation de couches d'organismes biologiques
EP0590746A1 (fr) * 1992-09-28 1994-04-06 W.R. Grace & Co.-Conn. Protéases pour inhiber et éliminer la formation d'un film biologique
WO1998026807A1 (fr) * 1996-12-18 1998-06-25 Novo Nordisk A/S Procede de traitement enzymatique d'un film biologique

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1423337A4 (fr) * 2001-09-07 2005-08-24 Advanced Biocatalytics Corp Reduction du film biologique dans les systemes de filtration a flux tangentiel
WO2004041988A1 (fr) * 2002-10-31 2004-05-21 Sanchez, Thierry Procede d'elimination du biofilm
FR2846665A1 (fr) * 2002-10-31 2004-05-07 Karine Marion Procede d'elimination du biofilm
DE10304331B4 (de) * 2003-02-04 2017-12-28 Henkel Ag & Co. Kgaa Enzymatische Entfernung von Biofilmen auf Haushaltsoberflächen
US12139701B2 (en) 2006-11-22 2024-11-12 Toyota Motor Corporation Biofunctional materials
US11236323B2 (en) 2006-11-22 2022-02-01 Toyota Motor Corporation Biofunctional materials
US11225654B2 (en) 2006-11-22 2022-01-18 Toyota Motor Corporation Biofunctional materials
US10781438B2 (en) 2006-11-22 2020-09-22 Toyota Motor Engineering & Manufacturing North America, Inc. Biofunctional materials
EP2099466A4 (fr) * 2006-12-01 2012-04-11 Laclede Inc Utilisation d'enzymes hydrolytiques et oxydatives pour dissoudre un biofilm dans les oreilles
EP2939687A1 (fr) * 2006-12-01 2015-11-04 Laclede, Inc. Utilisation d'enzymes hydrolytiques pour dissoudre un biofilm dans les oreilles
US8597927B2 (en) 2007-12-20 2013-12-03 Danisco Us Inc. Enzymatic prevention and control of biofilm
US10767141B2 (en) 2010-06-21 2020-09-08 Toyota Motor Engineering & Manufacturing North America, Inc. Thermolysin for easy-cleaning of insect body stains
US12195704B2 (en) 2010-06-21 2025-01-14 Toyota Motor Engineering & Manufacturing North America, Inc. Compositions for facilitating biological stain removal
US10988714B2 (en) 2010-06-21 2021-04-27 Regents Of The University Of Minnesota Methods of facilitating removal of a fingerprint from a substrate or a coating
US11015149B2 (en) 2010-06-21 2021-05-25 Toyota Motor Corporation Methods of facilitating removal of a fingerprint
US12134751B2 (en) 2010-06-21 2024-11-05 Regents Of The University Of Minnesota Bioactive protein-polymer compositions for stain removal
US11692156B2 (en) 2010-06-21 2023-07-04 Toyota Motor Corporation Bioactive protein-polymer compositions for stain removal
US9388370B2 (en) * 2010-06-21 2016-07-12 Toyota Motor Engineering & Manufacturing North America, Inc. Thermolysin-like protease for cleaning insect body stains
US20120301946A1 (en) * 2010-06-21 2012-11-29 Toyota Motor Corporation Thermolysin for easy-cleaning of insect body stains
US11254898B2 (en) 2010-06-21 2022-02-22 Toyota Motor Corporation Bioactive protein-polymer compositions
US11624044B2 (en) 2010-06-21 2023-04-11 Toyota Motor Corporation Compositions for facilitating biological stain removal
US10683529B2 (en) * 2011-06-24 2020-06-16 Realco Kit for detecting biofilms
US11566149B2 (en) 2011-09-09 2023-01-31 Toyota Motor Corporation Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
US11542410B2 (en) 2011-09-09 2023-01-03 Toyota Motor Corporation Coatings containing enzyme for stable self-cleaning of organic stains
US11535773B2 (en) 2011-09-09 2022-12-27 Toyota Motor Corporation Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
US11597853B2 (en) 2011-09-09 2023-03-07 Toyota Motor Corporation Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
US12060497B2 (en) 2011-09-09 2024-08-13 Toyota Motor Corporation Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
US10563094B2 (en) 2011-09-09 2020-02-18 Toyota Motor Engineering & Manufacturing North America, Inc. Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
EP3505609A1 (fr) 2017-12-29 2019-07-03 Itram Higiene, S.L. Composition détergente pour le contrôle et l'élimination de biofilms
WO2021207679A1 (fr) 2020-04-10 2021-10-14 Liberty Biosecurity, Llc Compositions de polypeptides et utilisations associées
WO2021207687A1 (fr) 2020-04-10 2021-10-14 Liberty Biosecurity Llc Compositions de polypeptides et utilisations associées
WO2022171120A1 (fr) * 2021-02-10 2022-08-18 Novozymes A/S Produit de nettoyage enzymatique amélioré pour membranes et son procédé de nettoyage
CN117279706A (zh) * 2021-02-10 2023-12-22 Bl 科技公司 用于膜的加强的酶清洁剂及其清洁方法

Also Published As

Publication number Publication date
ES2162593B1 (es) 2002-07-01
ES2162593A1 (es) 2001-12-16
AU7525800A (en) 2001-07-31

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