Method for rapidly detecting bacterial spores in industrial process
阅读说明：本技术 在工业工艺中快速检测细菌孢子的方法 (Method for rapidly detecting bacterial spores in industrial process ) 是由 L·伦德 L·赖斯 于 2017-07-12 设计创作，主要内容包括：描述一种通过测量随时间变化的微生物代谢活性来检测细菌孢子存在的方法。通过检测指示孢子萌发的代谢活性的爆发,将孢子与营养细胞和其它微生物区分开来。这种方法可用于在典型工作班次的时间范围内检测诸如造纸系统的商业工艺系统中的细菌孢子。(A method for detecting the presence of bacterial spores by measuring the metabolic activity of the microorganism over time is described. Spores are distinguished from vegetative cells and other microorganisms by detecting bursts of metabolic activity indicative of spore germination. This method can be used to detect bacterial spores in commercial process systems, such as paper making systems, over the time frame of a typical work shift.)
1. A method of detecting bacterial spores in a sample and distinguishing them from vegetative cells and other microorganisms, comprising:
preparing a sample for testing for the presence of bacterial spores;
measuring a baseline level of metabolic activity of a microorganism in the sample;
incubating the sample under conditions that initiate germination;
monitoring the level of subsequent metabolic activity of the microorganism in the sample; and
after about 5 to 8 hours of incubation, the sample is tested for the presence of a burst of microbial metabolic activity.
2. The method of claim 1, wherein the metabolic activity of the microorganism is determined by measuring Adenosine Triphosphate (ATP).
3. The method of claim 1, wherein the metabolic activity of the microorganism is detected by a metabolic dye.
4. The method of claim 1, wherein the preparing comprises:
collecting a sample comprising an unknown amount of spores, vegetative cells, or both spores and vegetative cells;
if solid, decomposing the sample; and
the sample is placed in a tray or container.
5. The method of claim 4, wherein the coating is applied from a hard surface, a liquid, a slurry (starch, carbonate, clay, or TiO)2) Or collecting the sample in a paper product.
6. The method of claim 5, wherein the hard surface is selected from the group consisting of food and beverage processing equipment, piping, tanks, evaporators, nozzles, dairy processing equipment, milk storage tanks, milk carts, milking equipment, countertops, cooking appliance surfaces, bathroom surfaces such as washbowl and toilet handles, light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient pens, grab bars, surgical instruments, factory paper interior equipment including piping, tanks, headbox, cutoff towers, economizer blades, and forming screens.
7. The method of claim 6, wherein the liquid is selected from the group consisting of process water, feed water, cooling tower water, treated and untreated wastewater, paper stock, thin and thick stock, white water, suction box water, tray water, fruit and vegetable sink water, protein process water, hydroponic or seafood culture water, and agricultural water.
8. The method of claim 6, wherein the paper product is selected from the group consisting of paper products such as finished paper products and finished board products for food contact and non-food contact grades; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers; a food can; aluminum and PET beverage containers; a bag, a film, and modified atmosphere packaging.
9. The method of any of claims 2, wherein the measuring comprises:
adding luciferase and luciferin to the sample; and
light emission was measured in Relative Light Units (RLU).
10. The method of any one of claims 1 to 5 and 9, wherein the incubating comprises:
providing a nutrient liquid medium and a germination enhancer to the sample; and optionally adding a biocide neutralizing agent,
incubating the sample at a temperature of about 30 ℃ to about 45 ℃.
11. The method of claim 10, wherein the nutrient broth is a 2X nutrient broth and the germination enhancer is L-alanine.
12. The method of claim 2, wherein the monitoring comprises adding luciferase and luciferin to the sample and measuring light emission at multiple time points during 8 hour incubation.
13. The method of claim 12, wherein the measuring occurs once per hour.
14. The method of claim 2, wherein the measuring and monitoring comprises measuring ATP levels by HPLC.
15. The method according to any one of claims 1 to 5, wherein the burst of microbial metabolic activity comprises measuring at least 10 times higher microbial metabolic activity than the initial measurement in the sample and determining the presence of spores in the sample.
16. The method of any one of claims 1 to 15, further comprising inactivating vegetative cells in the sample.
17. The method of claim 16, wherein the inactivating comprises heating the sample at a temperature of about 80 ℃ to about 110 ℃ for about 5 to 15 minutes.
18. The method of any one of claims 1 to 17, further comprising selecting an antimicrobial treatment based on the presence or absence of spores, vegetative cells, or both spores and vegetative cells.
19. The method of claim 18, wherein the antimicrobial treatment is selected from the group consisting of chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, peracids, UV, extreme heat, and radiation when spores are detected.
20. The method of claim 18, wherein the antimicrobial treatment is selected from the group consisting of isothiazoline, glutaraldehyde, dibromonitrilopropionamide, carbamate, quaternary ammonium compounds, sodium hypochlorite, chlorine dioxide, peracetic acid, ozone, chloramines, bromine-sulfonates, bromine-chlorine-dimethyl hydantoin, dichloro-dimethyl hydantoin, monochloramine, sodium hypochlorite used in combination with ammonium salts and stabilizers including dimethyl hydantoin amino acids, cyanuric acid, succinimide and urea, and combinations thereof, when only vegetative cells are detected.
21. The method of claim 18, further comprising applying the antimicrobial treatment to the hard surface, liquid, or paper product.
22. The method of any one of claims 1 to 21, wherein the presence or absence of bacterial spores in the sample can be determined within 8 hours of preparing the sample and spores are distinguished from vegetative cells.
Background
Endospores are dormant, tough, non-reproductive structures produced by specific bacterial species in the firmicutes phylum. When a bacterial cell is stressed or starved of nutrients, an endospore or spore is produced. Endospores have a very low metabolic rate and therefore cannot be detected by methods typically used for rapid detection of vegetative bacterial cells. Further, spores are extremely difficult to kill as they evolve to survive harsh conditions such as UV, heat, disinfectants, dryness, and starvation. Upon exposure to favorable conditions and nutrients, the spores germinate to produce vegetative cells.
Spore-forming bacteria are problematic because they cause human and animal diseases, deterioration of foods and beverages, and persistence of biofilms. Spore-forming bacterial strains of particular interest are those of the genera Bacillus and Clostridium. Both are gram-positive, rod-shaped bacteria, which include species that are harmful to humans. Anthrax toxin is produced by bacillus anthracis, and food poisoning is caused by bacillus cereus. Clostridium botulinum causes botulism (also known as botulinum toxin), clostridium difficile causes diarrhea, clostridium perfringens causes food poisoning, and clostridium tetani causes tetanus. Bacillus cereus is one of the most problematic bacteria because it has been identified as being more resistant to germicidal chemicals used to decontaminate environmental surfaces.
Bacillus cereus is often diagnosed as the cause of gastrointestinal disorders and has been recognized as the cause of the onset of a variety of food-borne diseases. Bacillus cereus is easy to survive in the environment because of its rapid sporulation ability. Such bacteria can directly and indirectly contaminate food products. Bacillus cereus can directly contaminate raw milk via feces and soil and can survive the intestinal tract and pasteurization process of cattle. The presence of bacillus cereus spores in liquid and food packaging can indirectly contaminate. Spores present in materials that come into direct contact with food products can migrate into the food product, causing spoilage.
Given the negative impact of human intake of these bacteria, governmental agencies have enacted standards or guidelines aimed at reducing the presence of spores. Current spore detection methods take 48 hours to complete. The most common method of testing for bacterial spores is electroplating. This two-day delay is impractical for many industries. In the case of product testing, a two-day delay requires the quarantine of a large number of products until the test results are complete. This is a problem, for example, in the paper or board industry.
Likewise, in testing food and beverage processing equipment, the equipment is only periodically removed for cleaning and cannot be kept offline for two days in practice. While awaiting a test result to diagnostically identify the presence of spores on the surface of a ward after a patient who has been infected with Clostridium had been discharged from the hospital, the hospital could not leave the ward empty for two days. It is also impractical to maintain the quantity of food or water under quarantine, particularly where the water or liquid is constantly changing during waiting (e.g., cooling tower water, beverages, milk during milk processing) where the food may deteriorate.
The present disclosure has been made in this context.
Disclosure of Invention
The present invention relates generally to methods for rapidly detecting bacterial spores and distinguishing them from vegetative cells.
In one aspect, the invention provides a method of detecting bacterial spores in a sample and distinguishing them from vegetative cells and other microorganisms. Samples were prepared for testing for the presence of bacterial spores. A baseline level of metabolic activity of the microorganism in the sample is measured. The sample is incubated under conditions that initiate germination, and the level of subsequent metabolic activity of the microorganism in the sample is monitored. After incubation for about 5 to about 8 hours, the samples are tested for the presence of a burst of microbial metabolic activity.
In some aspects, the method of detecting bacterial spores in a sample and distinguishing them from vegetative cells and other microorganisms includes additional steps. In some embodiments, preparing the sample comprises collecting a sample comprising an unknown amount of spores, vegetative cells, or both spores and vegetative cells. The sample is disintegrated and placed in a tray or container.
In some aspects, the coating is applied from a hard surface, liquid, slurry (starch, carbonate, clay, or TiO)2) Or paperSamples were collected from the articles. In some embodiments, samples are collected from hard surfaces, such as food and beverage processing equipment, pipes, tanks, evaporators, nozzles, dairy processing equipment, milk storage tanks, milk carts, milking equipment, countertops, cooking appliance surfaces, bathroom surfaces (such as washbowl and toilet handles), light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient fences, grab bars, surgical instruments, equipment within a paper mill (including pipes, tanks, headbox, cutoff towers, economizer blades, and forming nets). In other embodiments, the sample is taken from a liquid, such as process water, feed water, cooling tower water, treated and untreated wastewater, paper stock, thin and thick stock, white water, suction box water, tray water, fruit and vegetable sink water, protein process water, hydroponic or seafood culture water, and agricultural water. In other embodiments, the sample is taken from a paper product such as a finished paper product and a finished board product for food contact and non-food contact grades; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers; a food can; aluminum and PET beverage containers; bags, films and modified atmosphere packaging.
In some embodiments, the vegetative cells in the sample are inactivated. In such embodiments, the vegetative cells can be inactivated by heating the sample at a temperature of about 80 ℃ to about 110 ℃ for about 5 to 15 minutes.
In some embodiments, the metabolic activity of the microorganism is determined by measuring Adenosine Triphosphate (ATP). In such embodiments, ATP may be measured by adding luciferase and luciferin to a sample and measuring light emission in Relative Light Units (RLU). ATP can also be measured by HPLC. In other embodiments, the metabolic activity of the microorganism is detected by a metabolic dye.
In some embodiments, incubating the sample involves providing a nutrient liquid medium and a germination enhancer to the sample. Optionally, a biocide neutralizing agent is added. Incubating the sample at a temperature of about 30 ℃ to about 45 ℃. In some aspects, the nutrient broth is a 2X nutrient broth and the germination enhancer is L-alanine.
In some aspects, the microbial metabolic activity of a sample is monitored by adding luciferase and luciferin to the sample and measuring light emission at multiple time points during an 8 hour incubation. In some embodiments, the measurement occurs once per hour.
In some aspects, after incubation, an outbreak of microbial metabolic activity is detected when at least 10-fold higher microbial metabolic activity is measured in the sample than the initial measurement of the sample. Detection of this burst results in a determination that spores are present in the sample. In some embodiments, the method can determine the presence or absence of bacterial spores in a sample within 8 hours of preparing the sample, and distinguish the spores from vegetative cells.
In some aspects, the method further comprises selecting the antimicrobial treatment based on the presence or absence of spores, vegetative cells, or both spores and vegetative cells. In this aspect, the antimicrobial treatment may be selected from the group consisting of chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, peracids, UV, extreme heat, and radiation when spores are detected. When only vegetative cells are detected, the antimicrobial treatment is selected from the group consisting of isothiazoline, glutaraldehyde, dibromonitrilopropionamide, carbamate, quaternary ammonium compounds, sodium hypochlorite, chlorine dioxide, peracetic acid, ozone, chloramine, bromine-sulfonate, bromine-chlorine-dimethyl hydantoin, dichloro-dimethyl hydantoin, monochloramine, sodium hypochlorite used in combination with ammonium salts and stabilizers including dimethyl hydantoin amino acids, cyanuric acid, succinimide and urea, and combinations thereof. In some embodiments, the method further comprises applying the selected antimicrobial treatment to a hard surface, a liquid, or a paper product.
In one embodiment, a method of detecting bacterial spores and distinguishing them from vegetative cells and other microorganisms begins by taking a sample from a hard surface, liquid, or paper product that has an unknown amount of vegetative cells and spores. The samples can be taken from hard surfaces (such as food and beverage processing equipment, plumbing, storage tanks, evaporators, spray nozzles, dairy processing equipment, milk storage tanks, milk carts, milking equipment, countertops, cooking appliance surfaces, bathroom surfaces (such as washbowl and toilet handles), light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient fences, grab bars, surgical instruments, equipment within a paper mill (including plumbing, tanks, headbox, cutoff towers, economizer blades, and forming nets), samples can also be taken from liquids, such as process water, influent water, cooling tower water, treated and untreated wastewater, paper stock, thin stock and thick stock, white water, suction tank water, tray water, fruit and vegetable sink water, protein process water, seafood or aquaculture water, and agricultural water A paper product of the article; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers; a food can; aluminum and PET beverage containers; bags, films and modified atmosphere packaging. The vegetative cells in the sample are inactivated by heating the sample at a temperature of about 80 ℃ to about 110 ℃, about 85 ℃ to about 105 ℃, or about 90 ℃ to about 100 ℃ for about 5 to 15 minutes, 7 to 13 minutes, or 9 to 11 minutes. The baseline level of metabolic activity is determined by measuring the level of ATP in the sample. ATP levels were measured by adding luciferase and luciferin to the sample and measuring light emission with RLU. The sample is incubated with 2X nutrient broth and an L-alanine germination enhancer at a temperature of about 30 ℃ to about 45 ℃ until germination is initiated. The microbial metabolic activity of the samples was monitored by adding luciferase and luciferin to the samples and measuring light emission at various time points during 8 hours incubation. After about 5 to about 8 hours of incubation, the presence of a burst of metabolic activity of the microorganism that is at least 10-fold higher than the baseline level indicates the presence of spores. The absence of a burst of microbial metabolic activity indicates the absence of spores. If spores are detected, an antimicrobial treatment is selected to treat the spores and apply them to a hard surface, liquid, or paper product. If no spores are detected, an antimicrobial treatment is selected to treat the vegetative cells and apply them to a hard surface, liquid or paper product.
Drawings
FIG. 1 is a graph of ATP production over time in non-heat treated, non-germination enhancing agent, and non-nutrient industrial water samples.
FIG. 2 is a graph of ATP production over time in an industrial water sample without heat treatment, with germination enhancers, and without nutrients.
FIG. 3 is a graph of ATP production over time in an industrial water sample without heat treatment, with germination enhancers, and with nutrients.
FIG. 4A is a graph of ATP (expressed as RLU) production over time in a sample of industrial water with heat treatment, with germination enhancers, and with nutrients.
FIG. 4B is a graph of ATP production (expressed as the quotient of RLU production over time) in a sample of industrial water with heat treatment, with germination enhancer, and with nutrients.
FIG. 5 is a time course graph of ATP production in 7 industrial water samples that were heat treated, with germination enhancers, and with nutrients.
Detailed Description
The present disclosure relates to methods of detecting bacterial spores. In particular, embodiments relate to a method of rapidly distinguishing the presence of bacterial spores from the presence of other microorganisms and determining the source of spore contamination. In addition, the present disclosure distinguishes between the vegetative state and the spore state of spore forming bacteria.
The following definitions are provided to determine how to interpret the terms used in this application, and in particular how to interpret the claims. The organization of these definitions is for convenience only and is not intended to limit any definition to any particular category.
As used herein, the term "bacterial spore" or "endospore" refers to a structure produced by some species of bacteria (such as bacillus and clostridium species). Spores are capable of keeping bacteria dormant under harsh conditions such as extreme temperatures, drought, and chemical treatments.
As used herein, the term "germination" refers to the growth of vegetative cells from a dormant bacterial spore. Germination occurs when spores are exposed to conditions favorable for vegetative cell growth.
As used herein, the term "vegetative bacterial cell" refers to a bacterial cell that is actively growing, exhibits metabolic activity, and divides.
As used herein, the term "biocide" refers to a chemical substance or microorganism that is intended to destroy or neutralize any harmful microorganism by chemical or biological means. Biocides can include preservatives, insecticides, disinfectants and pesticides, which are used to control organisms that are harmful to human or animal health or cause damage to natural or manufactured goods.
As used herein, the term "sporicide" refers to a physical or chemical agent or process having the ability to cause a reduction of more than 90% (1-log reduction) of the spore population of Bacillus cereus or Bacillus subtilis. Preferably, the sporicidal compositions of the present disclosure provide a reduction of greater than 99% (2-log reduction), more preferably greater than 99.99% (4-log reduction), and most preferably greater than 99.999% (5-log reduction).
The term "quarantine" refers to the isolation and isolation of objects that may or may not be contaminated or infected by microorganisms.
As used herein, the term "rapid detection" refers to a method of detecting bacteria and bacterial spores in less than 48 hours. Preferably, "rapid detection" means detection of bacteria in less than 24 hours. Most preferably, "rapid detection" means detection of bacteria in less than 12 hours.
As used herein, the term "process water" is water used in connection with technical plants and production processes. Process water is considered non-potable and is used to facilitate the manufacturing process.
The term "adenosine triphosphate" (ATP) refers to a molecule used to transport chemical energy within a cell. ATP comprises adenine, ribose and three phosphate groups. ATP decomposes into Adenosine Diphosphate (ADP) and phosphate to release energy.
As used herein, the term "microorganism" refers to a single-or multi-celled microscopic organism. These organisms may include bacteria, viruses, fungi and algae.
As used herein, the term "metabolic activity" refers to a chemical reaction that occurs in a living body.
As used herein, the term "bioluminescence" refers to the light produced and emitted by a living organism. Luciferase catalyses the oxidation of luciferin, producing light.
As used herein, the term "high performance liquid chromatography" (HPLC) refers to analytical chemistry techniques for separating, identifying, and quantifying each component in a mixture.
As used herein, the term "cation" refers to a positively charged ion.
As used herein, the term "dormant" refers to an organism that has a normal physical function of being suspended for a period of time.
As used herein, the term "colony forming unit" (CFU) refers to an estimate of the number of viable bacterial or fungal cells in a sample. Viable cells are capable of propagating under controlled conditions. CFU is provided as a measure of CFU/mL for liquids or CFU/g for solids.
As used herein, the term "relative light unit" (RLU) refers to the amount of light measured by a photometer.
As used herein, the term "germination conditions" refers to conditions that favor the activation, germination, and growth of bacterial spores (endospores).
In various industries, it is important to detect the presence of bacteria and bacterial spores. When human health is involved, the guidelines specify the maximum amount of bacteria that may be present. For example, "dairy commercial standards" provide requirements for the number of Colony Forming Units (CFU) that may be present per gram of paper or paperboard used for dairy products. Samples are cut from the paper or paperboard to be tested and placed in a sealed envelope. The samples were then cut into small cubes and stored in a sterile blender. Sterile phosphate dilution water was added to the shredded paper sample in the blender to help disintegrate the sample. Immediately after disintegration, the samples were transferred to one or more petri dishes. The melted agar was poured onto the sample in a petri dish and allowed to solidify. After coagulation, the plates were incubated at 36 ℃ for 48 hours. After incubation, the plates were checked for the presence and number of CFU's by colony counter.
Industry guidelines limit the number of Colony Forming Units (CFU) present on paper or paperboard products used with dairy products to less than 250 per gram. Some end users may require more or less strict compliance (<100- >1000 CFU/g). In order to comply with the spore guidelines of the dairy, food and healthcare industries, it is important to detect and properly treat bacteria that can form spores while they are in the most fragile, vegetative state.
Spores consist of a number of protective layers that make them resistant to oxidation and chemical attack. Killing spores requires higher biocide doses than vegetative cells. It is always more effective to apply biocides to active vegetative cells. Spore control programs must be strong enough to erode cells in a vegetative state and prevent sporulation. The dose must be high enough to kill vegetative cells before they develop into spores.
One method of detecting microorganisms relies on measuring the activity of the microorganisms. The activity of the microorganism can be measured using Adenosine Triphosphate (ATP) concentration as an activity index. The activity of microorganisms can also be measured using metabolic dyes including redox dyes (e.g., Resazurin and 2- (p-iodophenyl) -3- (p-nitrophenyl) 5-phenyltetrazolium chloride (INT)), fluorescent redox dyes (e.g., 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC)), and indicators of enzyme activity (e.g., carboxyacetic acid diacetate).
Adenosine Triphosphate (ATP) measurements have been used to detect microorganisms in various industries. Cells use ATP as an energy source and are an indicator of metabolic activity. ATP can be measured in vegetative bacteria, but the bacterial spores contain little or no ATP.
Typically, ATP levels are measured by a bioluminescent assay involving reaction with a luciferase quantified with a luminometer. Other methods include colorimetric or fluorometric assays using phosphorylation of glycerol and High Performance Liquid Chromatography (HPLC).
In the method using bioluminescence, luciferase and luciferin from firefly are mixed with a sample having a cation (such as magnesium) in the presence of oxygen. If ATP is present, a reaction between luciferase (substrate) and luciferin (catalyst) will be induced in an oxidation reaction that generates light. Light emission was measured with a photometer and reported in Relative Light Units (RLU). The amount of light produced is directly proportional to the metabolic activity of the microorganisms present, but does not indicate the number of organisms present. Luciferase/luciferin reactions are well known in the art and there is a need for commercial sources of reagents and protocols for their use. For example, various luciferase/luciferin reagents along with luciferase are available from commercial kits such as Promega Corp (Promega Corp.) (madison, wisconsin) and ultracellulomine technologies ltd (lumine) (fradriton, new-florelix). Commercially available luciferases include firefly luciferase (Photinus pyralis, "Ppy luciferase"). Purified methoprene is also commercially available from Promega.
According to the present disclosure, when spores are placed into a nutrient liquid medium containing a germination enhancer and incubated, the spores will germinate into vegetative bacteria. During germination, ATP production will increase rapidly and measurement of the ATP activity peak will provide an indication that there is spore germination. Furthermore, depending on the concentration of spores in the sample, the process takes place within a relatively short time of incubation of about 5 to 8 hours. Optionally, samples containing high levels of vegetative cells exhibit high ATP values early in the testing protocol, typically within the first hour. ATP in the sample may decrease as the incubation is extended due to the presence of residual biocide. The use of a biocide neutralization solution may be utilized.
Germination of spores can be directly observed by measuring the initial ATP in the sample before addition of the nutrients and germination enhancers, and then measuring ATP periodically over the determined incubation time. During this time, vegetative cells produce a steady level of ATP, while spores exhibit an explosion in ATP production, which can be measured in the assay for 5 to 8 hours, depending on the concentration of spores present in the sample. Spore concentrations in the range of 100-200CFU/mL will produce an ATP burst between 7 and 8 hours, while high spore concentrations at 1,000-100,000CFU/mL will show an ATP burst at about 5 hours.
In some samples, high levels of vegetative bacterial cells may obscure the ATP readings of the germinating spores. To alleviate this, an optional heating step of 10 to 15 minutes at 80 to 95 ℃ may be included at the start of the assay. This heating step inactivates the vegetative cells, thereby eliminating their effect on ATP levels. Meanwhile, spores can survive at high temperature in a dormant state. After addition of nutrients and germination enhancers, the spores will then produce an ATP burst (if present).
In order to implement such a method for the rapid detection of bacterial spores in an industrial process, it is necessary to first prepare a sample which is to be tested for the presence of bacterial spores. The sample may be collected from any source that may be contaminated with bacterial spores. The sample may be contaminated with bacterial spores, with vegetative bacterial cells, or uncontaminated with both bacterial spores and vegetative bacterial cells, or contaminated with both bacterial spores and vegetative bacterial cells. The sample may be taken from a hard surface, slurry (starch, carbonate, clay or TiO)2) Raw fiber, food or beverage product, liquid or packaging or cardboard product samples.
Non-limiting examples of facilities having hard surfaces include food and beverage plants, dairy plants, farms and dairy plants, breweries, ethanol plants, full-service and fast-food restaurants, grocery stores, warehouses and retail stores, commercial or office spaces, hotels, motels, hospitals, paper mills, industrial manufacturing plants (including automotive plants), cooling towers, water treatment plants, oil and gas plants and pipelines, and drilling platforms. Examples of hard surfaces include food and beverage processing equipment (including pipes, tanks, evaporators, spouts, etc.), dairy processing equipment, milk tanks, milk carts, milking equipment, countertops, cooking utensil surfaces, bathroom surfaces (such as washbowl and toilet handles), light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient pens, grab bars, surgical instruments, equipment within a paper mill (including pipes, tanks, headbox, shut off towers, economizer blades, forming nets, etc.).
Samples on hard surfaces may be taken from sterile swabs or other sterile devices that may be used to collect microorganisms from hard surfaces, such as: medical adhesive tape, cotton cloth, cellulose cloth, etc. If the surface is dry, a solvent may be applied with the swab to dissolve any bacterial residue that may be present and the residue suspended in the solvent for testing. Examples of solvents may include, but are not limited to: sterile water, sterile phosphate buffer, sterile Tris EDTA (TE) buffer, dilute detergent solutions (such as tween), and the like.
Non-limiting examples of liquids include process water, feed water, cooling tower water, treated and untreated wastewater, paper stock (thin and thick stock), white water, suction box water, tray water, fruit and vegetable sink water, protein (e.g., poultry, pork, red meat, seafood) process water, hydroponic or seafood culture water, and agricultural water. The liquid sample may be aliquoted from a potential source of contamination into sterile containers.
Non-limiting examples of food and beverage products include milk, beer, wine, drinking water, fruits and vegetables, proteins (such as poultry, pork, red meat or seafood), ready-to-eat meat, cheese, prepared foods, frozen foods, ice cream.
Non-limiting examples of packaging and products include: paper products (such as finished paper products and finished board products for food contact and non-food contact grades); drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers (e.g., yogurt containers, milk containers, deli containers); a food can; an aluminum or PET beverage container; bag or film or modified atmosphere package. The product may be tested before it leaves the manufacturing plant, for example at a paper mill, or at a point of use, such as at a food or beverage plant. Paper product samples can be tested according to the procedure described in dairy commercial standards (TAPPI test method T449 bacteriological examination of paper and paperboard, or microbiological examination of ISO8784 pulp and board).
According to some embodiments, the sample is subjected to an optional initial ATP measurement. As described above, the sample may be bound to luciferase and luciferin together with cation, oxygen and tris buffer or the like. The light produced by the reaction is measured in Relative Light Units (RLU). The measurement can be made with a photometer. Optionally, initial ATP measurements may be performed by HPLC. In some embodiments, the vegetative cells can be inactivated with an optional heating step. The vegetative cells can be inactivated by heating the sample for about 5 to about 15 minutes, about 7 to about 13 minutes, or about 9 to about 11 minutes. The cells can be heated at a temperature of about 80 ℃ to about 110 ℃, about 85 ℃ to about 105 ℃, or about 90 ℃ to about 100 ℃. Preferably, the sample is heated at a temperature of about 95 ℃ for 10 minutes. Heating the sample to 80 ℃ is a well-established method of eliminating vegetative cells from the sample, however, laboratory tests have shown that some vegetative cells survive at this level of heat.
In some embodiments, the sample is incubated under conditions that induce germination of the bacterial spores. This generally involves providing at least the spores with nutrients and appropriate temperature conditions. A nutrient broth including an optional germination enhancer may be added to the sample and incubated at a temperature of about 30 ℃ to about 45 ℃, about 35 ℃ to about 40 ℃, or about 36 ℃ to about 38 ℃ effective to initiate spore germination. Examples of commercially available nutrient broth include nutrient broth, tryptic soy broth, lysogenic broth, Luria broth, beef extract broth, and masterbatches. Effective germination enhancers include L-alanine, inosine, glucose, amino acids, and potassium bromide. Preferably, the germination enhancer is L-alanine. In one embodiment, the sample is incubated at 37 ℃ to induce spore germination.
ATP levels were monitored over time by taking ATP readings at specified time intervals while the samples were incubated. The light emission was measured with a photometer at intervals throughout the incubation period. A new sample is taken at each time interval and combined with the ATP reagent to generate each luminescence reading. ATP measurements may be collected every 2 hours, every 1 hour, every 30 minutes, every 15 minutes, or every 5 minutes. Preferably, ATP measurements are taken at least once every hour over a period of 5 to 8 hours. Optionally, the ATP level in the sample can be measured using HPLC. HPLC measurements can also be taken at least once every hour to monitor ATP levels. The same procedure can be used with dye treatments to detect microbial activity based on redox changes or metabolic activity. Visual evidence of a color change of the sample can be checked spectrophotometrically or by measuring fluorescence.
As discussed above, when a spore germinates, it produces an ATP burst due to an increase in metabolic activity. This burst of ATP production can be observed at different time points during the incubation, depending on the concentration of spores present, but will typically occur between 5 and 8 hours after the start of the incubation. Spore concentrations in the range of 100-200CFU/mL (colony forming units per mL) will produce ATP bursts between 7 and 8 hours, while high spore concentrations at 1,000-100,000CFU/mL will show signals around 5 hours. If vegetative cells are present in the sample (and not heat inactivated), a steady level of ATP production will be detected throughout the incubation period. Typical measurements of vegetative cells may range from about 5,000 to 200,000 RLU. However, germination of bacterial spores will exhibit higher amounts of ATP, with RLU measurements at least 500-fold higher than the initial measurements. Depending on the spore concentration present in the sample, a typical ATP burst can be measured in the range of 10 to 500 times higher RLU than the initial measurement. Thus, the presence of an ATP burst indicates the presence of spores in the sample, while the absence of an ATP burst indicates the absence of spores in the sample. If no ATP is measured in the sample, no microorganisms are present at all.
Once the presence of spores in the sample has been determined, an antimicrobial treatment is selected. If spores are detected, the process can be selected from: hypochlorite, chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, monochloramine, peracetic acid, 2-dibromo-3-nitrosopropionamide (DBNPA), 5-Dimethylhydantoin (DMH), alcohols, peracids, and combinations thereof.
In paper mills, sodium hypochlorite (bleach) is effective in processes and water systems with pH between 6.0 and 7.5 and requires a contact time of at least 20 minutes. In the effective pH range, most of the chlorine is present in the form of hypochlorous acid, which is easily consumed by the oxidizer requirements. In high demand systems, high doses may need to be applied to achieve the desired target residual. Free chlorine is effective against spores when the target residue is reached at doses between 0.4 and 0.5 ppm. Residual free chlorine above 0.5ppm can negatively impact machine efficiency as it can oxidize dyes, OBA, felt and machine surfaces. Therefore, good control of the feed system is recommended to minimize the residue required on the wet end. Sodium hypochlorite can be continuously applied to the mixing tank. For a flow break tank, sodium hypochlorite must be applied in combination with a non-oxidizing biocide (such as glutaraldehyde).
Chlorine dioxide is also effective in
Monochloramine (MCA), available and registered as a biocide, is effective in systems with pH values of 7 to 9 and is less likely to be consumed by the demand for oxidants than bleach. MCA is produced by mixing a source of ammonia with chlorine under controlled conditions. This is usually generated in situ by reacting diluted sodium hypochlorite with an ammonium sulphate solution at a pH of 9 to 10 to produce a final MCA concentration of 3,000 to 6,000 ppm. MCA is not a good sporicide, but it may be very effective in controlling a broad spectrum of bacteria including spore forming bacteria in a vegetative state. It is critical to maintain consistent biological control in the feed system and the influent water. MCA does not respond rapidly to process abnormalities and therefore replenishment with glutaraldehyde is recommended. In the paper mill, a stock tank with good mixing is the ideal feed point for the MCA. MCA may be injected into the white water, but the residual concentration should be maintained between 2 and 3ppm to minimize the potential for corrosion. Good control of the feed system and water intake will minimize the treatment required on the wet end and improve the compatibility of the machine.
Peracetic acid is delivered at equilibrium in the form of a solution of peracetic acid (PAA), hydrogen peroxide, and acetic acid. While high doses of hydrogen peroxide are effective in killing spores, PAA is considered to be the major active ingredient in equilibrium solutions. PAA will not lead to the formation of AOX and can be used where local regulations limit the use of chlorine. PAA is most effective in systems with pH values between 5 and 7, possibly requiring higher doses to achieve the effect of pH above 7. Although considered a strong oxidant, PAA may take longer contact times than bleach or chlorine dioxide and require higher dosages. PAA can be used in combination with chlorine dioxide but can neutralize the hypochlorite program. If the feed points are carefully considered to avoid mixing of the two, then PAA and hypochlorite can only be used to treat the same machine. PAA is most effective when complementary to DBNPA (2,2 dibromo-3-nitrotripropyl acrylamide) in the same process stream. The target concentration for PAA was 5.0ppm for 1 hour of contact time and 15ppm for 20 to 60 minutes of contact time.
Chlorine is stabilized with non-biocidal precursor chemicals such as dimethylhydantoin, urea, sulfamic acid, sodium sulfamate, ammonium carbamate, ammonium sulfate, ammonium chloride, and ammonium bromide. For spore control with DMH stabilized chlorine, it is recommended to use Cl2DMH (5, 5-dimethylhydantoin) is a balance between durability and fast kill in the case of 4: 1.
Typically, a non-oxidant is required as a supplement to the oxidant program. Glutaraldehyde has an effect on both the spore-forming bacteria and the vegetative cells of the bacterial spores. It is effective in systems with a pH in the range of 6.0 to 9.0.
Non-chemical treatments like extreme heat and UV radiation may also be used. Targeting spore-forming bacteria expands the list of effective biocides to include DBNPA, isothiazolines, quaternary amines, etc., when they are in their vegetative state. Once the appropriate treatment has been selected, it is applied to the part of the process where the spore-forming bacteria are present in a vegetative state when no spores are detected. This may include hard surfaces, food or beverage products, liquids, packaging, or paper products at the point of production where contamination has been detected. Since the process is completed in no more than 8 hours, bacterial contamination can be rapidly eliminated, thereby preventing downstream sporulation. In addition, the equipment can be disposed of while offline for cleaning, or the product can be unscrambled for multiple shifts, and production problems can be corrected more quickly. After application of the treatment, the next round of testing should be performed to ensure that spores have been eliminated. The ability to work with the right chemistry in the right place in production can save cost and time.
In order to more fully understand the present disclosure, the following examples are given to illustrate some embodiments. These examples and experiments are to be understood as illustrative and not restrictive. All parts are by weight unless otherwise indicated.
Examples of the invention
Example 1: comparison of ATP production in a mixed population of vegetative cells and spores under various conditions.
ATP levels in samples containing a mixture of vegetative cells and spores were measured using the lumineultra quenchgon 21 industrial ATP kit. A sample of industrial water from a paper mill was determined to have a mixture of vegetative cells and bacteria. Confirmation and quantification of the two bacterial forms was determined by plating on R2A agar. ATP production was measured over the course of 6 hours under various conditions.
The time course of ATP production in the absence of a heating step to eliminate vegetative bacteria, germination enhancers, and nutrients is similar to that shown in fig. 1. In the absence of the heating step and nutrients but in the presence of the germination enhancer, the time course for ATP production is similar to that shown in fig. 2. The time course of ATP production in the presence of germination enhancer and nutrients but in the absence of a heating step is similar to that shown in fig. 3. ATP production increased steadily within 6 hours in the presence of nutrients and germination enhancers. When a heating step is included prior to addition of germination enhancer and nutrients, the ATP production time course is similar to that shown in fig. 4a (RLU) and fig. 4B (quotient of RLU production over time).
ATP produced by the vegetative cells was removed by thermal inactivation, and only ATP produced by the spores was observed. Addition of nutrients and germination enhancers accelerates germination of spores and also promotes production of ATP in vegetative cells. For eliminating ATP produced by vegetative cells, a heat inactivation step is necessary in order to be able to detect ATP produced by germinating spores. The data were plotted as time-varying RLU manufacturers to facilitate easy comparison of multiple samples.
Example 2: detection of spores in process water samples.
Seven process water samples were collected from a paper mill having two paper machines. Each sample had an unknown amount of vegetative cells and spores. The samples were heat treated prior to the addition of nutrients and germination enhancers. ATP production was measured hourly over 6 hours using the lumineutra queenchgone 21 industrial ATP kit (fig. 5).
Spores were determined to be present in three of the seven samples: seal
The above specification, examples and data provide a complete description of the manufacture and use of the composition. Since many embodiments of the invention can be made without departing from the spirit and scope of the disclosure, the invention resides in the claims hereinafter appended.
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