WO2012145185A1

WO2012145185A1 - Bacteriocins as biocides for industrial uses

Info

Publication number: WO2012145185A1
Application number: PCT/US2012/032729
Authority: WO
Inventors: Victor ORDONEZ
Original assignee: Vh Biotechnology, Inc.
Priority date: 2011-04-09
Filing date: 2012-04-09
Publication date: 2012-10-26

Abstract

The present invention provides a novel biocide composition and a method using a biocide composition for attacking and killing microorganisms with subsequent growth inhibition, preventing the build-up of microbial slime that is generated in industrial water systems, such as open or closed water-loops in pulp and paper mills, cooling-water systems, oil field water systems, and fuel storage systems, by adding to the water a combination of bacteriocins. The present invention also provides a novel biocide composition and a method for using a biocide composition for replacing chlorination of drinking water in poultry farms, in order to minimize the formation of biofilm, or to help retard the spread of infectious agents through contaminated water in the drinkers.

Description

BACTERIOCINS AS BIOCIDES FOR INDUSTRIAL USES

Inventor: Victor Ordonez

RELATED APPLICATIONS

[0001 ] Benefit of priority under 35 U.S.C. §1 19(e) is claimed to U.S. Provisional Application No. 61473746,filed on April 09, 201 1 , to Victor Ordonez entitled "Bacteriocins as natural biocides in industrial operating-water systems". The subject-matter of the above noted provisional application is incorporated by reference in its entirety by reference thereto. TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates generally to the use of at least one bacteriocin with bactericide and/or bacteriostatic properties for elimination of microorganisms and prevention of slime build-up on surfaces of water-bearing systems, in particular of industrial process-water systems. Dealing with a complex microbial population on industrial process-water systems, a broad spectrum of bacteriocins is preferred. Various bacteriocins can be produced via fermentation by using different strains of bacteria, particularly lactic acid bacteria, under different process conditions. Extra-cellular bacteriocins produced by mean of various single- strain bacterial fermentations can be mixed together in order to obtain a broad spectrum bacteriocin product.

STATE OF THE ART PRIOR TO THE INVENTION

[0003] In their natural environment, microorganisms often grow as populations attached to surfaces in complex structures called microbial slime or biofilm. These biofilms are principally aggregates of bacteria encased in a mucoid polysaccharide structure which attaches the community to a surface. Once established, biofilm populations cause a number of reactions, many considered detrimental, like damage to industrial equipment, contamination of water or food, pharmaceutical and medical products, energy loss through inefficient energy transfer, medical infections and antibiotic resistance.

[0004] Industrial operating-water systems, such as open or closed water-loops in pulp and paper mills, cooling-water systems, oil field water systems, fuel storage systems and water drinking systems typically have copiotrophic (nutrient-rich) water which is circulated at ambient temperatures which are favorable of rapid microbial growth. These conditions can result in large biofilm formations on the surfaces of water-bearing systems promoting several problems such as loss of process efficiency, scale formation and particulate deposition or microbial influenced corrosion.

[0005] The types of microorganisms that cause slime formation in industrial operating-water environments are typically the heavily encapsulated fast growing bacteria, such as species of Pseudomonas, Aerobacter, Alcaligenes, Arthrobacter, Proteus, Bacillus, and others. For example, bacteria found most often in paper and board machine slime include species of Flavobacterium, Clavibacter, Sphaerotilus, and Leptothrix (Johnsrud, 1 997). Desjardins & Beaulieu (2003) reported over 100 bacterial isolates in a pulp & paper mill, being the major proportion Pseudomonas, Bacillus and Psedoxanthomonas.

[0006] In pulp and paper mills, large quantities of water are recirculated in cycle systems called "white water loops" (primary or secondary loops). The white water provides an ideal environment for the growth of microorganisms. This problem is more severe in mills dealing with recycled fiber. In paper mills, biofilm formation produces off-smells, spots and holes in the produced paper and may cause severe production delays in the case of web breaks. In addition, there are multiple inconveniences and economical disadvantages caused by the downtime due to elimination of dirt and slime detachment at the paper machine. Therefore there is a need for reducing said downtime in pulp and paper mills.

[0007] In cooling-water systems, as used in power-generating plants, refineries, chemical plants, air conditioning systems and other commercial and industrial operations, the biofilm deposits increase of the frictional resistance in pipes and lead to a reduced heat exchange efficiency, damage to the joints of pipelines and corrosion within the piping system. This is because cooling water systems are commonly contaminated with airborne organisms entrained by air/water contact in cooling towers, as well as waterborne organisms from the systems' makeup water supply. The water in such systems is generally an excellent growth medium for these organisms. If not controlled, the biofilm biofouling resulting from such growth can plug towers, block pipelines and coat heat transfer surfaces with layers of slime, and thereby prevent proper operation and reduce equipment efficiency. [0008] In the oil industry, the development and operation of an oilfield goes through several distinct phases, all of which can be affected by unwanted microbial growth in produced waters and drilling fluids. Thus, an ongoing challenge in today's fuel industry is the presence and rapid growth of bacterial related problems and their effect on a variety of aqueous handling situations in well production, disposal, stimulation and remediation, as well as on surface water handling operations.

[0009] In oil field water systems such as waterflooding, which is an enhanced oil recovery technique by which water is pumped underground via one or more injection wells in order to push the remaining oil towards a production well. Chemicals such as surfactants, polymers, caustic, microemulsions, and the like can optionally be injected together with or separately from the water. The polymers used in well treatment fluids are subjected to an environment leading to bacterial growth. The growth of the bacteria on polymers used in such fluids can materially alter the physical characteristics of the fluids. For example, bacterial action can degrade the polymer, leading to loss of viscosity and subsequent ineffectiveness of the fluids. Fluids that are especially susceptible to bacterial degradation are those that contain polysaccharide and/or synthetic polymers. Unless precautions are taken to inhibit microbial growth, waterflooding can seriously diminish the value of the crude oil, therefore it is generally recommend the use of a chemical biocide to limit the growth of microorganisms and biofilm formation in the oil industry. US2008032903A1 describes the use of 2,5-dimethyl-1 ,3,5-thiadiazinane-2-thione as a chemical biocide in gas and oil field well stimulation fluids that can control bacterial contamination and have minimal interaction with the polymer and/or oxygen scavenger. Another alternative is described in US Patent 4507212, wherein unsaturated nitrile compounds, e.g., acrylonitrile, are used as biocides to inhibit undesired bacterial growth in injection water used in enhanced oil recovery methods.

[0010] In fuel storage systems severe operational difficulties can be encountered when distillate fuels contain minute traces of water contamination, which is primarily a result of condensation within a storage tank, be it bulk storage or vehicular. This water falls into the fuel where it sinks to the bottom providing a growth media for microorganisms that feed on hydrocarbon fuels, producing biological contamination such as biofilm formation. Microbial growth will occur in the boundary layer between fuel and water in the tank. [001 1 ] Said microbial growth can also result in souring of the crude oil in a reservoir, which is caused by the reduction of inorganic sulfate compounds to sulfides by certain microorganisms. Plugging of the reservoir, wells, and related equipment can even result if such growth is substantial. In addition, equipment will quickly corrode if the metal is exposed to byproducts of microbial metabolism, particularly hydrogen sulfide.

[0012] The introduction of biofuels such as biodiesel into standard hydrocarbon diesel has resulted in a surge of incidents involving microbial contamination, which can cause the accumulation of harmful sludge in fuel filters and physical damage to tanks, pipelines, hoses and other components. Biodiesel can provide the perfect environment for bacteria to flourish as it has the potential to increase the amount of water held in suspension within the fuel, which in turn creates a rich food source for bacteria to grow and the constant need to clean blocked filters. Biodiesel blends that contain recovered greases and fats may see an increase in microbial contamination and oxidation stability issues. With Ethanol many of the same concerns that we have addressed with biodiesel can occur in blends of unleaded gasoline and ethanol.

[0013] Some of the main consequences of microbial growth and biofilm formation in the oil industry, and more specifically in the introduction of biofuels, in fuel storage systems and in oil field water systems such as waterflooding, are the followings (Gaylarde, et. al., 1999):

- Blockage of pipes, valves, filters and incorrect readings.

- Increased water content

- Sludge formation

- Surfactant production, causing oil/water emulsification, entry of cells into the oil phase and coalesce malfunction.

- Corrosion of storage tanks and lines

- Production of suspended solids in the fuel

- Breakdown of hydrocarbons

- Shortened filter life

- Fouling of injectors

- Increased sulfur content of fuel

- Shortened life of engine parts

- Penetration of protective tank linings [0014] These operational problems have been reported by domestic heating oil suppliers, railroads, commercial airlines and at military installations (US4166725). Common treatment for these operational problems in aqueous handling situations in the petroleum industry, is the use of standard chemical biocides with bacteriostatic and bactericide effects. Standard chemical biocides, by their nature, are toxic and may present a hazard to the fuel handlers, to personnel working in areas where fuel volatilization may occur and to organisms in the environment receiving wastes from the system.

[0015] Another specific example of problems caused by the growth of microorganisms and the biofilm formation in drinking water systems is found at poultry farms. Good water helps the bird digestion process, the transport of the nutrients in the body, the regulation of the body temperature and the elimination of waste. However, the water itself can be a source of contamination when its microbiological condition is not optimal. Contaminated water can impede a good digestion or a good absorption of additives like medicines, vaccines, vitamins, etc. The administration of these additives via the drinking water will create a polysaccharide layer in the system or biofilm, in which microorganisms will develop and ultimately contaminate the birds. Dirty water supply lines hide all kind of microorganisms in the biofilm. Chlorination of drinking water of poultry farms minimizes the level of said microorganism and also prevents or minimizes the formation of the biofilm. It may also help retard the spread of the infectious agents through contaminated water in the drinkers. The preferred method to sanitize the drinking water at poultry farms is the high-test hypochlorite (HTH). However, some countries do not allow the use of chlorine in poultry processing because of the possible production of trihalomethanes (THM) such as chloroform that might be formed in reaction with tissue. Moreover, chlorine is unable to remove the biofilm, it increases the pH of the water; but also it will be inactivated by organic matter (as all halogens do). Also, excessive chlorine in drinking water affects poultry health; it also affects negatively body weight gain of birds and eggshell quality because toxic substances can build up in fat and muscle tissues and hens can export toxic substances into eggs. Therefore, reducing the biofilm formation (mesophilic microorganism count and coliform bacteria count) improve the water quality and have the potential to influence positively in the health and productivity of poultry and the quality of processed poultry products, particularly eggs. [0016] Within this objective, specific methods and tools have been developed to control, kill, prevent, and/or reduce the microbial growth and the correspondent development of microbial slime on the surfaces of the industrial operating-water equipments. Historically, deposition of the bacterial slime has been treated by the addition to industrial waters of chemical biocides. The purpose of chemical biocides is to destroy or arrest the growth of some of the many organisms present in the water to thereby prevent or retard the formation of microbial slime. However, those chemical biocides are effective mainly against floating microorganisms and thus cannot adequately control large populations of attached bacteria encased in the biofilm. Additionally, chemical biocides raise many concerns from an ecological point of view and, because of their toxicity, pose considerable dangers when handled. Great care is required when handling chemical biocides and appropriate protective clothing and equipment should be used, in addition, disposal of used or unwanted chemical biocides must be undertaken carefully to avoid serious and potentially long-lasting damage to the environment. Particularly, chemical biocides agents have been used since the 1 800s in industrial operating-water systems, not only in the pulp and paper industry but also in cooling-water systems, petroleum industry, and drinking water sanitizing systems. Chemicals used as biocides have included amines, nitrile compounds, chlorinated phenolics, cooper salts, organo- sulphur compounds and quaternary ammonium slats, all of which are relatively toxic to humans. Many of the early active ingredients were used indiscriminately, with little regard for workers' health and safety or environmental concerns. Specifically, the huge doses of chemical biocides required to rid industrial operating-water systems of established microbial slime have been environmentally undesirable. The primary problem has been that the chemical biocide level required to control biofilm formation usually leaves residual levels that make the water too toxic for consumption.

[0017] In recent years, registration of chemical biocides has become more difficult, since increased emphasis is now placed on characteristics such as health and safety issues, and fate in the environment. Therefore, emphasis was given to the development of natural biocides which were harmless to human beings and to the natural environment. For this reason, alternative ways of eliminating biofilm were being evaluated such as for example the use of specific enzymes (Patents No.4994390 and No. 5238572), bacteriophages, competing organisms, biological complex formers, and biodispersants (Johnsrud, 1997).

[0018] Microorganisms produce an extraordinary array of microbial defense systems. These include broad-spectrum classical antibiotics, metabolic byproducts, such as the lactic acids produced by lactobacilli, lytic enzymes such as lysozymes, numerous types of protein exotoxins and bacteriocins, which are loosely defined as biologically active protein moieties with a bactericidal mode of action.

[0019] Nowadays, bacteriocins have been used as natural biocides against biofilm formation for controlling contamination on hospital countertops and in catheter lock solutions, also as skin disinfectants, for eliminating dental plaque and in contact lens storage case disinfection. WO2006/053445 describes the use of bacteriocins as antimicrobial peptides possessing cationic and amphipathic properties which allow interactions with the membrane of living cells in a treatment against cancer.

[0020] Until now there is no mention in the scientific literature nor on the specialized literature nor any patent concerning the use of bacteriocins as natural biocides for elimination and/or prevention not only of microbial growth but also of microbial biofilm build-up on surfaces of water-bearing systems, in particular of industrial process-water systems.

DESCRIPTION OF THE INVENTION

[0021 ] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0022] In accordance with the purposes of this invention, as embodied and broadly described therein, this invention relates to a biocide composition and the method for use said biocide composition as a natural biocide against microbial growth and/or biofilm formation comprising (a) bacteriocins as first active agent, preferably derived from lactic acid bacteria, or (b) a composition comprising a bacteriocin preferably derived from lactic acid bacteria and a second or additional agent select from the group comprising a stabilizer, an enzyme, an antibiotic, etc.

[0023] As used herein, the term "active agent" is defined to mean a substance or compound which (irrespective of its relative quantity) help directly in achieving its performance objectives.

[0024] As used in the present invention, the term "biocide composition" as used herein means a chemical substance or a substance from a microorganism which can deter, render harmless, or exert a controlling effect on any harmful organism/microorganism by chemical or biological means. The biocidal or microbiocide composition of the present invention is also employed as anti-fouling agent or disinfectant against microbial growth and/or biofilm formation.

[0025] As used in the present invention, the term "microbial growth" means the increase in biomass production, cell production, and cell survival of microorganisms such as bacteria, algae, diatoms, plankton, and fungi.

[0026] A preferred embodiment of the invention refers to a biocide composition and the method for use said biocide composition for eliminating and/or preventing microbial growth and/or biofilm formation.

[0027] As used in the present invention, the term "biofilm" or "biofilm formation" refers to a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides in various configurations, also referred to as slime or microbial slime in the present invention. A more preferred definition is bacterial biofilm or bacterial slime. A biofilm may be a pure culture derived from a single type of bacteria or (more commonly) a mixed culture of multiple bacteria. Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life in natural, industrial and hospital settings. Biofilms are usually found on solid substrates submerged in or exposed to an aqueous solution or on surfaces of water-bearing systems.

[0028] A preferred embodiment of the invention refers to a biocide composition and the method for use said biocide composition for eliminating and/or preventing microbial growth and/or biofilm formation at an industrial operating-water system, characterized in that the biocide composition comprises bacteriocins as first active agent, preferably derived from lactic acid bacteria.

[0029] The following industrial operating-water systems have been identified as potential users of the biocide composition and the method for use said biocide composition claimed in the present invention: papermaking industry system (pulp and paper mills), cooling water system, oil or fuel industry, and more specifically in the introduction of biofuels, in fuel storage systems and in oil field water systems such as waterflooding, sanitizing drinking water system (in a preferred embodiment in a poultry farm) and waste water system. Additionally, other industries are also potential users of the biocide composition and the method for use said biocide composition claimed in the present invention: steel producers, coating producers, nuclear and power industry, oil refinery, oil and gas production, petrochemistry, harbour maintenance, plastic producers, producers of chemicals for water treatment, naval construction, medicine (sterilization, implants, instruments), dentistry, food and drug industry, electronics (ultra clean laboratories), and air conditioning (building, industry, cars).

[0030] A more preferred embodiment of the invention refers to a biocide composition and the method for use said biocide composition exhibiting bactericidal and/or bacteriostatic properties against bacterial species and/or bacterial biofilm formation, preferably at an industrial operating-water system, characterized in that the biocide composition comprises bacteriocins as first active agent, preferably derived from lactic acid bacteria.

[0031 ] As used herein, the term "bactericide or bactericidal" is defined to mean having a destructive killing action upon bacteria. As used herein, the term "bacteriostatic" is defined to mean having an inhibiting action upon the growth of bacteria.

[0032] Another aspect of the invention relates to a biocide composition and the method for use said biocide composition against microbial growth and/or biofilm formation comprising at least one bacteriocin as first active agent, preferably when the bacteriocin derives from a Gram-positive bacteria and/or Gram-negative bacteria bacteriocin enriched fermentation extract. In a preferred embodiment bacteriocins derived from a Batch Fermentation Process As used herein, the term "Batch Fermentation Process" describes the process wherein a tank of fermenter or batch is filled with the prepared mash of raw materials to be fermented. The temperature and pH for microbial fermentation is properly adjusted, and occasionally nutritive supplements are added to the prepared mash. The mash is usually steam sterilized in a pure or mixed culture process. The inoculum of a culture is added to the fermenter or batch, from a separate culture vessel. The cultures employed in the present invention could be pure or mixed, comprising Gram-positive bacteria and/or Gram-negative bacteria. In a more preferred embodiment of the invention in order to obtain the fermentation pool comprising bacteriocins the culture employed in the fermentation process comprises Gram- positive bacteria and more preferably comprises lactic acid bacteria.

[0033] The biocide composition and the method for use said biocide composition against microbial growth and/or biofilm formation comprising at least one bacteriocin, are characterized in that said bacteriocins, preferably obtained as a result of the fermentation process, are also found as a product of almost every bacterial species examined to date, and within a species tens or even hundreds of different kinds of bacteriocins are present (Riely & Chavan, 2007).

[0034] A more precise definition of a bacteriocin is provided by Galvez et. al. (2007) and Berjeaud and Cenatiempo (2004): a ribosomally synthesized proteinaceous compound released extracellularly by Gram-positive as well as Gram-negative bacteria that can be shown to interfere with the growth of other bacteria and to which the producer cell expresses a degree of specific immunity.

[0035] As a group, bacteriocins of the present invention are heterogeneous. Generally bacteriocins are classified largely based on their molecular weight differences. Some bacteriocins are peptides consisting of only 19 to 37 amino acids, whereas others are large peptides with molecular weights of up to 90,000. Some small bacteriocins contain unusual amino acids originating from modifications of conventional amino acids after translation. The activity spectrum of bacteriocins can be narrow and confined to inhibition of closely related species, or it can be relatively broad and include many different bacterial species (Joerger, 2003).

[0036] Bacteriocins produced by Gram-positive bacteria are classified in four groups (Riley and Chavan, 2007; Chung, 2003):

[0037] Group I bacteriocins are known as lantibiotics. They are small peptides with molecular weight below 2 kDa, with dehydrated residues such as dehydroalanine, dehydrobutyrine, lanthionine, and β-methyl lanthionine, introduced by posttranslational modifications. Nisin is the most studied lantibiotic bacteriocins, along with lactocin S.

[0038] Group II bacteriocins are small heat stable peptides with molecular weight in the range of 2-6 kDa. Majority of bacteriocins produced by Lactobacillus species belongs to this group. Group II bacteriocins include pediocin PA-1 , lactococcin A, curvacin A, lactacin B, lactocin S, and sakacin P. Lactacin F is a well-known two- component bacteriocin produced by Lb. johnsonnii.

[0039] Group III bacteriocins are large heat-labile proteins. Helveticin J (37kda), acidophilin, or caseicin (40kda) are large thermolabile bacteriocins with similarity to colicins produced by Escherichia coli.

[0040] Group IV bacteriocins are proteins complexed with lipid or carbohydrate moiety. Plantaricin S and lactocin 27 belong to group IV bacteriocins.

[0041 ] As used herein the term "bacteriocin" refers also to "extracellular bacteriocin protein", "bacteriocin produced by means of various single-strain bacterial fermentations" and as a group of heterogeneous bacteriocins obtained from a mixed fermentation process.

[0042] A large number of Gram positive bacteriocins have been isolated and characterized from lactic acid bacteria (LAB-bacteriocins) and some have acquired a status as potential antimicrobial agents because of their antagonistic affect against important pathogens. LAB-bacteriocins comprise a heterogeneous group of physicochemically diverse ribosomally-synthesized peptides or proteins showing a narrow or broad antimicrobial activity spectrum against Gram-positive bacteria. Most LAB-bacteriocins act on sensitive cells by destabilization and permeabilization of the cytoplasmic membrane through the formation of transitory poration complexes or ionic channels that cause the reduction or dissipation of the proton motive force (PMF). The most important LAB-bacteriocins are nisin, diplococcin, acidophilin, bulgarican, helveticins, lactacins and plantaricins.

[0043] As used herein lactic acid bacteria (LAB) comprise a clade of Gram- positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. These bacteria, usually found in decomposing plants and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. Bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms. The industrial importance of the LAB is further evidenced by their generally recognized as safe (GRAS) status. The genera that comprise the LAB are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Teragenococcus, Vagococcus, and Weisella; these belong to the order Lactobacillales.

[0044] A more preferred embodiment of the invention refers to a biocide composition and the method for use said biocide composition, wherein bacteriocins are derived from Gram-positive bacteria fermented extract, preferably from acid lactic bacteria species, more preferably from the group consisting of Streptococcus spp., Lactococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp., Bifidobacterium spp. and Enterococcus spp.

[0045] Bacteriocins produced by Gram-negative bacteria are classified as nicins and colicins, which are produced by Esherichia coli. Most of the Gram positive bacteriocins often show a much broader spectrum of bactericidal activity than bacteriocins produced by Gram-negative bacteria.

[0046] Bacteriocins used herein comprise: acidophilin, agrocin, alveicin, aureocin, bulgarican, carnocin, caseicin, colicin, curvaticin (curvacin A), diplococcin, divercin, enterocin, enterolysin, epidermin, erwiniocin, glycinecin, halocin, helveticin J, lactococcin (lactococcin A), lacticin (lactacin B, lactacin F), lactocin (lactocin S, lactocin 27), leucoccin, mesentericin, nisin, pediocin (pediocin PA-1 ), plantaricin (plantaricin S), sakacin (sakacin P), subtilin, sulfolobicin, thuricin 17, trifolitoxin, vibriocin, and warnerin.

[0047] The following non-restrictive list provides the name of a bacterial source along with the bacteriocins that the strain can produce under specific and/or particular fermentation parameters by applying classic microbiological techniques in order to obtain a bacteriocin enriched fermentation extract as the one obtained in the present invention in order to obtain the biocide composition against microbial growth and/or biofilm formation and which characterized also the method for use said biocide composition.

Lactobacillus helveticus Helveticin

Lactobacillus plantarum Pediocin, Plantaricin

Lactobacillus casei Caseicin

Leuconostoc mesenteroides Mesentericin

[0048] Bacteriocins covered by this patent can also be produced by genetic engineering methods particularly named Recombinant DNA Technology. Following this technique, small fragments of DNA obtained by a bacteriocin producer organism can be isolated. These fragments can then be spliced into a smaller genome in order to have a less complex environment in which the activity caused by this piece of genetic code can be screened and analyzed in order to find the sequence that encodes the bacteriocin activity. In this way, genes that code for bacteriocin activity can be isolated and transported to new host. Eventually, the host can overexpress the bacteriocin protein to result in a commercially viable product.

[0049] As used herein, the term "bacteriocin" also refer to a polypeptide produces by a microbial cell (e.g. a bacteria), wherein such polypeptide possesses microbicidal (e.g. bactericidal or bacteriostatic) activity. A major difficulty in bacteriocin research and applications is obtaining accurate quantification of the bacteriocin activity or the microbicidal activity of bacteriocins using bioassays which are already known by those skilled in the art are based on the quantification of the inhibition produced in a sensitive microorganism. These types of assays are the most widely used techniques for quantitative determination of bacteriocins. Although numerous other methods which are already known by those skilled in the art have also been described such as ELISA, ATP-bioluminometry, radiometry, conductance measurements, or even sophisticated bioassays based on self- induction of the nis promoter and bioluminescence, they have not gained wide acceptance because of requirements for dedicated equipment, supplies and skills, and more over because the results produced by such methods cannot necessarily be correlated with antimicrobial activity. Therefore, growth inhibition techniques which are already known by those skilled in the art are still the most commonly used in everyday trials for obtaining accurate quantification of the bacteriocin activity or the microbicidal activity of bacteriocins. Multiple procedures based on growth inhibition are described in the literature, which are already known by those skilled in the art, relying on tests performed either in solid, e.g. the plate agar diffusion assay, or in liquid medium, e.g. turbidometry. The agar diffusion assay, in which inhibition zones are produced in plates in a procedure similar to that of antibiograms, is undoubtedly the most commonly used despite the inconveniences and limitations of its application. In addition, diffusion-related difficulties of the active substance represent another important limitation of agar diffusion assays. An expert in the art already knows that the performance of the method for obtaining accurate quantification of the bacteriocin activity or the microbicidal activity of bacteriocins, which is laborious and time-consuming, depends largely on human ability and judgment and precision cannot be obtained when inhibition zones are unclear or not perfectly circular.

[0050] A more preferred embodiment of the invention is a biocide composition and the method for use said biocide composition comprising at least one additional agent, preferably an enzyme or an enzyme variant thereof having the relevant enzyme activity, and more preferably lysozyme, protease, amylase, lipase, cellulase and hemicellulase.

[0051 ] Another preferred embodiment of the invention is a biocide composition and the method for use said biocide composition wherein the additional agent is a stabilizer well known in the art to protect proteins for the shell life of the product, such as highly water soluble sodium and/or potassium salts.

[0052] In a more preferred embodiment of the present invention, the biocide composition and the method for use said biocide composition are characterized in that the biocide composition can also comprise other active agents, such as additional algicides, fungicides, corrosion inhibitors, scale inhibitors, complexing agents, surfactants, enzymes, nonoxidizing biocides and other compatible products which will lend greater functionality to the biocide composition of the present invention.

[0053] In a preferred embodiment of the invention the biocide composition and the method for use said biocide composition are characterized in that the application form is selected from the group consisting of: liquid form, solid form and powdered form. [0054] Another aspect of the present invention refers the biocide composition and the method for use said biocide compositions are characterized in that at least one bacteriocin is substantially pure and/or recombinant.

[0055] As used herein substantially pure bacteriocin means a bacteriocin which consists substantially of only one bacteriocin with definite physical and chemical properties and a definite composition. In addition, when referring to recombinant bacteriocin, it means a bacteriocin produced by the combining of genetic material from more than one origin.

[0056] Another aspect of the present invention refers to a method for eliminating and/or preventing microbial growth and/or biofilm formation at industrial operating- water systems comprising adding to the industrial water the biocide composition comprising bacteriocins as first active agent.

[0057] A preferred aspect of the present invention refers to a method for eliminating and/or preventing microbial growth and/or biofilm formation wherein the industrial operating-water system is selected from the group consisting of:

a) papermaking system,

b) cooling water system,

c) oil field water system,

d) fuel storage system, and

e) sanitizing drinking water system.

[0058] A more preferred aspect of the present invention refers to a method for eliminating and/or preventing microbial growth and/or biofilm formation, wherein at least one bacteriocin derives from Gram-positive bacteria and/or Gram-negative bacteria fermentation extract, preferably wherein the Gram-positive bacteria is a lactic-acid bacteria.

[0059] Another preferred aspect of the present invention refers to a method for eliminating and/or preventing microbial growth and/or biofilm formation, wherein said bacteriocin is derived from a lactic acid bacteria species selected from the group consisting of Streptococcus spp., Lactococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp., Bifidobacterium spp. and Enterococcus spp.

[0060] The biocide composition and the method for use said biocide composition object of the present invention are characterized in that when added to the industrial water system the bacterial count, preferably expressed in CFU/g or CFU/ml, is reduced as an indicator of the reduction in the microbial growth and/or biofilm formation.

[0061 ] As refer in the present invention the term "bacterial count" concerns to the bacterial control or measurement of microbial or bacterial growth and biofilm formation using standard methods already known for an expert in the field, preferably measuring the mesophilic microorganism count and/or the coliform bacteria count. Said measurement preferably includes two different parameters: changes in cell mass and changes in cell numbers. The methods for measuring the bacterial cell mass preferably involve both direct and indirect techniques selected from the group consisting of: direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation, direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content, indirect measurement of chemical activity such as rate of 02 production or consumption, CO2 production or consumption, etc. In addition, in order to measure the bacterial count, the present invention also refers to turbidity measurement which employs a variety of instruments to determine the amount of light scattered by a suspension of bacterial cells.

BRIEF DESCRIPTION OF THE FIGURES

[0062] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one embodiment of the invention and together with the description, serve to explain the principles of the invention.

[0063] Figure 1 shows how after the third month the bacterial count at the wire pit in a recycling paper mill is reduced due to the addition of the composition comprising a bacteriocin enriched fermentation extract.

[0064] Figure 2 shows how the total bacterial count at the machine chest of the recycling mill is reduced during the next fifth months due to the addition of the composition comprising a bacteriocin enriched fermentation extract.

[0065] Figure 3 shows how the total bacterial count at the head box of the recycling mill is reduced during the next month due to the addition of the composition comprising a bacteriocin enriched fermentation extract. EMBODIMENTS OF THE INVENTION

[0066] The present invention is additionally illustrated by the following Examples that, together with the Figures described previously illustrate the experimental methodology used for its development. It is understood that the skilled in the art will be able to understand the modifications, variations and changes that can be implemented within the scope of the present invention.

EXAMPLES

[0067] The amount of biocide composition according to the present invention will vary depending upon the severity of the microbial produced slime problem.

Example 1.

[0068] In order to show the efficacy of the biocide composition and the method for use the biocide composition for eliminating microorganisms and preventing slime/biofilm build-up on surfaces of water-bearing systems of the present invention, the biocide composition was used in a pulp and paper mill in order to reduce bacterial counts in the white water loop.

1.1 Bacterial control at a tissue mill.

[0069] The biocide composition was added to the wire pit, and the bacterial count was quantified at day 0, 5, 10. Table I shows how the bacterial count expressed in CFU/g, is reduced at the wire pit after the addition of the biocide composition.

Table I. Data at the wire pit in a recycling paper mill, starting dosage of the biocide composition.

1.2. Downtime reduction due to elimination of dirt and slime detachment at the paper machine by using the composition comprising the biocide composition at an old corrugated cardboard (OCC) mill.

[0070] This mill produces approximately 90 t/day of liner and medium package papers by recycling OCC. The raw material is 100 % recycled paper. It operates under a 100 % water closed loop. Under those conditions the white water loop presented a high bacteria count expressed in CFU/ml. The biocide composition was added to paper mill 1 (PM 1 ) and paper mill 2 (PM 2) in order to eliminate the dirt detachment and the slime detachment. The downtime reduction is quantified as a reduction % in time (hr/day) compared to the control situation wherein there was no addition of the biocide composition. Table I I A. shows the downtime reduction due to elimination of dirt detachment, while B. shows the downtime reduction due to elimination of slime detachment. Table I I. A. Downtime reduction due to elimination of dirt detachment:

Reduction from 5,58 hr/day to 0,41 hr/day on PM 1 (Reduction 93 %) Reduction from 2,67 hr/day to 0,22 hr/day on PM 2 (Reduction 92 %)

Table I I. B. Downtime reduction due to elimination of slime detachment:

Reduction from 0,53 hr/day to 0,14 hr/day on PM 1 (Reduction 74 %)

Reduction from 0,83 hr/day to 0,25 hr/day on PM 2 (Reduction 70 %)

[0071 ] The addition of the biocide composition to PM 1 and PM 2 showed a 93 % and a 92 % respectively in the downtime reduction due to elimination of dirt detachment. And the addition of the biocide composition to PM 1 and PM 2 showed a 74 % and 70 % respectively in the downtime reduction due to elimination of slime detachment.

1.3. Bacterial Control at a Tissue Mill using the biocide composition.

[0072] The biocide composition was added to the wire pit, and the bacterial count is measured and expressed in CFU (x10⁶) during 20 months. First three months represent the control measurement of the bacterial count in the wire pit, during said months there was no addition of the biocide composition. After the third month, the biocide composition was added. Figure 1 shows how after the third month the bacterial count at the wire pit is reduced due to the addition of the biocide composition. 1.4. An OCC recycling mill using the biocide composition.

[0073] The biocide composition was added to the recycling mill, and the total bacterial count or average is measured at the machine chest and expressed in CFU/ML (x10⁶) during 6 months. First month represent the control measurement of the bacterial count in the recycling mill, during said month there was no addition of the biocide composition. After the first control month, the composition was added. Figure 2 shows how the total bacterial count at the machine chest of the recycling mill is reduced during the next fifth months due to the addition of the biocide composition.

1.5. An OCC recycling mill starting the use of the biocide composition.

[0074] The biocide composition was added to the recycling mill, and the total bacterial count or average is measured at the head box and expressed in CFU/ML (x1 0⁶) during 31 days. The measurements are taken at the following dates:

February 15, 2010 (0 days),

March 2, 2010 (15 days),

March 3, 2010 (16 days),

March 4, 2010 (17 days),

March 5, 2010 (18 days),

March 8, 2010 (21 days),

March 9, 2010 (22 days),

March 10, 201 0 (23 days),

March 12, 201 0 (25 days),

March 16, 201 0 (29 days), and

March 18, 201 0 (31 days).

[0075] First date represent the starting control measurement of the bacterial count in the recycling mill, during said month there was no addition of the biocide composition of the present invention comprising a bacteriocin enriched fermentation extract. After the first control date, the composition was added and the bacterial count was measured during the following month. Figure 3 shows how the total bacterial count at the head box of the recycling mill is reduced during the next month due to the addition of the biocide composition. Example 2.

[0076] The biocide composition was tested in a poultry farm in order to replace high-test hypochlorite (HTH) dosage to sanitize drinking water. Two tanks with raw drinking water were used for this test, one tank was treated with the composition of the present invention: sample 1 and the other tank with standard HTH: sample 2. In order to establish the effectiveness of the composition of the present invention sanitizing the drinking water, two control parameters were measured as indicators of water quality before and after the treatment: the mesophilic microorganism count and the coliform bacteria count. See Table III for dosages and time reaction..

Table I II

[0077] After the treatment, the mesophilic microorganism count and the coliform bacteria count were 0 for both samples. Presented lab results show that the biocide composition object of the present invention is as effective as HTH to sanitize drinking water at poultry farms.

Example 3.

[0078] The biocide composition was tested in the fuel industry, as a fuel additive on samples of gasoline and diesel "contaminated" with water contaminated with microbial growth and/or biofilm formation. Said contaminated fuel samples experiment a microbial contamination which can be extrapolate in other oil field water systems such as waterflooding, in fuel storage systems or in biofuels.

[0079] In order to eliminate and/or prevent microbial growth and/or biofilm formation, the test was done in 100 ml samples of gasoline and diesel. Two bottle samples were prepared for each type of fuel. One liter of "contaminated" water was prepared by pouring 200 grams of soil in 1000 cc of water. The water-soil mix was agitated during five minutes, then filtered and the water filtrate was used to contaminate the fuel samples. Each sample of 100 ml fuel was mixed with 100 ml of contaminated water. Each bottle was left for 80 hours at 34 degrees Celsius for microbial incubation. One sample of contaminated gasoline and diesel was left as a "blanc" for comparison purposes, in order to evaluate "base line" microbial contamination. The other sample was treated with the enzymatic biocide at a dose of 10 ppm. The biocide was dosed and left for 15 hours. Finally, the samples were analyzed for mesophilic bacteria CFU as an indicator for microbial growth and/or biofilm formation. See Table IV for test results.

Table IV

[0080] The biocide composition shows excellent results in reducing microbial growth and/or biofilm formation, having a removal rate of mesophilic bacterial counts of 99.95 % in the diesel fuel sample and 89 % in the gasoline sample. The biocide composition is a green and safe alternative to standard biocides used for eliminate and/or prevent microbial growth and/or biofilm formation in the petroleum industry as a fuel additive.

[0081 ] While certain novel features of the present invention have been shown and described, it will be understood that various omissions, substitutions and changes in the forms and details of the device illustrated and in its operation can be made by those skilled in the art without departing from the spirit of the invention.

Claims

1 . A biocide composition for use against microbial growth and/or biofilm formation comprising at least one bacteriocin as first active agent.

2. The biocide composition according to claim 1 wherein it is applied for eliminating and/or preventing microbial growth and/or biofilm formation at an industrial operating-water system.

3. The biocide composition according to claim 2, wherein the industrial operating- water system is selected from the group consisting of:

a) papermaking system,

b) cooling water system,

c) oil field water system,

d) fuel storage system, and

e) sanitizing drinking water system.

4. The biocide composition according to claim 2, wherein at least one bacteriocin derives from Gram-positive bacteria and/or Gram-negative bacteria fermentation extract.

5. The biocide composition according to claim 4, wherein the Gram-positive bacteria is a lactic-acid bacteria.

6. The biocide composition according to claim 5, wherein said bacteriocin is derived from a lactic acid bacteria species selected from the group consisting of Streptococcus spp., Lactococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp., Bifidobacterium spp. and Enterococcus spp.

7. The biocide composition according to claim 2, wherein the bacteriocin is selected from the group consisting of: acidophilin, grocin, alveicin, aureocin, bulgarican, carnocin, caseicin, colicin, curvaticin, diplococcin, divercin, enterocin, enterolysin, epidermin, erwiniocin, glycinecin, halocin, helveticin, lactococcin, lacticin, lactocin, leucoccin, mesentericin, nisin, pediocin, plantaricin, sakacin, subtilin, sulfolobicin, thuricin, trifolitoxin, vibriocin, and warnerin.

8. The biocide composition according to claim 2, wherein it comprises at least one additional agent.

9. The biocide composition according to claim 8, wherein the additional agent is an enzyme or an enzyme variant thereof having the relevant enzyme activity.

10. The biocide composition according to claim 9, wherein the enzyme is select from the group consisting of: lysozyme, protease, amylase, lipase, cellulase and hemicellulase.

1 1 . The biocide composition according to claim 8, wherein the additional agent is a stabilizer.

12. The biocide composition according to claim 1 1 , wherein the stabilizer is highly water soluble sodium and/or potassium salts.

13. The biocide composition according to claim 2, wherein the application form is selected from the group consisting of: liquid form, solid form and powdered form.

14. The biocide composition according to claim 2, wherein at least one bacteriocin is substantially pure.

15. The biocide composition according to claim 2, wherein at least one bacteriocin is a recombinant bacteriocin.

16. A method for eliminating and/or preventing microbial growth and/or biofilm formation at industrial operating-water systems comprising adding to the industrial water the biocide composition defined in claim 1 .

17. The method for eliminating and/or preventing microbial growth and/or biofilm formation according to claim 16, wherein the industrial operating-water system is selected from the group consisting of:

a) papermaking system,

b) cooling water system,

c) oil field water system,

d) fuel storage system, and

e) sanitizing drinking water system.

18. The method for eliminating and/or preventing microbial growth and/or biofilm formation according to claim 16, wherein at least one bacteriocin derives from Gram-positive bacteria and/or Gram-negative bacteria fermentation extract.

19. The method for eliminating and/or preventing microbial growth and/or biofilm formation according to claim 16, wherein the Gram-positive bacteria is a lactic-acid bacteria.

20. The method for eliminating and/or preventing microbial growth and/or biofilm formation according to claim 16, wherein said bacteriocin is derived from a lactic acid bacteria species selected from the group consisting of Streptococcus spp., Lactococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp., Bifidobacterium spp. and Enterococcus spp.