阅读说明：本技术 用于检测细菌孢子的快速方法 (Rapid method for detecting bacterial spores ) 是由 涂文利 陈跖 于 2017-07-12 设计创作，主要内容包括：本发明提供了通过测量二磷酸腺苷(ATP)的经时产生,用于检测细菌孢子的存在的方法。通过将单磷酸腺苷(AMP)和二磷酸腺苷(ADP)转换为ATP来检测孢子。(The present invention provides methods for detecting the presence of bacterial spores by measuring the production of adenosine diphosphate (ATP) over time. Spores were detected by converting Adenosine Monophosphate (AMP) and Adenosine Diphosphate (ADP) to ATP.)

1. A method of detecting and distinguishing bacterial spores from vegetative cells and other microorganisms in a sample, the method comprising:

preparing a sample to be tested for the presence of bacterial spores;

measuring an initial amount of Adenosine Triphosphate (ATP);

enriching the sample;

extracting Adenosine Monophosphate (AMP) and Adenosine Diphosphate (ADP) from the sample;

converting AMP and ADP to ATP with an enzyme; and

measuring a final amount of ATP, wherein an increase in ATP concentration from the initial ATP measurement to the converted ATP measurement indicates the presence of spores.

2. The method of claim 1, further comprising isolating spores by heating the sample at about 80 ℃ for about 5 to 60 minutes or at about 100 ℃ for about 5 to 30 minutes.

3. The method of claim 1 or claim 2, wherein the preparing comprises:

collecting a sample containing an unknown number of spores, vegetative cells, or both spores and vegetative cells; and

if solid, the sample is disintegrated.

4. The method of claim 3, wherein the sample is collected from a hard surface, a liquid, or a paper product.

5. The method of claim 4, wherein the hard surface is selected from the group consisting of: food and beverage processing equipment, pipes, tanks, evaporators, nozzles, dairy processing equipment, milk tanks, milk carts, milking equipment, countertops, cooking surfaces, bathroom surfaces such as sinks and toilet handles, light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient guardrails, grab bars, surgical instruments, equipment in a paper mill including pipes, cabinets, head boxes, broke towers, reclaimer blades, and forming lines.

6. The method of claim 4, wherein the liquid is selected from the group consisting of: process water, feed water, cooling tower water, treated and untreated wastewater, paper furnish (thin and thick stock), white water, suction box water, tray water, fruit and vegetable sink water, protein processing water, hydroponic or seafood farming water, and agricultural water.

7. The method of claim 4, wherein the paper product is selected from the group consisting of: paper products for food contact and non-food contact grades, such as finished paper products and finished paperboard products; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers; food can; an aluminum or PET beverage container; bag or film or modified atmosphere package.

8. The method of any one of claims 1 to 7, wherein the enriching comprises increasing the concentration of microorganisms in the sample by centrifugation, glass beads, magic beads, or filtration.

9. The method of any one of claims 1 to 8, wherein the extracting comprises heat treating the sample with a solvent, an acid, or.

10. The method of claim 9, wherein the extracting further comprises treating the sample with a detergent or surfactant.

11. The method of any one of claims 1 to 9, wherein the spores are extracted using a temperature of 65 ℃ or greater for 1 to 10 minutes.

12. The method of any of claims 1-3 and 8-10, wherein the converting comprises:

treating the sample with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP;

treating the sample with myokinase to convert ATP and AMP to ADP; and

treating the sample with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP.

13. The method of any one of claims 1-3 and 8-11, wherein the adenine nucleotide is converted to ATP from pyruvate kinase, myokinase, and phosphoenolpyruvate within about 10 minutes to 24 hours.

14. The method of any of claims 1-3 and 8-12, wherein the measuring comprises:

adding luciferase and luciferin to the sample; and

light emission was measured in Relative Light Units (RLU).

15. The method of any one of claims 1-3 and 8-12, wherein the measuring comprises measuring ATP levels by HPLC.

16. The method of any one of claims 1-3 and 8-15, further comprising selecting an antimicrobial treatment based on the presence or absence of spores, vegetative cells, or both spores and vegetative cells.

17. The method of claim 16, wherein the antimicrobial treatment is selected from chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, peracids, UV, extreme heat, radiation when spores are detected.

18. The method of claim 16, wherein when only vegetative cells are detected, the antimicrobial treatment is selected from the group consisting of: an isothiazoline; glutaraldehyde; dibromonitrilopropionamide; a carbamate; a quaternary ammonium compound; sodium hypochlorite; chlorine dioxide; peroxyacetic acid; ozone; chloramine; bromo sulfamate; bromo-chloro-dimethylhydantoin; dichlorodimethyl hydantoin; monochloramine, sodium hypochlorite used in combination with an ammonium salt and stabilizers including dimethylhydantoin amino acids, cyanuric acid, succinimide, urea; and combinations thereof.

19. The method of claim 16, further comprising applying the antimicrobial treatment to the hard surface, liquid, or paper product.

20. The method of any one of claims 1-19, wherein the concentration of spores in the sample can be determined within 2 to 8 hours of sample collection.

Background

Endospores are dormant, tough, non-reproductive structures produced by specific bacterial species in the Firmicute (Firmicute). When a bacterial cell in a vegetative state is exposed to stress or lacks nutrition, an endospore or spore is produced. Endospores have a very low metabolic rate and therefore cannot be detected by the methods commonly used for rapid detection of vegetative bacterial cells. Further, spores are extremely difficult to kill because they are designed to survive harsh conditions, such as UV, heat, disinfectants, drying, and starvation. After exposure to favorable conditions and nutrients, the spores germinate to produce vegetative cells.

Spore-forming bacteria are problematic because they cause disease in humans and animals, spoilage in foods and beverages, and promote the perpetuation 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 harmful to humans. Anthrax toxin is produced by bacillus anthracis (b.anthracosis), and food poisoning is caused by bacillus cereus (b.cereus). Clostridium botulinum (c.botulinum) causes botulism (also known as botulinum toxin), clostridium difficile (c.difficile) causes diarrhea, clostridium perfringens (c.perfringens) causes food poisoning, and clostridium tetani (c.tetani) causes tetanus. Bacillus, Paenibacillus (Paenibacillus) and Brevibacillus (Brevibacillus) bacteria can cause problems in food packaging board products. Bacillus cereus is one of the most problematic bacteria because it has been identified as being increasingly resistant to germicidal chemicals used to decontaminate environmental surfaces.

Bacillus cereus is often diagnosed as the cause of gastrointestinal disorders and has been proposed as the cause of several outbreaks of food-borne diseases. Bacillus cereus is readily viable in the environment due to its rapid sporulation ability. Such bacteria can directly and indirectly contaminate food. Bacillus cereus can directly contaminate raw milk via feces and soil and can survive the intestinal tract and pasteurization process of cattle. Indirect contamination can result from the presence of Bacillus cereus spores in liquid and food packaging. Spores present in materials that come into direct contact with food can cause the spores to migrate into the food, causing spoilage.

In view of the negative impact of these bacteria on human intake, government agencies have established standards or guidelines aimed at reducing the presence of spores. Current spore detection methods require 48 hours to complete. The most common method of testing bacterial spores is plate technology. This two-day delay is impractical for many industries. In the case of product testing, a two-day delay requires isolation 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, when testing food and beverage processing equipment, the equipment is only removed periodically for cleaning and, in fact, cannot be kept offline for two days. Hospitals cannot empty a patient room for two days while waiting for test results designed to identify the presence of spores on surfaces in the patient room after a patient who has been infected with clostridium difficile has been discharged. Moreover, it is impractical to keep a certain amount of food or water isolated, especially where food may spoil while waiting or where water or fluid is constantly changing (e.g., cooling tower water, beverages, milk in a milk process).

Spore forming bacteria can have a negative impact on the quality of the liquid packaging board product and on the machine production. An insufficient diagnosis of spore contamination of liquid packaging board products during production can lead to economic losses.

It is against this background that the present disclosure has been made.

Disclosure of Invention

In general, the present disclosure relates to methods for rapidly detecting and distinguishing bacterial spores from vegetative cells.

In one aspect, the invention features a method of detecting and distinguishing bacterial spores from vegetative cells and other microorganisms in a sample. The method begins with preparing a sample to be tested for the presence of bacterial spores. The sample is enriched and the initial amount of ATP is measured. Adenosine Monophosphate (AMP) and Adenosine Diphosphate (ADP) are extracted from the sample, and the enzyme converts AMP and ADP to ATP. Measuring a final amount of ATP, wherein an increase in ATP concentration from the initial ATP measurement to the converted ATP measurement indicates the presence of spores in the sample.

In some aspects, the method of detecting and distinguishing bacterial spores from vegetative cells and other microorganisms in a sample begins with preparing a sample to be tested for the presence of bacterial spores. The sample is enriched and the initial amount of ATP is measured. AMP and ADP are extracted from the sample and the enzyme converts AMP and ADP to ATP. Measuring a final amount of ATP, wherein an increase in ATP concentration from the initial ATP measurement to the converted ATP measurement indicates the presence of spores in the sample. The method may comprise further steps. In some embodiments, the sample is prepared by collecting a sample containing an unknown number of spores, vegetative cells, or both spores and vegetative cells. If the sample is a solid, it is disintegrated. Samples may be collected from hard surfaces, liquids, or paper products. The hard surface may be food and beverage processing equipment, tubing, tanks, evaporators, nozzles, dairy processing equipment, milk tanks, milk carriers, milking equipment, countertops, cooking surfaces, bathroom surfaces such as sinks and toilet handles, light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient guards, grab bars, surgical instruments, equipment in a paper mill including tubing, cabinets, head boxes, broke towers, recovery equipment (savall) blades, and forming lines. The liquid may be process water, feed water, cooling tower water, treated and untreated wastewater, paper furnish (thin and thick stock), white water, suction box water, tray water, fruit and vegetable sink water, protein processing water, hydroponic or seafood farming water, and agricultural water. Also, the paper products may be paper products for food contact and non-food contact grades, such as finished paper products and finished paperboard products; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers; food can; an aluminum or PET beverage container; bag or film or modified atmosphere package. Isolating spores from the sample by heating the sample at 80 ℃ for about 5-60 minutes, about 10-30 minutes, or about 15-20 minutes; or alternatively, the sample is heated at 100 ℃ for about 5-30 minutes, about 10-30 minutes, or about 15-20 minutes. AMP and ADP are extracted from the sample with a solvent, acid, heat (e.g., 65 ℃ or higher for 1-10 minutes), detergent, or surfactant. Converting AMP and ADP to ATP by: the sample is treated with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP, the sample is treated with myokinase to convert ATP and AMP to ADP, and the sample is treated with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP. Adenine nucleotides are converted to ATP by pyruvate kinase, myokinase and phosphoenolpyruvate within about 10 minutes to 24 hours. By adding luciferase and luciferin to the sample and measuring light emission in Relative Light Units (RLU), or measuring ATP levels in the sample by HPLC. The antimicrobial treatment is selected and applied based on the presence or absence of spores, vegetative cells, or both spores and vegetative cells. If spores are present, the antimicrobial treatment is selected from chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, peracids, UV, extreme heat, and radiation. When vegetative cells are present only, the antimicrobial treatment is selected from: an isothiazoline; glutaraldehyde; dibromonitrilopropionamide; a carbamate; a quaternary ammonium compound; sodium hypochlorite; chlorine dioxide; peroxyacetic acid; ozone; chloramine; bromo sulfamate; bromo-chloro-dimethylhydantoin; dichlorodimethyl hydantoin; monochloramine, sodium hypochlorite used in combination with an ammonium salt and stabilizers including dimethylhydantoin amino acids, cyanuric acid, succinimide, urea; and combinations thereof. In some embodiments, the method further comprises applying the selected antimicrobial treatment to the hard surface, liquid, or paper product. The method can detect spores within 2-8 hours after sample collection.

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 some embodiments, spores are isolated from a sample by heating the sample at about 80 ℃ for about 5 to 60 minutes or at about 100 ℃ for about 5 to 30 minutes. In some embodiments, enriching the sample involves increasing the concentration of the microorganisms in the sample by centrifugation, glass beads, magic beads, or filtration.

In some aspects, AMP and ADP are extracted from the sample using a solvent, acid, heat, detergent, or surfactant. In embodiments where the sample is treated with heat, a temperature of 65 ℃ or greater is applied for 1 to 10 minutes.

In some embodiments, AMP and ADP are converted to ATP by: the sample is treated with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP, the sample is treated with myokinase to convert ATP and AMP to ADP, and the sample is treated with pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP. In some aspects, the adenine nucleotide is converted to ATP by pyruvate kinase, myokinase, and phosphoenolpyruvate within about 10 minutes to 24 hours.

In some aspects, the measuring of ATP levels in the sample is accomplished by adding luciferase and luciferin to the sample, and measuring light emission in Relative Light Units (RLU). In other aspects, ATP levels are measured by HPLC.

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 some embodiments, the antimicrobial treatment is selected from chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, peracids, UV, extreme heat, radiation when spores are detected. In other embodiments, when only vegetative cells are detected, the antimicrobial treatment is selected from: an isothiazoline; glutaraldehyde; dibromonitrilopropionamide; a carbamate; a quaternary ammonium compound; sodium hypochlorite; chlorine dioxide; peroxyacetic acid; ozone; chloramine; bromo sulfamate; bromo-chloro-dimethylhydantoin; dichlorodimethyl hydantoin; monochloramine, sodium hypochlorite used in combination with an ammonium salt and stabilizers including dimethylhydantoin amino acids, cyanuric acid, succinimide, urea; and combinations thereof. In some embodiments, the method further comprises applying the selected antimicrobial treatment to the hard surface, liquid, or paper product.

In some aspects, the method can determine the concentration of spores in the sample within 2 to 8 hours of sample collection.

Detailed Description

The present disclosure relates to methods of detecting bacterial spores. In particular, embodiments relate to methods 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 terms are used in this patent application and in particular how the claims are to be interpreted. The organization of the 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 certain bacterial species, such as bacillus and clostridium species. Spores cause bacteria to remain dormant under harsh conditions, such as extreme temperatures, drought, and chemical treatments.

As used herein, the term "end use spoilage event" refers to when a microorganism has grown for a time sufficient to spoil a product.

As used herein, the term "germination" refers to the growth of vegetative cells from dormant bacterial spores. 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 and dividing.

As used herein, the term "biocide" refers to a chemical substance or microorganism that is expected to destroy or neutralize any pest by chemical or biological means. Biocides may include preservatives, insecticides, disinfectants and pesticides, which are used to control organisms that are harmful to human or animal health or that cause damage to natural or manufactured products. Biocides are antimicrobial agents or chemical compositions that can prevent microbial contamination or spoilage caused by microorganisms.

As used herein, the term "sporicide" refers to any substance used to kill or neutralize bacterial spores. As used in this disclosure, the term "sporicide" refers to a physical or chemical agent or process having the ability to cause greater than a 90% reduction (1-log reduction) in a spore population of bacillus cereus or bacillus subtilis within 10 seconds at 60 ℃. Preferably, the sporicidal compositions of the present disclosure provide a reduction in such population 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) within 10 seconds at 60 ℃.

The term "isolation" refers to the separation and isolation of organisms or objects contaminated or infected with a pathogen.

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" refers to detection of bacteria in less than 12 hours. Most preferably, "rapid detection" refers to detection of bacteria in less than 4 hours.

As used herein, the term "process water" is water used in connection with technical equipment and production processes. Process water is not considered 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 contains 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 microscopic organism that is single or multicellular. These organisms may include bacteria, viruses, fungi and algae.

As used herein, the term "bioluminescence" refers to the generation and emission of light by a living organism. Luciferase catalyses the oxidation of luciferin, producing light.

As used herein, the term "high performance liquid chromatography" (HPLC) refers to an analytical chemistry technique 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 having a cessation of normal physiological function 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 as measured by a luminometer.

As used herein, the term "germination conditions" refers to conditions that favor the activation, germination, and outgrowth of bacterial spores (endospores).

Detecting the presence of bacteria and bacterial spores is important in many industries. When human health is involved, guidelines specify the maximum amount of bacteria that can be present. For example, "dairy commercial standards (Dairymen's Standard)" provide requirements regarding the number of Colony Forming Units (CFU) of bacteria that may be present per gram of paper or paperboard to be used in dairy products. The samples were cut from the paper or paperboard to be tested and placed in sealed envelopes. The samples were then cut into small cubes and placed in a sterile stirrer. Sterile phosphate dilution water was added to the paper samples shredded in the blender to aid in disintegration of the samples. 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 curing, the plates were incubated at 36 ℃ for 48 hours. After incubation, plates were checked for the presence and number of CFUs with a 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 capable of sporulation when the bacteria are in their most susceptible nutritional state.

Spores consist of a number of protective layers that make them resistant to oxidation and chemical attack. Higher biocide dosages are required to kill spores compared to vegetative cells. It is always more effective to apply the biocide to actively vegetative cells. The spore control program must be effective enough to attack the cells in a vegetative state and prevent sporulation. The dose must be high enough to kill the vegetative cells before they develop into spores.

Adenosine Triphosphate (ATP) measurements have been used to detect microorganisms in various industries. ATP is used by cells as an energy source and is an indicator of metabolic activity. ATP can be measured in vegetative bacteria, but bacterial spores contain little or no ATP.

ATP levels are typically measured by a bioluminescent assay involving reaction with luciferase which is quantified with a luminometer. Other methods include colorimetric or fluorometric assays that utilize phosphorylation of glycerol, and High Performance Liquid Chromatography (HPLC).

In methods utilizing bioluminescence, luciferase and luciferin from fireflies are mixed with a sample having a cation (e.g., magnesium) in the presence of oxygen. If ATP is present, it will cause a reaction between luciferase (substrate) and luciferin (catalyst) in an oxidation reaction that generates light. Light emission was detected with a luminometer and reported in Relative Light Units (RLU). The amount of light produced is proportional to the metabolic activity of the microorganisms present, but is not indicative of the amount of organisms present. Luciferase/luciferin reactions are well known in the art, and there are commercial sources for the necessary reagents and protocols for their use. For example, several luciferase/luciferin reagents are available along with luciferase in commercial kits from, for example, Promega Corp. (Madison, WI) and lumineltra (Fredericton, New Brunswick). Commercially available luciferases include firefly luciferases ("Ppy luciferases"). Purified luciferin was also commercially available from Promega.

As mentioned above, although vegetative bacterial cells produce large amounts of ATP, spores produce very little ATP. About 80% of Adenine Nucleotides (AN) in vegetative cells are ATP, while in spores this figure is 1% or less. However, spores contain large amounts of Adenine Nucleotides (AN), such as AMP and ADP. AMP and ADP cannot be detected by luminescence techniques, but AMP and ADP can be extracted from bacterial spores. The addition of certain enzymes converts AMP and ADP to ATP. The ATP can then be quantified using luminescence techniques. These ATP measurements can be used to estimate spore counts within the sample. Surprisingly, this method allows the number of spores present in a sample to be quantified in as little as one hour.

In order to implement such a method for the rapid detection of bacterial spores in an industrial process, a sample to be tested for the presence of bacterial spores must first be prepared. The sample may be collected from any source that may be contaminated with bacterial spores. The sample may be contaminated with bacterial spores, vegetative bacterial cells, none, or both. The sample may be taken from a hard surface, a slurry, raw fiber, a food or beverage product, a liquid or packaging or a cardboard product sample.

Non-limiting examples of facilities having hard surfaces include food and beverage plants, dairy plants, farms and dairy stores, breweries, ethanol plants, full-service and quick-service restaurants, grocery stores, warehouse and retail stores, commercial or office locations, hotels, motels, hospitals, paper mills, industrial manufacturing plants including automotive plants, cooling towers, water treatment plants, oil and gas refineries, oil and gas fields and pipelines, and drilling platforms. Examples of hard surfaces include food and beverage processing equipment including pipes, cans, evaporators, nozzles, and the like; dairy processing equipment, milk tanks, milk carriers, milking equipment, countertops, cooking surfaces, bathroom surfaces such as sinks and toilet handles, light switch panels, door handles, call buttons, telephone handles, remote controls, desktops, patient guardrails, grab bars, surgical instruments, equipment within a paper mill including pipes, cabinets, head boxes, broke towers, reclaimer blades, forming lines, and the like.

Samples on hard surfaces can be taken using sterile swabs or other sterile devices that can be used to collect microorganisms from hard surfaces, such as: medical tapes, cotton cloth, cellulose cloth, and the like. If the surface is dry, a solvent may be applied with the swab to dissolve any bacterial residue that may be present and suspend the residue 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 furnish (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 farming water, agricultural water. The liquid sample may be aliquoted from a potential source of contamination into a sterile container.

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 packages and products include: paper products for food contact and non-food contact grades, such as finished paper products and finished paperboard products; drapes for surgical or medical use; an aseptic packaging container; plastic food and beverage containers (e.g., yogurt containers, milk containers, deli containers); food cans (e.g., soup cans); an aluminum or PET beverage container; bag or film or modified atmosphere package. The products may be tested before they leave the manufacturing facility, for example at a paper mill, or at a point of use, for example 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 evaluation of Paper and paperboard Paper board), or ISO8784 Paper Pulp and paperboard microbiological evaluation (ISO8784 Pulp and board microbiological evaluation)).

The initial ATP level of the sample was measured and recorded as "ATP raw". ATP measurements were performed by luminescence methods. As described above, the sample may be combined with luciferase and luciferin together with cation, oxygen and tris buffer or similar buffers. The light produced by the reaction is measured in Relative Light Units (RLU). The measurement may be performed with a luminometer. Alternatively, ATP measurements may be performed by HPLC.

The sample is heated to destroy the vegetative cells. In some embodiments, the sample is heated at 80 ℃ for about 5 to 60 minutes, preferably about 10 to 30 minutes, and more preferably about 15 to 20 minutes. In other embodiments, the sample is heated at 100 ℃ for about 5 to 30 minutes, preferably about 10 to 30 minutes, and more preferably about 15 to 20 minutes. ATP levels were measured as viable spore forming bacteria and recorded as "ATP heat".

The spores in the sample are enriched by one or more of centrifugation, glass beads, magic beads and filtration techniques. In some embodiments, the spores are extracted by subjecting the spore sample to a temperature of about 60 ° to 65 ℃ for about 1 to 30 minutes, preferably 1 to 20 minutes, and most preferably 5 to 15 minutes.

The enriched sample is then treated with solvents, acids or heat, including but not limited to n-propanol, nitrites, trichloroacetic acid, ultrasound, detergents, protein degrading agents, denaturants, surfactants and divalent metals.

The extracted sample is then treated with Pyruvate Kinase (PK), Myokinase (MK) and phosphoenolpyruvate (PEP) to convert ADP and AMP to ATP. This transition occurs in 10 minutes to 24 hours. Preferably, the switching occurs within 15 minutes to 2 hours. Most preferably, the switching occurs within 15 minutes. The following chemical equation summarizes the conversion process:

after the conversion is complete, another ATP measurement of the sample is performed. ATP levels of spore forming bacteria were recorded as "ATP switches". "

A significant difference between the measurements of "ATP raw" and "ATP heated" indicates that the vegetative cells are destroyed. The level of "ATP conversion" is generally increased compared to "ATP heating". The magnitude of the increase in ATP levels is indicative of the presence of spore forming bacteria. If only vegetative cells are present, the "ATP switch" measurement is not significantly increased by comparison with the "ATP heat" measurement.

Once it has been determined that spores are present in the sample, an antimicrobial treatment is selected. If spores are detected, the treatment may be selected from the following: oxidizing biocides chlorine dioxide, ozone, glutaraldehyde, sodium hypochlorite, monochloramine, peracids, and mixtures thereof; non-oxidizing biocides include, but are not limited to, alcohols, isothiazolines, Dibromonitrilopropionamide (DBNPA), quaternary ammonium compounds, Bronopol (BNPD), bis-trichloromethyl sulfone, methylene bis-thiocyanate, 1- (3-chloropropenyl) -3,5, 7-triaza-1-azoniaadamantane, tetrakis (hydroxymethyl) phosphonium sulfate (THPS), (thiazolyl) benzimidazole, and combinations thereof. In order to achieve bactericidal efficacy, the concentration of biocide should be higher than the amount of treatment used as biocide; typically from 0.1ppm to 5000ppm in the liquid, or from 0.001% to 0.5% for surface, solid or slurry treatment.

Non-chemical treatments such as 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, and the like, when they are in their vegetative state. Once the appropriate treatment has been selected, when no spores are detected, it is applied to the part of the process in which the spore-forming bacteria are present in a vegetative state. This may include hard surfaces at the production site where contamination has been detected, food or beverage products, liquids, packaging or paper products.

Since the process takes no longer than 8 hours to complete, bacterial contamination can be rapidly eliminated to prevent downstream sporulation. In addition, the treatment can be done while the equipment is offline for cleaning, or without the need to isolate the product for multiple shifts, and production problems can be corrected more quickly. After treatment application, subsequent test cycles should be performed to ensure that elimination of spores has occurred. The ability to work with the right chemicals at the right place in production results in cost and time savings.

In order to more fully understand the disclosure, the following examples are given to illustrate some embodiments. These examples and experiments are to be construed as illustrative and not restrictive. Unless stated to the contrary, all parts are by weight.

Examples of the invention

Example 1: converting AMP and ADP to ATP for detection of bacterial spores

Bacillus subtilis was used as a test spore forming bacterial strain. The Bacillus subtilis culture was cultured on plates with TSB agar at 36. + -. 2 ℃ for 24 hours. The plate was scraped and the culture was suspended in sterile water, transferred to a flat bottle (bottle flat) and incubated at 32 ± 2 ℃ for 24 hours. The samples were then centrifuged at 5000rpm for 5 minutes. Excess fluid above the pellet was removed, the pellet was washed with sterile phosphate buffer, and centrifuged again. The pellet was resuspended in phosphate buffer to a volume of 50ml and recorded as "raw". Spore suspensions were prepared by soaking the harvested samples in a water bath at 80 ℃ for 15 minutes and recorded as "spore suspensions". The samples were treated at 60 ℃ for 10 minutes before being treated with the extraction reagent. The prepared spore sample was divided into two parts. The first fraction was undiluted, while the second fraction was diluted 10-fold. Total aerobic count (TABC) and ATP were measured and are listed in Table 1.

Table 1: ATP and TABC levels in endospores prepared prior to transformation

The 2 nd sample was diluted 10 times the 1 st sample.

Spore samples were then extracted by different methods including n-propanol, centrifugation and sonication. ATP was measured and is listed in table 2. ATP levels did not increase after the extraction procedure.

Table 2: ATP levels in samples treated with different extraction methods

After extraction, the sample was treated with Pyruvate Kinase (PK), Myokinase (MK) and phosphoenolpyruvate (PEP) to convert ADP and AMP to ATP. ATP is measured by luminescence methods. The level of ATP was measured and is listed in table 3. ATP levels increased significantly after conversion in samples extracted with centrifugation and n-propanol.

Table 3: converted ATP levels

When spore suspensions were prepared, the ATP readings in the samples were measured at 1298RLU, while the spore counts were 1.6 x 10⁵cfu/ml. Therefore, the ATP reading is not correlated with the presence of spore forming bacteria. The results indicate that ATP readings significantly increased after extraction with n-propanol and conversion of AN to ATP. The ATP readings of 28194 and 9918RLU correspond to a spore plate count of 2.5X 10, respectively⁵cfu/ml and 2X 10⁴cfu/ml。

Example 2: spore detection by ATP conversion

A bacillus subtilis spore suspension was prepared as described in example 1, except that: after 24 hours of incubation at 32 ± 2 ℃, the cultures were placed in a refrigerator for 9 days and then heated at 80 ℃ for 20 minutes to prepare spore suspensions that produced longer periods of time. As described in example 1, the spore suspension was not treated at 60 ℃ prior to the extraction procedure.

Table 4: ATP and TABC of the produced endospores

The spore suspension is then extracted and converted by enzymes. The results for TABC and ATP levels are listed in Table 5. ATP levels were significantly increased by TCA and n-propanol extraction compared to the no extraction treatment process.

Table 5: results of ATP testing in samples after conversion

Spore suspensions of different loadings were prepared as described in example 2, and after extraction and conversion, ATP levels increased to 17894RLU compared to 655RLU in the spore suspension without treatment.

Table 6: results of ATP testing in samples after conversion

In summary, the present method can rapidly identify spore forming bacteria by bringing low levels of ATP readable in dormant spores to significant levels that can be correlated with plate counts through the enrichment, extraction and conversion processes.