Method and system for rapid detection of saccharomycetes

Abstract

A method of preparing a sample for rapid quantification of the determination of biological contaminants. The method includes adding a sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the sample to produce a first supernatant and a first precipitate, discarding the first supernatant, and dissolving the first precipitate in a first volume of buffer to produce a first suspension volume. The method further includes centrifuging the first suspension volume to produce a second supernatant and a second precipitate, discarding the second supernatant, and dissolving the second precipitate in a second volume of buffer to produce a final sample suspension. The method further includes adding the final sample suspension to an assay for rapid quantification of microbial contaminants.

Description

Cross-reference to related applications

[0001] This application is filed as a PCT international patent application and claims priority to U.S. Provisional Patent Application Serial No. 63 / 589,831, filed on October 12, 2023, the subject matter of which is incorporated herein by reference in its entirety. Background Technology

[0002] Juice is transported long distances by tanker from suppliers to processing plants, making it susceptible to microbial spoilage, especially during warmer months. High external temperatures cause internal temperatures to rise, creating ideal conditions for microbial growth. Every year, this problem impacts the juice industry, affecting inventory and causing financial losses.

[0003] Traditional microbiological plate culture methods for counting yeast in fruit juice typically take several days to produce results. This timeframe is too long, considering that the juice needs to be accepted by processing plants for rapid processing or rejected outright if it spoils during transport. Other methods include assessing the juice's taste, odor, and appearance, but these provide limited information about the product's microbiological status. Summary of the Invention

[0004] The examples presented herein relate to a method for preparing a sample for the rapid quantitative determination of biocontaminants. The method includes adding a sample to a glass dish and inserting the dish into a centrifuge. The method further includes centrifuging the sample to produce a first supernatant and a first precipitate, discarding the first supernatant, and dissolving the first precipitate in a first volume of buffer to produce a first suspension volume. The method further includes centrifuging the first suspension volume to produce a second supernatant and a second precipitate, discarding the second supernatant, and dissolving the second precipitate in a second volume of buffer to produce a final sample suspension. The method also includes adding the final sample suspension to a rapid quantitative determination of microorganisms.

[0005] In other embodiments of this document, the sample is fruit juice. In still other embodiments of this document, the sample is one or more of stone fruit juice or berry juice. In other embodiments of this document, the sample includes at least one of apple juice, pear juice, sand pear juice, or quince juice. In other embodiments of this document, the sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

[0006] In other embodiments of this invention, the method further includes diluting the sample. In yet another embodiment of this invention, sample dilution is performed in at least one of the following ways: before adding the sample to the receiver, before inserting the receiver into a centrifuge dish, and / or after centrifuging the sample. In yet another embodiment of this invention, the sample is diluted in buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, or 1000:1. In yet another embodiment of this invention, the sample is diluted in buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1. In yet another embodiment of this invention, the sample comprises citrus juice. In yet another embodiment of this document, the sample includes at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0007] In other embodiments herein, the buffer is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer. In other embodiments herein, the assay is an adenosine triphosphate (ATP) assay. In further embodiments herein, the assay is a luciferase system.

[0008] In other embodiments of this invention, the method further includes culturing the final sample suspension during the assay. In yet another embodiment of this invention, culturing the final sample suspension includes an incubation period. In still another embodiment of this invention, the incubation period is selected from the group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0009] In other embodiments of this invention, the method further includes stirring the final sample suspension. In other embodiments of this invention, stirring the final sample suspension includes vortexing the final sample suspension. In yet another embodiment of this invention, stirring the final sample suspension is performed in at least one of the following ways: before and simultaneously with culturing the final sample suspension during the assay.

[0010] In other embodiments of this invention, the method further includes using a photometer to read the final suspension to obtain results, wherein the results are measured in relative optical units (RLU). In yet another embodiment of this invention, the method further includes interpreting the results by converting the relative optical units (RLU) output by the photometer into colony forming units (CFU) measurements.

[0011] In other embodiments herein, the biocontaminant is at least one of bacteria, mold, mildew, yeast, and virus. In still other embodiments herein, the biocontaminant is a microorganism.

[0012] In other embodiments of this document, the vessel is one of a flask, a small glass bottle, a tube, and a vial.

[0013] Other examples presented herein relate to a method for rapidly quantifying biocontaminants in liquid samples. The method includes adding a liquid sample to a vessel and inserting the vessel into a centrifuge. The method further includes centrifuging the liquid sample to produce a first precipitate and resuspending the first precipitate in a first volume of buffer to produce a first suspension volume. The method further includes centrifuging the first suspension volume to produce a second precipitate and resuspending the second precipitate in a second volume of buffer to produce a final sample suspension. The method also includes determining the bioluminescence produced in the final sample suspension in proportion to the presence of the biocontaminant, measuring the bioluminescence produced in the final sample suspension, and converting the bioluminescence measurement into a quantification of the biocontaminant.

[0014] In other embodiments of this document, determining the final sample suspension includes culturing the final sample suspension. In yet another embodiment of this document, the culturing of the final sample suspension is performed for less than one hour. In still another embodiment of this document, the culturing of the final sample suspension is performed for less than 30 minutes. In yet another embodiment of this document, the culturing of the final sample suspension is performed for 10 minutes.

[0015] In other embodiments of this document, the method further includes stirring the final sample suspension. In still other embodiments of this document, stirring the final sample suspension includes vortexing the final sample suspension. In other embodiments of this document, stirring the final sample suspension is performed in at least one of the following ways: before and simultaneously with culturing the final sample suspension in the assay.

[0016] In other embodiments of this document, the method also includes determining the source of rejected liquid samples based on the quantification of biological contaminants.

[0017] In other embodiments of this document, the liquid sample is fruit juice. In still other embodiments of this document, the liquid sample is one or more of stone fruit juice or berry juice. In still other embodiments of this document, the liquid sample includes at least one of apple juice, pear juice, sand pear juice, or quince juice. In other embodiments of this document, the liquid sample includes at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

[0018] In other embodiments of this invention, the method further includes diluting the liquid sample. In other embodiments of this invention, sample dilution is performed before the sample is added to the receiver, before the receiver is inserted into a centrifuge dish, and before the sample is centrifuged. In other embodiments of this invention, the sample is diluted in buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, or 1000:1. In still other embodiments of this invention, the sample is diluted in buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1.

[0019] In other embodiments herein, the sample comprises citrus juice. In other embodiments herein, the sample comprises at least one of orange juice, lemon juice, lime juice, and grapefruit juice. In some embodiments, the sample comprises a mixed fruit juice, such as pineapple juice, or a berry juice such as strawberry, raspberry, or blackberry.

[0020] In other embodiments herein, the buffer solution is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer. In other embodiments herein, the assay is an adenosine triphosphate (ATP) assay. In yet another embodiment herein, the assay is a luciferase system. In other embodiments herein, the biocontaminant is at least one of bacteria, mold, mildew, yeast, and virus. In yet another embodiment herein, the biocontaminant is yeast. In other embodiments herein, the vessel is one of a flask, a small glass bottle, a tube, and a vial.

[0021] Other examples presented herein relate to a method for determining whether a sample contains less than a threshold amount of biocontaminants. The method includes adding a sample to a glass dish and inserting the dish into a centrifuge. The method further includes centrifuging the sample to produce a first precipitate, dissolving the first precipitate in a first volume of buffer solution to produce a first suspension volume, centrifuging the first suspension volume to produce a second precipitate, and dissolving the second precipitate in a second volume of buffer solution to produce a final sample suspension. The method also includes determining that the final sample suspension produces bioluminescence proportional to the presence of the biocontaminant, measuring the bioluminescence produced in the final sample suspension, converting the bioluminescence measurement into a quantification of the biocontaminant, and comparing the quantification of the biocontaminant to a threshold amount of the biocontaminant.

[0022] Various additional aspects of the invention will be set forth in the following description. These aspects may relate to individual features and combinations thereof. It should be understood that both the foregoing general description and the following detailed description are exemplary and illustrative only, and do not limit the broad inventive concept upon which the embodiments disclosed herein are based. Attached Figure Description

[0023] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate several aspects of this disclosure. A brief description of the drawings is as follows:

[0024] Figure 1 A flowchart illustrating an example method for preparing samples for the rapid quantitative determination of biological pollutants is provided.

[0025] Figure 2 A flowchart illustrating an exemplary method for preparing a diluted sample for the rapid quantitative determination of biological pollutants is provided.

[0026] Figure 3 A flowchart illustrating an example method for performing quantitative determination on a prepared sample is provided.

[0027] Selected definition

[0028] As used herein, “weight percentage”, “wt%”, “percentage by weight”, “% by weight” and their variations refer to the concentration of a substance, which is the weight of the substance divided by the total weight of the composition and multiplied by 100.

[0029] As used herein, “vol%”, “percentage by volume”, “vol%”, and their variations refer to the concentration of a substance, which is the volume of the component divided by the total weight of the solution and multiplied by 100. It should be understood that where “percentage”, “%”, etc. are not specified herein, they should generally be understood as “vol%”, “vol%”, etc.

[0030] As used in this article, "g" means gram; "L" means liter; "mg" means milligram (10⁻³ g); "mL" means milliliter (10⁻³ liter); and "μl" means microliter (10⁻⁶ liter).

[0031] The term "about" is used in conjunction with numerical values ​​to include normal variation in measurement results as would be expected by those skilled in the art, and is understood to have the same meaning as "approximately" and to cover typical error margins, such as ±10% of the stated value. The term "about" also covers amounts that vary due to different equilibrium conditions of the composition resulting from a particular initial composition. Whether or not modified by the term "about," the claims include equivalents of the quantity.

[0032] It should be noted that, unless otherwise expressly stated, the singular forms “a” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, a reference to a composition containing “a compound” includes having two or more compounds that are the same as or different from each other. It should also be noted that, unless otherwise expressly stated, the term “or” is generally used in its meaning, including “and / or”.

[0033] For the sake of brevity and conciseness, any range of values ​​set forth in this specification considers all values ​​within that range and should be interpreted as supporting claims that describe any subranges having endpoints that are real values ​​within the specified range under discussion. As an illustrative example of assumption, the ranges 1 to 5 disclosed in this specification should be considered to support claims of any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0034] The term "substantially free" can refer to any component lacking or substantially lacking in the compositions or methods of this disclosure. When "substantially free" is mentioned, it means that the component was not intentionally added to the compositions or methods of this disclosure. The use of the term "substantially free" allows trace amounts of the component to be included in the compositions or methods of this disclosure because they are present in another component. However, it is recognized that when a composition is referred to as "substantially free" of a component, only trace or minimal amounts of that component are permitted. Furthermore, if a composition is referred to as "substantially free" of a component and if the component is present in trace or minimal amounts, the term should be understood as not affecting the effectiveness of the composition. It should be understood that a composition of this disclosure may be substantially free of an ingredient if it is not expressly included herein or if its possible inclusion is not stated herein. Similarly, the express inclusion of an ingredient allows for its express exclusion, thereby allowing the composition to be substantially free of the expressly stated ingredient.

[0035] The processes, methods, treatments, formulations, and compositions disclosed herein may comprise, consist substantially of, or consist of the components and ingredients of the invention, as well as other components described herein. As used herein, “consistent substantially of” means that the methods and compositions may include additional steps, components, or ingredients, provided that such additional steps, components, or ingredients do not substantially alter the essential and novel characteristics of the claimed process and composition. Detailed Implementation

[0036] The examples presented herein relate to a rapid method for quantifying the amount of biocontaminants in a sample in less than about three hours, or less than about two hours, or less than about one hour, for example, to identify the risk of impending spoilage and / or fermentation. Advantages of the disclosed method include the ability to quickly decide whether to accept or reject new products.

[0037] Numerous commercially available rapid tests are currently available for quantifying live microorganisms in beverages. However, these tests cannot produce suitable results using fruit juice as a matrix. These tests are often characterized as unsuitable for highly acidic, non-filterable substrates, such as fruit juice. These substrates can pose challenges to the testing system, leading to inhibition or enhancement due to low pH or the presence of bioactive metabolites. While the examples discussed herein focus on fruit juice, it should be noted that the methods disclosed herein can also be advantageously used to analyze other substrates that may exhibit similar properties, such as high background noise from the matrix.

[0038] This document discloses systems and methods for the rapid quantification of yeasts and other microorganisms in substrates that would otherwise render the analysis impossible. As will be understood by those skilled in the art, the disclosed methods significantly alter the performance of rapid analysis and enable rapid quantification systems to be used effectively with historically problematic substrates.

[0039] Methods for quantifying microorganisms sometimes require an enrichment step, in which the concentration of microorganisms present in a sample is increased to a level that is easily measurable. For some microorganisms (such as bacteria), this enrichment period can be on the order of several hours. For other microorganisms (such as yeast), the enrichment step may require an incubation period of several hours to at least overnight.

[0040] To remove interfering components from the solution readings and reduce background readings, a series of sample centrifugations are performed, and the supernatant containing background-generating components is discarded. Furthermore, because conventional enrichment methods may be ineffective for evaluating juices, microorganisms present in larger samples are rapidly concentrated through a series of centrifugations, due to the desired time range for the analysis and the characteristics of the juice. Eliminating the need for an enrichment step significantly increases the speed of analysis, enabling efficient quantification at the field location, such as at the loading / unloading terminal upon receiving juice shipments. With the enrichment step eliminated, no culturer or other enrichment device is required, thus increasing the flexibility and speed of the analysis.

[0041] To reduce background noise from the matrix, the method disclosed herein incorporates a dilution step. After dilution, the sample is centrifuged to neutralize interference from matrix components. Finally, the sample is concentrated to eliminate the effects of the initial dilution. Once a sample is prepared according to the disclosed method, it can be efficiently analyzed using quantitative microbial assays. Even considering the potential incubation period after reagent introduction into the sample, sample preparation and analysis according to the disclosed method can still be performed within one hour, compared to the multiple hours required by methods requiring enrichment. In some embodiments, the total time from initial sample extraction to obtaining results is less than about 120 minutes, less than about 100 minutes, less than about 80 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes.

[0042] The method disclosed herein offers numerous advantages over conventional methods. As noted above, the total analysis time is significantly reduced, from several hours (typically at least 7 to 8 hours) to less than one hour, and in many cases, no more than 40 minutes. The method disclosed herein reduces the analysis time by eliminating the time-intensive enrichment of samples, which further reduces costs and increases analytical flexibility by eliminating the need for enrichment media or devices. The method disclosed herein enables the analysis of highly acidic and / or non-filterable substrates (such as fruit juice) to quantify microorganisms that would otherwise be inhibited or enhanced by the substrate characteristics. Furthermore, the method disclosed herein expands the range of quantifiable microorganisms beyond bacteria, including yeast. Finally, the disclosed method can detect psychrophilic yeasts, which cannot be detected by yeast plate counting methods performed at conventional temperatures.

[0043] Figure 1 A flowchart illustrating an example method 100 for preparing a sample for the rapid quantitative determination of biological contaminants is provided. Biological contaminants can be any known or identified cause of spoilage, but in many cases can be microorganisms. In the example, the biological contaminant is any of bacteria, mold, mildew, or yeast. In some specific examples, the biological contaminant is yeast.

[0044] At operation 102, a sample is added to a vessel. The sample may be drawn from the source to be tested, such as a can or cargo of juice received as a storage or distribution facility. The sample may be drawn manually by a technician or automatically, such as periodic quality control samples obtained from a production or packaging facility. In some embodiments, the sample is a liquid sample, and the volume of the sample to be drawn is determined based on the final determination to be performed on the prepared sample. For example, the volume of the liquid sample drawn is between 1 μl and 1 L, between 10 μl and 500 ml, between 50 μl and 100 ml, or between 100 μl and 5 ml. In other examples, the volume of the liquid sample drawn is less than 1 L, less than 500 ml, less than 300 ml, less than 100 ml, less than 1 ml, less than 500 μl, less than 100 μl, less than 50 μl, or less than 20 μl.

[0045] In this implementation, the vessel may be a centrifuge vessel. The centrifuge vessel can be any suitable vessel and can be selected to accommodate an available or desired centrifuge. In some examples, the vessel is a flask, vial, tube, vial, or Eppendorf tube. The centrifuge vessel will have an appropriate size for the volume of the liquid sample being drawn. For example, the centrifuge vessel may be 1L, 500ml, 100ml, 2ml, 1ml, 500μl, or 100μl.

[0046] In some embodiments, the sample is fruit juice. In some examples, the sample is one or more of stone fruit juice or berry juice. For example, the sample may include at least one of apple juice, pear juice, sand pear juice, or quince juice. In other examples, the sample may include at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice. The sample may be a combination of fruit juices. The sample may be of a single concentration or concentrated.

[0047] At operation 104, the sample is centrifuged for the first time, producing a first supernatant and a first precipitate. The time and settings of the first centrifugation (such as speed and / or force) can be determined based on factors such as sample size and available equipment. In some embodiments, the sample is centrifuged at 3,000 x g (relative centrifugal force (RCF)) for 10 minutes. Centrifugation can be performed for less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 3 minutes. Centrifugation can be performed between about 1000 × g and about 10,000 × g, between about 2000 × g and about 5000 × g, or between about 2500 × g and about 3500 × g. In some cases, the sample is removed immediately upon completion of centrifugation, and the method proceeds to operation 106. At operation 106, the first supernatant is discarded.

[0048] At operation 108, the first precipitate is dissolved in a first volume of buffer solution to produce a first suspension volume. In some examples, the buffer solution includes one or more of calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer. In some embodiments, the first volume of the buffer solution may be equal to the original sample volume. For example, 1 ml of sample may be drawn and centrifuged, and the first volume of the buffer solution may be 1 ml. In some embodiments, the dissolution of the first precipitate to produce the first suspension volume is enhanced by using stirring (e.g., vortexing).

[0049] At operation 110, the first suspension volume is centrifuged to produce a second supernatant and a second precipitate. The second centrifugation step can be determined based on the same factors as the first centrifugation step (operation 104). In some examples, the second centrifugation step can be matched with the first centrifugation sub-step. In some embodiments, the sample is centrifuged at 3,000 x g RCF for 10 minutes. In some cases, the sample is removed immediately upon completion of centrifugation, and the method proceeds to operation 112. At operation 112, the second supernatant is discarded.

[0050] At step 114, the second precipitate is dissolved in a second volume of buffer solution to produce a final sample suspension. The final sample suspension may use the same buffer solution as the first sample suspension. In some cases, a different buffer solution may be used instead. In some embodiments, the second volume of the buffer solution may be equal to the original sample volume and / or the first volume of the buffer solution. For example, 1 mL of sample may be drawn and centrifuged, and the second volume of the buffer solution may be approximately 100 μL, or approximately 200 μL, or approximately 300 μL, or approximately 400 μL, or approximately 500 μL, or approximately 1 mL, or any volume in between.

[0051] At step 116, the final sample suspension is added to the assay for rapid quantification of yeast. In an embodiment, the assay may alternatively be added to the final sample suspension or a combination thereof, such that some reagents are added to the sample suspension and the sample solution is added to some reagents. The assay can be any suitable assay for quantifying microorganisms or other biocontaminants. Some non-limiting examples of suitable assays include manual counting of colony-forming units, absorbance-based assays, microscopy, flow cytometry, methylene blue dye reduction test (MBRT), and start-of-growth time (SGT) methods. In an embodiment, a particular assay may be selected based on available equipment and time. In an example, an absorbance-based adenosine triphosphate (ATP) assay, such as a luciferase system, may be used.

[0052] In some cases, method 100 can be modified to include a dilution step to reduce background from the sample. Figure 2A flowchart illustrating an exemplary method 200 for preparing a diluted sample for the rapid quantitative determination of biological pollutants is provided.

[0053] Dilute the sample at step 201. Figure 2 In the example, the sample dilution step is shown before adding the sample to the vessel at operation 202, and this advantageously ensures that the uniformly diluted sample is centrifuged. In an embodiment, sample dilution may be performed before adding the sample to the vessel, before inserting the vessel into the centrifuge, or before centrifuging the sample.

[0054] In some embodiments, the sample is diluted at a ratio of 1:10. The dilution ratio can be selected based on the characteristics of the juice, such as strength and acidity. Some non-limiting examples of suitable dilution ratios are 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, or 1000:1. Other non-limiting examples of suitable dilution ratios include 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1.

[0055] At operation 202, the sample is added to a vessel. In this embodiment, the vessel may be a centrifuge dish. The sample may be drawn from the source to be tested, such as a tank or cargo of juice received as a storage or distribution facility. The sample may be drawn manually by a technician or automatically, such as periodic quality control samples obtained from a production or packaging facility.

[0056] In this implementation, the vessel can be a centrifuge vessel. The centrifuge vessel can be any suitable vessel and can be selected to accommodate an available or desired centrifuge. In some examples, the vessel is a flask, a small glass bottle, a tube, or a vial.

[0057] In some implementations, the sample is fruit juice. Implementations incorporating a dilution step may be particularly advantageous when sampling substrates with low pH, such as citrus juice. In some examples, the sample may include orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0058] At operation 204, the sample is centrifuged for the first time, producing a first supernatant and a first precipitate. The time and settings, speed and / or force of the first centrifugation can be determined based on factors such as sample size and available equipment. In some embodiments, the sample is centrifuged at 3,000 xg RCF for 10 minutes. In some cases, the sample is removed immediately upon completion of centrifugation, and the method proceeds to operation 206. At operation 206, the first supernatant is discarded.

[0059] At operation 208, the first precipitate is dissolved in a first volume of buffer solution to produce a first suspension volume. In some examples, the buffer solution includes one or more of calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer. In some embodiments, the first volume of the buffer solution may be equal to the original sample volume. For example, 1 ml of sample may be drawn and centrifuged, and the first volume of the buffer solution may be 1 ml.

[0060] At operation 210, the first suspension volume is centrifuged to produce a second supernatant and a second precipitate. The second centrifugation step can be determined based on the same factors as the first centrifugation step (operation 204). In some examples, the second centrifugation step can be matched with the first centrifugation sub-step. In some embodiments, the sample is centrifuged at 3,000 xg RCF for 10 minutes. In some cases, the sample is removed immediately upon completion of centrifugation, and the method proceeds to operation 212. At operation 212, the second supernatant is discarded.

[0061] At step 214, the second precipitate is dissolved in a second volume of buffer solution to produce a final sample suspension. The final sample suspension may use the same buffer solution as the first sample suspension. In some cases, a different buffer solution may be used instead. In some embodiments, the second volume of the buffer solution may be equal to the original sample volume and / or the first buffer solution volume. The second buffer solution volume may be adjusted according to the dilution ratio, or it may be statically reduced in the case of sample dilution. For example, 1 ml of sample may be drawn and centrifuged, and the first volume of the buffer solution may be 1 ml, while the second buffer solution volume may be reduced, for example, to 200 μl.

[0062] At step 216, the final sample suspension is added to the assay for rapid quantification of yeast. In an embodiment, the assay may alternatively be added to the final sample suspension or a combination thereof, such that some reagents are added to the sample suspension and the sample solution is added to some reagents. The assay can be any suitable assay for quantifying microorganisms or other biocontaminants. Some non-limiting examples of suitable assays include manual counting of colony-forming units, absorbance-based assays, microscopy, flow cytometry, MBRT, and SGT methods. In an embodiment, a particular assay may be selected based on available equipment and time. In an example, absorbance-based ATP assays, such as luciferase systems, may be used.

[0063] Figure 3A flowchart illustrating an example method 300 for performing a quantitative determination on a prepared sample is provided. The sample may be prepared according to method 100 or method 200. As noted above, the determination may be any suitable determination for quantifying microorganisms or other biocontaminants. Example method 300 relates to a specific example of an ATP determination based on relative light (e.g., using a photometer), which includes determining the final sample suspension to produce bioluminescence proportional to the presence of the biocontaminant according to the operations and considerations discussed below, measuring the bioluminescence produced in the final sample suspension, and converting the bioluminescence measurement result into a quantification of the biocontaminant.

[0064] At step 302, the final sample suspension is added to the assay. Step 302 can be used with... Figure 1 Operation 116 or Figure 2 This is related to operation 216. In some embodiments, the assay may include a single reagent or a premixed combination of reagents. Adding a sample to the assay may include mixing reagents or combining a first premixed combination of reagents with additional reagents or a second or more combinations of reagents. Adding a sample to the assay may include, for example, mixing the sample with the assay reagents by vortexing.

[0065] The volume of the final sample suspension added to the assay can be determined based on a number of factors, including the size of the original sample drawn, the concentration of the original sample drawn, the sample preparation (e.g., diluted or undiluted), the size of the container used for the assay, the concentration of the reagents used or available, etc.

[0066] At operation 304, the final sample suspension is cultured during the assay. The culture can be performed at a predetermined temperature (e.g., 30°C) for a predetermined incubation period. The incubation period is determined based on factors such as sample size and the reagents used. In some embodiments, the incubation period is typically less than one hour, or even less than 30 minutes, to suit the rapid nature of the assay. Some non-limiting examples of suitable incubation periods include 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0067] In some embodiments, the culture at operation 304 incorporates additional elements to mix the assay during culture. In some embodiments, the final sample suspension is stirred, such as by vortexing, during the culture performed in conjunction with the assay. Stirring may be performed before and / or simultaneously with the culture of the final sample suspension in the assay.

[0068] At operation 306, the assay result is measured. In some embodiments, depending on the assay and / or reagents used, the measurement can be performed immediately after the incubation period ends. In this example, the measurement result includes measuring the bioluminescence produced in the final sample suspension after the incubation period. The measurement result can be performed using any suitable tool. For example, a photometer is used to measure low light (such as bioluminescence) produced in relative light units (RLU).

[0069] At operation 308, the results are converted. In this example, the measurement results are converted from RLU output by the photometer to colony forming units (CFU) measurements. RLU values ​​themselves are not the final results of a bioluminescence assay. They must be correlated with the number of CFUs to obtain the actual number of biocontaminant units or microorganisms in the sample. This conversion is typically calibrated to the biocontaminant to be quantified using a standard curve. For example, bacteria are typically smaller than average yeast, and the associated CFU values ​​for bacteria will not provide accurate results to quantify yeast in the sample, and vice versa.

[0070] At operation 310, the final disposal of the sample is determined based on the final quantification of the biocontaminant identified at operation 308. In some embodiments, the final quantification may be compared to a predetermined threshold. The predetermined threshold may be associated with standards such as food safety standards. If the quantification exceeds the threshold, the sample may be rejected, and the source (e.g., can, batch, etc.) may also be rejected. In some embodiments, the threshold may be a predetermined number of RLUs. For example, in various embodiments, thresholds of 500 RLUs, 1000 RLUs, or 2000 RLUs may be implemented. If the quantification does not exceed the threshold, the sample may be accepted, and the source may also be accepted.

[0071] In the implementation plan, standards and thresholds may be related to sensory standards and the measured microbial content. For example, the sample may be further evaluated by taste and odor to determine the impact on the sample from the measured microbial count.

[0072] Preferred aspects and specific embodiments of this disclosure have been described, and modifications and equivalents of the disclosed concepts will readily occur to those skilled in the art. However, such modifications and equivalents are intended to be included within the scope of the appended claims.

[0073] Example

[0074] Example 1

[0075] Using MicroSnap ™ The Total detection device, according to embodiments of this disclosure, quantifies yeast in orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice. MicroSnap™ Total is a rapid bioluminescent assay for the detection and counting of bacteria, available from Hygiena, Camario, California. ® Hygiena Limited Liability Company ® It was acquired by LLC, Camarillo, California.

[0076] Make MicroSnap ™ The detection device reached approximately room temperature.

[0077] Dilute each of the above juices by adding 1 ml of juice to 9 ml of Butterfield buffer, resulting in a 10 ml diluted mixture of each juice. Butterfield buffer is a common phosphate buffer (pH 7.2) commercially available from many companies, such as Remel Butterfield Phosphate Buffer (catalog number: R112520) from Thermo Fisher Scientific. Thoroughly mix the resulting 10 ml diluted mixtures of each juice.

[0078] Add 1 ml of each 10 ml dilution mixture to a microcentrifuge tube. Centrifuge each 1 ml tube for each dilution at 3000 × g (RCF) for 10 minutes. Immediately remove each microcentrifuge tube containing the diluted juice mixture from the centrifuge and quickly discard the supernatant by decanting out all liquid and retaining the sediment.

[0079] Add 1 ml of sterile Butterfield buffer to each microcentrifuge tube and vortex the tube to resuspend the precipitate. Centrifuge each 1 ml tube for each dilution a second time at 3000 × g (RCF) for 10 minutes. Immediately remove each microcentrifuge tube containing the diluted juice mixture from the centrifuge and quickly discard the supernatant by decanting out all liquid and retaining the precipitate.

[0080] Add 200 µl of sterile Butterfield buffer to each microcentrifuge tube and vortex the tube to resuspend the precipitate. After vortexing (resuspending the precipitate in 200 µl of buffer), add 200 µl of the solution from each microcentrifuge tube to MicroSnap. ™ In the total detection device. In a preferred embodiment, due to the time sensitivity of the procedure, only 4 samples are tested in duplicate (8 tubes) at a time.

[0081] Damaging MicroSnap ™Seal the top of the liquid ampoule of the total detection device and mix the liquid with the sample below. Vortex the mixture, then squeeze the top of the device again to ensure all liquid is at the bottom of the tube. Add each MicroSnap... ™ The device was incubated at 30°C for 10 minutes.

[0082] Open Ensure just before the 10-minute incubation period ends. ™ Photometer (available from Hygiena, Camario, California) ® (Obtained by a limited liability company). Heat the photometer for 15 seconds.

[0083] Immediately after 10 minutes of incubation, from MicroSnap ™ The sample was removed from the Total detection device, and the MicroSnap was used. ™ The total detection device is placed inside the photometer. Press "OK" on the photometer to display the results in relative light units (RLU). In a preferred embodiment, for two samples of the same juice, the difference between the results must be within 35% of each individual: Difference % = (Higher reading - Lower reading) / Lower reading. If both readings are within 35%, the average of the two readings, expressed in RLU, is taken.

[0084] The average RLU readings for each juice are then correlated with colony-forming units of different concentrations of yeast. In an embodiment, the correlation table may be specific to a particular type of device or model or model number.

[0085] Example 2

[0086] Using MicroSnap ™ The Total detection device quantifies yeast in apple juice and cranberry juice according to embodiments of this disclosure.

[0087] Make MicroSnap ™ The detection device reached approximately room temperature.

[0088] Add 1 ml of each of the above juices (undiluted) to a microcentrifuge tube. Centrifuge each 1 ml tube for each juice at 3000 × g (RCF) for 10 minutes. Immediately remove each microcentrifuge tube containing the juice from the centrifuge and quickly discard the supernatant by decanting out all liquid and retaining the precipitate.

[0089] Add 1 ml of sterile Butterfield buffer to each microcentrifuge tube and vortex the tube to resuspend the precipitate. Centrifuge each 1 ml tube for each juice a second time at 3000 × g (RCF) for 10 minutes. Immediately remove each microcentrifuge tube containing the juice from the centrifuge and quickly discard the supernatant by decanting out all liquid and retaining the precipitate.

[0090] Add 1 ml of sterile Butterfield buffer to each microcentrifuge tube and vortex the tube to resuspend the precipitate. After vortexing (resuspending the precipitate in 1 ml buffer), add 200 μl from each microcentrifuge tube to MicroSnap. ™ In the device. In a preferred embodiment, due to the time sensitivity of the procedure, only 4 samples are tested in duplicate (8 tubes) at a time.

[0091] Damaging MicroSnap ™ Seal the top of the device on the liquid ampoule and mix the liquid with the sample below. Vortex the mixture, then squeeze the top of the device again to ensure all liquid is at the bottom of the tube. Add each MicroSnap... ™ The device was incubated at 30°C for 10 minutes.

[0092] Open Ensure just before the 10-minute incubation period ends. ™ Photometer (available from Hygiena, Camario, California) ® (Obtained by a limited liability company). Heat the photometer for 15 seconds.

[0093] Immediately after 10 minutes of incubation, from MicroSnap ™ The sample was removed from the device, and the MicroSnap was... ™ The apparatus is placed in a photometer. Press "OK" on the photometer to display the results in relative light units (RLU). In a preferred embodiment, for two samples of the same juice, the difference between the results must be within 35% of each individual: Difference % = (Higher reading - Lower reading) / Lower reading. If both readings are within 35%, the average of the two readings, expressed in RLU, is taken.

[0094] The average RLU readings for each juice are then correlated with colony-forming units (CFU) of different concentrations of yeast. In an embodiment, the correlation table may be specific to a particular type of device, model, or model number. As an example, such a table is provided in Table 1 below. The correlation table can be used to generate a graph from which a best-fit line can be determined and used to evaluate the RLU readings of the sample.

[0095] Table 1

[0096]

[0097] Terms and Conditions

[0098] The following numbered clauses define further exemplary aspects and features of this disclosure:

[0099] Clause 1. A method for preparing a sample for the rapid quantitative determination of a biological contaminant, the method comprising: adding the sample to a glass dish; inserting the glass dish into a centrifuge; centrifuging the sample to produce a first supernatant and a first precipitate; discarding the first supernatant; dissolving the first precipitate in a first volume of buffer solution to produce a first suspension volume; centrifuging the first suspension volume to produce a second supernatant and a second precipitate; discarding the second supernatant; dissolving the second precipitate in a second volume of the buffer solution to produce a final sample suspension; and adding the final sample suspension to a rapid quantitative determination of yeast.

[0100] Clause 2. The method according to Clause 1, wherein the sample is fruit juice.

[0101] Clause 3. The method according to Clause 1 or 2, wherein the sample is one or more of stone fruit juice or berry juice.

[0102] Clause 4. The method according to Clause 3, wherein the sample comprises at least one of apple juice, pear juice, sand pear juice or quince juice.

[0103] Clause 5. The method according to Clause 3, wherein the sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

[0104] Clause 6. The method according to any one of Clauses 1 to 5, wherein the method further comprises diluting the sample.

[0105] Clause 7. The method according to Clause 6, wherein the dilution of the sample is performed in at least one of the following circumstances: before the sample is added to the receiver, before the receiver is inserted into the centrifuge dish, and / or before the sample is centrifuged.

[0106] Clause 8. The method according to Clause 6, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1 or 1000:1.

[0107] Clause 9. The method according to Clause 8, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:1.

[0108] Clause 10. The method according to Clause 6, wherein the sample comprises citrus juice.

[0109] Clause 11. The method according to Clause 10, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0110] Clause 12. The method according to any one of Clauses 1 to 11, wherein the buffer is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic acid buffer.

[0111] Clause 13. The method according to any one of Clauses 1 to 12, wherein the determination is an adenosine triphosphate determination.

[0112] Clause 14. The method according to Clause 13, wherein the assay is a luciferase system.

[0113] Clause 15. The method according to Clause 13, the method further comprising culturing the final sample suspension in the determination.

[0114] Clause 16. The method according to Clause 15, wherein culturing the final sample suspension includes a culture period.

[0115] Clause 17. The method according to Clause 16, wherein the incubation period is selected from the group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes.

[0116] Clause 18. The method according to Clause 15, the method further comprising stirring the final sample suspension.

[0117] Clause 19. The method according to Clause 18, wherein stirring the final sample suspension includes vortexing the final sample suspension.

[0118] Clause 20. The method according to Clause 18, wherein stirring of the final sample suspension is performed in at least one of the following circumstances: before and / or simultaneously with culturing the final sample suspension in the assay.

[0119] Clause 21. The method according to Clause 15, the method further comprising using a photometer to read the final suspension to obtain a result, wherein the result is measured in relative light units (RLU).

[0120] Clause 22. The method according to Clause 21 further includes interpreting the results by converting the relative light units (RLU) output by the photometer into colony forming units (CFU) measurements.

[0121] Clause 23. The method according to Clause 15, wherein the total time for preparing, culturing and analyzing the sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes and less than 40 minutes.

[0122] Clause 24. The method according to any one of Clauses 1 to 23, wherein the biological contaminant is at least one of bacteria, mold, mildew, and yeast.

[0123] Clause 25. The method according to Clause 24, wherein the biological contaminant is yeast.

[0124] Clause 26. The method according to any one of Clauses 1 to 25, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

[0125] Clause 27. A method for rapidly quantifying a biocontaminant in a liquid sample, the method comprising: adding the liquid sample to a glass dish; inserting the glass dish into a centrifuge; centrifuging the liquid sample to produce a first precipitate; dissolving the first precipitate in a first volume of buffer solution to produce a first suspension volume; centrifuging the first suspension volume to produce a second precipitate; dissolving the second precipitate in a second volume of the buffer solution to produce a final sample suspension; determining the final sample suspension to produce bioluminescence proportional to the presence of the biocontaminant; measuring the bioluminescence produced in the final sample suspension; and converting the bioluminescence measurement result into a quantification of the biocontaminant.

[0126] Clause 28. The method according to Clause 27, wherein determining the final sample suspension includes culturing the final sample suspension.

[0127] Clause 29. The method according to Clause 28, wherein the culture of the final sample suspension is performed for less than one hour.

[0128] Clause 30. The method according to Clause 29, wherein the final sample suspension is cultured for less than 30 minutes.

[0129] Clause 31. The method according to Clause 30, wherein the final sample suspension is cultured for 10 minutes.

[0130] Clause 32. The method according to Clause 27 or 28, the method further comprising stirring the final sample suspension.

[0131] Clause 33. The method according to Clause 32, wherein stirring the final sample suspension comprises vortexing the final sample suspension.

[0132] Clause 34. The method according to Clause 32, wherein stirring of the final sample suspension is performed in at least one of the following circumstances: before and / or simultaneously with culturing the final sample suspension in the assay.

[0133] Clause 35. The method according to Clause 27 or 28, wherein the total time for preparing, culturing and determining the sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes and less than 40 minutes.

[0134] Clause 36. The method according to any one of Clauses 26 to 35, the method further comprising determining the source of rejection of the liquid sample based on the quantification of the biological contaminant.

[0135] Clause 37. The method according to any one of Clauses 26 to 36, wherein the liquid sample is fruit juice.

[0136] Clause 38. The method according to Clause 37, wherein the liquid sample is one or more of stone fruit juice or berry juice.

[0137] Clause 39. The method according to Clause 38, wherein the liquid sample comprises at least one of apple juice, pear juice, sand pear juice, or quince juice.

[0138] Clause 40. The method according to Clause 38, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

[0139] Clause 41. The method according to any one of Clauses 26 to 40, the method further comprising diluting the liquid sample.

[0140] Clause 42. The method according to Clause 41, wherein the dilution of the sample is performed in at least one of the following circumstances: before the sample is added to the receiver, before the receiver is inserted into the centrifuge dish, and before the sample is centrifuged.

[0141] Clause 43. The method according to Clause 41, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1 or 1000:1.

[0142] Clause 44. The method according to Clause 43, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:1.

[0143] Clause 45. The method according to Clause 41, wherein the sample comprises citrus juice.

[0144] Clause 46. The method according to Clause 45, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

[0145] Clause 47. The method according to any one of Clauses 27 to 46, wherein the buffer is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic acid buffer.

[0146] Clause 48. The method according to any one of Clauses 27 to 47, wherein the determination is an adenosine triphosphate determination.

[0147] Clause 49. The method according to Clause 48, wherein the assay is a luciferase system.

[0148] Clause 50. The method according to any one of Clauses 27 to 49, wherein the biological contaminant is at least one of bacteria, mold, mildew, yeast, and virus.

[0149] Clause 51. The method according to Clause 50, wherein the biological contaminant is yeast.

[0150] Clause 52. The method according to any one of Clauses 27 to 51, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

[0151] Clause 53. A method for determining whether a sample contains less than a threshold amount of a biocontaminant, the method comprising: adding the sample to a glass dish; inserting the glass dish into a centrifuge; centrifuging the sample to produce a first precipitate; dissolving the first precipitate in a first volume of buffer solution to produce a first suspension volume; centrifuging the first suspension volume to produce a second precipitate; dissolving the second precipitate in a second volume of the buffer solution to produce a final sample suspension; determining that the final sample suspension produces bioluminescence proportional to the presence of the biocontaminant; measuring the bioluminescence produced in the final sample suspension; converting the bioluminescence measurement result into a quantification of the biocontaminant; and comparing the quantification of the biocontaminant with the threshold amount of the biocontaminant.

[0152] Clause 54. The method according to Clause 53, wherein the total time from adding the sample to the vessel to determining whether the sample contains less than the threshold amount of the biological contaminant is one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

[0153] Clause 55. A method for rapidly quantifying a biocontaminant in a liquid sample, the method comprising: diluting the liquid sample with a first volume of buffer solution; adding the diluted liquid sample to a glass dish; inserting the glass dish into a centrifuge; centrifuging the diluted liquid sample to produce a first precipitate; dissolving the first precipitate in a second volume of the buffer solution to produce a first suspension volume; centrifuging the first suspension volume to produce a second precipitate; dissolving the second precipitate in a third volume of the buffer solution to produce a final sample suspension; determining that the final sample suspension produces bioluminescence proportional to the presence of the biocontaminant; measuring the bioluminescence produced in the final sample suspension; and converting the bioluminescence measurement result into a quantification of the biocontaminant.

[0154] Clause 56. The method according to Clause 55, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1 or 1000:1.

[0155] Clause 57. The method according to Clause 56, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:1.

[0156] Clause 58. The method according to Clause 55, wherein the sample is citrus juice.

[0157] Clause 59. The method according to Clause 58, wherein the sample comprises at least one of orange juice, lemon juice, lime juice and grapefruit juice.

[0158] Clause 60. The method according to Clause 55, wherein the sample comprises pineapple juice.

[0159] Clause 61. The method according to any one of Clauses 55 to 60, wherein the buffer is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer and sulfonic acid buffer.

[0160] Clause 62. The method according to any one of Clauses 55 to 61, wherein the determination is an adenosine triphosphate determination.

[0161] Clause 63. The method according to Clause 62, wherein the assay is a luciferase system.

[0162] Clause 64. The method according to any one of Clauses 55 to 63, wherein the biological contaminant is at least one of bacteria, mold, mildew, yeast, and virus.

[0163] Clause 65. The method according to Clause 64, wherein the biological contaminant is yeast.

[0164] Clause 66. The method according to any one of Clauses 55 to 65, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

[0165] Clause 67. The method according to any one of Clauses 55 to 66, wherein determining the final sample suspension comprises culturing the final sample suspension.

[0166] Clause 68. The method according to Clause 67, wherein the culture of the final sample suspension is performed for less than one hour.

[0167] Clause 69. The method according to Clause 68, wherein the final sample suspension is cultured for less than 30 minutes.

[0168] Clause 70. The method according to Clause 69, wherein the final sample suspension is cultured for 10 minutes.

[0169] Clause 71. The method according to Clause 67, the method further comprising stirring the final sample suspension.

[0170] Clause 72. The method according to Clause 71, wherein stirring the final sample suspension comprises vortexing the final sample suspension.

[0171] Clause 73. The method according to Clause 71, wherein stirring of the final sample suspension is performed in at least one of the following circumstances: before and / or simultaneously with culturing the final sample suspension in the assay.

[0172] Clause 74. The method according to any one of Clauses 67 to 73, the method further comprising determining the source of rejection of the liquid sample based on the quantification of the biological contaminant.

[0173] Clause 75. The method according to any one of Clauses 67 to 74, wherein the liquid sample is fruit juice.

[0174] Clause 76. The method according to Clause 55, wherein the liquid sample is one or more of stone fruit juice or berry juice.

[0175] Clause 77. The method according to Clause 76, wherein the liquid sample comprises at least one of apple juice, pear juice, sago pear juice, or quince juice.

[0176] Clause 78. The method according to Clause 76, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

[0177] Clause 79. The method according to Clause 55, wherein the total time for quantifying the biological contaminant in the liquid sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

Claims

1. A method for preparing a sample for the rapid quantitative determination of biological pollutants, the method comprising: Add the sample to the vessel; Insert the container into the centrifuge; The sample is centrifuged to produce a first supernatant and a first precipitate; Discard the first supernatant; The first precipitate is dissolved in a first volume of buffer solution to produce a first suspension volume; The volume of the first suspension is centrifuged to produce a second supernatant and a second precipitate; Discard the second supernatant; The second precipitate is dissolved in a second volume of the buffer solution to produce the final sample suspension; as well as The final sample suspension was added to the assay for rapid quantitative yeast.

2. The method according to claim 1, wherein the sample is fruit juice.

3. The method according to claim 1 or 2, wherein the sample is one or more of stone fruit juice or berry juice.

4. The method according to claim 3, wherein the sample comprises at least one of apple juice, pear juice, sand pear juice, or quince juice.

5. The method of claim 3, wherein the sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

6. The method according to any one of claims 1 to 5, wherein the method further comprises diluting the sample.

7. The method of claim 6, wherein diluting the sample is performed in at least one of the following situations: before adding the sample to the receiver, before inserting the receiver into the centrifuge dish, and / or before centrifuging the sample.

8. The method of claim 6, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1 or 1000:

1.

9. The method of claim 8, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:

1.

10. The method of claim 6, wherein the sample comprises citrus juice.

11. The method of claim 10, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

12. The method according to any one of claims 1 to 11, wherein the buffer solution is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer.

13. The method according to any one of claims 1 to 12, wherein the determination is an adenosine triphosphate determination.

14. The method of claim 13, wherein the assay is a luciferase system.

15. The method of claim 13, further comprising culturing the final sample suspension during the determination.

16. The method of claim 15, wherein culturing the final sample suspension includes a culture period.

17. The method of claim 16, wherein the incubation period is selected from the group consisting of: 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes.

18. The method of claim 15, further comprising stirring the final sample suspension.

19. The method of claim 18, wherein stirring the final sample suspension comprises vortexing the final sample suspension.

20. The method of claim 18, wherein stirring the final sample suspension is performed in at least one of the following situations: before and / or simultaneously with culturing the final sample suspension in the assay.

21. The method of claim 15, further comprising using a photometer to read the final suspension to obtain a result, wherein the result is measured in relative light units (RLU).

22. The method of claim 21, further comprising interpreting the results by converting the relative light units (RLU) output by the photometer into colony forming units (CFU) measurements.

23. The method of claim 15, wherein the total time for preparing, culturing and analyzing the sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes and less than 40 minutes.

24. The method according to any one of claims 1 to 23, wherein the biological contaminant is at least one of bacteria, mold, mildew, yeast, and virus.

25. The method of claim 24, wherein the biological contaminant is yeast.

26. The method according to any one of claims 1 to 25, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

27. A method for rapid quantification of biological contaminants in a liquid sample, the method comprising: Add the liquid sample to the vessel; Insert the container into the centrifuge; The liquid sample is centrifuged to produce a first precipitate; The first precipitate is dissolved in a first volume of buffer solution to produce a first suspension volume; The volume of the first suspension is centrifuged to produce a second precipitate; The second precipitate is dissolved in a second volume of the buffer solution to produce the final sample suspension; The final sample suspension was measured to produce bioluminescence proportional to the presence of the biological contaminant; The bioluminescence produced in the final sample suspension was measured. as well as The bioluminescence measurement results are converted into the quantification of the biopollutant.

28. The method of claim 27, wherein determining the final sample suspension comprises culturing the final sample suspension.

29. The method of claim 28, wherein the culture of the final sample suspension is performed for less than one hour.

30. The method of claim 29, wherein the final sample suspension is cultured for less than 30 minutes.

31. The method of claim 30, wherein the final sample suspension is cultured for 10 minutes.

32. The method according to claim 27 or 28, the method further comprising stirring the final sample suspension.

33. The method of claim 32, wherein stirring the final sample suspension comprises vortexing the final sample suspension.

34. The method of claim 32, wherein stirring the final sample suspension is performed in at least one of the following situations: before and / or simultaneously with culturing the final sample suspension in the determination.

35. The method according to claim 27 or 28, wherein the total time for preparing, culturing and determining the sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes and less than 40 minutes.

36. The method according to any one of claims 27 to 35, the method further comprising determining the source of rejection of the liquid sample based on the quantification of the biological contaminant.

37. The method according to any one of claims 27 to 36, wherein the liquid sample is fruit juice.

38. The method of claim 37, wherein the liquid sample is one or more of a stone fruit juice or a berry juice.

39. The method of claim 38, wherein the liquid sample comprises at least one of apple juice, pear juice, sand pear juice, or quince juice.

40. The method of claim 38, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

41. The method according to any one of claims 26 to 40, the method further comprising diluting the liquid sample.

42. The method of claim 41, wherein diluting the sample is performed in at least one of the following conditions: before adding the sample to the receiver, before inserting the receiver into the centrifuge dish, and before centrifuging the sample.

43. The method of claim 41, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1 or 1000:

1.

44. The method of claim 43, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:

1.

45. The method of claim 41, wherein the sample comprises citrus juice.

46. ​​The method of claim 45, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, grapefruit juice, and pineapple juice.

47. The method according to any one of claims 27 to 46, wherein the buffer solution is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer.

48. The method according to any one of claims 27 to 47, wherein the determination is an adenosine triphosphate (ATP) determination.

49. The method of claim 48, wherein the assay is a luciferase system.

50. The method according to any one of claims 27 to 49, wherein the biological contaminant is at least one of bacteria, mold, mildew, yeast, and virus.

51. The method of claim 50, wherein the biological contaminant is yeast.

52. The method according to any one of claims 27 to 51, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

53. A method for determining whether a sample contains less than a threshold amount of biological contaminants, the method comprising: Add the sample to the vessel; Insert the container into the centrifuge; The sample is centrifuged to produce a first precipitate; The first precipitate is dissolved in a first volume of buffer solution to produce a first suspension volume; The volume of the first suspension is centrifuged to produce a second precipitate; The second precipitate is dissolved in a second volume of the buffer solution to produce the final sample suspension; The final sample suspension was measured to produce bioluminescence proportional to the presence of the biological contaminant; The bioluminescence produced in the final sample suspension was measured. The bioluminescence measurement results are converted into the quantification of the biological pollutants; as well as The quantitative amount of the biological pollutant is compared with the threshold amount of the biological pollutant.

54. The method of claim 53, wherein the total time from adding the sample to the vessel to determining whether the sample contains less than the threshold amount of the biological contaminant comprises one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

55. A method for rapid quantification of biological contaminants in a liquid sample, the method comprising: The liquid sample is diluted with a first volume of buffer solution; Add the diluted liquid sample to the vessel; Insert the container into the centrifuge; The diluted liquid sample was centrifuged to produce a first precipitate; The first precipitate is dissolved in the second volume of the buffer solution to produce a first suspension volume; The volume of the first suspension is centrifuged to produce a second precipitate; The second precipitate is dissolved in a third volume of the buffer solution to produce the final sample suspension; The final sample suspension was measured to produce bioluminescence proportional to the presence of the biological contaminant; The bioluminescence produced in the final sample suspension was measured. as well as The bioluminescence measurement results are converted into the quantification of the biopollutant.

56. The method of claim 55, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 1:1000, 1:500, 1:100, 1:50, 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, or 1000:

1.

57. The method of claim 56, wherein the sample is diluted in the buffer at a ratio selected from the group consisting of: 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 and 15:

1.

58. The method of claim 55, wherein the sample comprises citrus juice.

59. The method of claim 58, wherein the sample comprises at least one of orange juice, lemon juice, lime juice, and grapefruit juice.

60. The method of claim 55, wherein the sample comprises pineapple juice.

61. The method according to any one of claims 55 to 60, wherein the buffer solution is selected from the group consisting of: calcium buffer, carbonate buffer, hydroxymethyl buffer, nitrate buffer, phosphate buffer, and sulfonic acid buffer.

62. The method according to any one of claims 55 to 61, wherein the determination is an adenosine triphosphate (ATP) determination.

63. The method of claim 62, wherein the assay is a luciferase system.

64. The method according to any one of claims 55 to 63, wherein the biological contaminant is at least one of bacteria, mold, mildew, yeast, and virus.

65. The method of claim 64, wherein the biological contaminant is yeast.

66. The method according to any one of claims 55 to 65, wherein the vessel is one of a flask, a small glass bottle, a tube, and a vial.

67. The method according to any one of claims 55 to 66, wherein determining the final sample suspension comprises culturing the final sample suspension.

68. The method of claim 67, wherein the culture of the final sample suspension is performed for less than one hour.

69. The method of claim 68, wherein the final sample suspension is cultured for less than 30 minutes.

70. The method of claim 69, wherein the final sample suspension is cultured for 10 minutes.

71. The method of claim 67, further comprising stirring the final sample suspension.

72. The method of claim 71, wherein stirring the final sample suspension comprises vortexing the final sample suspension.

73. The method of claim 71, wherein stirring the final sample suspension is performed in at least one of the following circumstances: before and / or simultaneously with culturing the final sample suspension in the assay.

74. The method according to any one of claims 67 to 73, the method further comprising determining the source of rejection of the liquid sample based on the quantification of the biological contaminant.

75. The method according to any one of claims 67 to 74, wherein the liquid sample is fruit juice.

76. The method of claim 55, wherein the liquid sample is one or more of stone fruit juice or berry juice.

77. The method of claim 76, wherein the liquid sample comprises at least one of apple juice, pear juice, sand pear juice, or quince juice.

78. The method of claim 76, wherein the liquid sample comprises at least one of blueberry juice, raspberry juice, goji berry juice, acai berry juice, aronia berry juice, and strawberry juice or cranberry juice.

79. The method of claim 55, wherein the total time for quantifying the biological contaminant in the liquid sample includes one of less than 120 minutes, less than 100 minutes, less than 80 minutes, less than 60 minutes, less than 50 minutes, and less than 40 minutes.

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