Selective detection of guaiacol-producing bacteria using liquid media

Abstract

The present disclosure relates to methods, compositions, and kits for determining whether a liquid contains a guaiacol-producing bacterium. In embodiments, a method comprises the steps of (a) mixing a media that changes color with a change in pH with a volume of a liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample, (b) placing the sample in a sterile container, (c) incubating the sample in the sterile container at about 30°C to about 60°C, (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c), and (e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d). In embodiments, the liquid that is potentially contaminated with one or more guaiacol-producing bacteria is an acidic fruit juice. In embodiments, the guaiacol-producing bacteria are guaiacol-positive thermophilic acidophilic bacteria.

Description

SELECTIVE DETECTION OF GUAIACOL-PRODUCING BACTERIA USING LIQUID MEDIACROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 634,828 filed on April 16, 2024, the entire contents of which are incorporated herein by reference.INTRODUCTION

[0002] Thermophilic Acidophilic Bacteria (TAB) are a group of non-pathogenic bacteria commonly found to spoilage liquid, e.g., beverages. These bacteria belong to the Alicyclobacillus genus and are characterized for being mostly Gram positive, rod-shaped, heat- resistant spore-forming, acidophilic and thermophilic bacteria. The genus Alicyclobacillus compromises a set of different species that can be grouped attending to their ability to produce guaiacol from vanillic acid. This metabolic trait can also vary depending on the strains of the same species, e.g., guaiacol-positive and guaiacol -negative strains.

[0003] Guaiacol-positive TAB create a risk in the beverage sector, especially to the juice industry. Juice products with guaiacol-positive Alicyclobacillus contamination may contaminate the final products, leading to significant economic losses for manufacturers and a negative impact on brand reputation. These bacteria have the potential to negatively affect the final product due to the production of the off-note guaiacol, a phenolic compound that imparts undesirable smoke-like, medicinal-like or antiseptic-like flavor to the juice. Moreover, the heat resistant spores can survive pasteurization and it has been observed that very low number of cells / spores can lead to spoilage of products. These bacteria are able to grow in a wide range of temperatures, allowing them to grow after product packaging for commercialization and end point sale.

[0004] Accordingly, detecting Thermophilic Acidophilic Bacteria (TAB) in liquids, e.g., beverages, is crucial for ensuring product safety and quality, particularly in industries such as dairy and fruit juice production where these bacteria can thrive. Conventional methods for TAB detection involve culture-based techniques, relying on selective media and incubation at elevated temperatures and low pH conditions to isolate viable bacterial colonies. However, these methods often suffer from limitations such as long incubation periods, low sensitivity, and difficulty in distinguishing TAB from other microorganisms present in complex beverage matrices.

[0005] Historically, detection of guaiacol-positive TAB can be achieved by traditional methods such as cultivation on agar plates, or molecular methods such as Real Time - Polymerase Chain Reaction (RT-PCR) or Random Amplification of Polymorphic DNA (RAPD). There are three different protocols to analyze TAB in the laboratory, established by the International Fruit and Vegetable Juice Association (TFU). These protocols include testing using agar plates but testing using agar plates can be tedious as only small aliquots of product can be tested per plate and replicates must be performed. For example, current methodology requires the analysis of 1 ml of juice, previously heat shocked at 80°C for 10 minutes. To test that volume, spread plates of 0.1 mL needs to be performed 10 times, which is laborious. Traditional methods such as plating also involve an extra step to confirm the presence of guaiacol-positive TAB. For example, confirmation of the production of guaiacol has to be confirmed by Guaiacol test kits, or other more complex methods, such as Gas Chromatography - Mass Spectrometry (GC-MS).

[0006] RT-PCR is a sensitive method for detecting guaiacol-positive TAB, but optimization of the DNA extraction must be customized depending on the matrix / juice, and it requires an enrichment step to obtain a sufficient number of cells for the analysis. Also, this testing method requires skills and efficiency in molecular biology and the related equipment. Moreover, primer-based detection of guaiacol production might be not accurate as changes affecting the genes involved in guaiacol production can contribute to disruption in the detection, such as SNPs, INDELs, or DNA recombinations.

[0007] Challenges with conventional methods for TAB detection extend beyond technical limitations to encompass the dynamic nature of microbial populations in beverages. Beverages can serve as dynamic environments where microbial communities evolve over time, influenced by factors such as processing conditions, storage temperature, and the presence of competing microorganisms. The efficacy of conventional detection methods may vary depending on the stage of beverage production and the microbial ecology of the specific product. Moreover, the presence of certain inhibitory compounds or matrix components in beverages can further complicate the isolation and enumeration of TAB, necessitating the development of robust detection strategies capable of overcoming these challenges while maintaining accuracy and efficiency. Consequently, conventional approaches may underestimate the prevalence of TAB or fail to detect specific strains, posing risks to product integrity and product perception by the consumer.

[0008] The present disclosure addresses the need for a rapid, sensitive, and accurate test to detect guaiacol-producing bacteria that requires little skill or equipment to complete. Thepresent disclosure provides a faster method for detecting guaiacol-producing bacteria because in an embodiment of the present disclosure only 1 ml of non-filterable product (such as juice or other fruit ingredients) can be tested directly in one test tube, thereby requiring less media (e.g., 9 ml of liquid media compared to 18 ml of solid media for an agar plate). Additional benefits of embodiments of the present disclosure include but are not limited to: positive contamination is detectable in less than three days; the disclosed media is selective, namely, it can select for guaiacol-producing bacteria (e.g., guaiacol-positive TAB); it is low cost and safe because the reagents are low cost, widely available, and non-hazardous; easily scalable without losing specificity; and no molecular biology or GC-MS skills are required.SUMMARY

[0009] The present disclosure addresses the need for a rapid, sensitive, and accurate test to detect benzoic acid derivative resistant bacteria, e.g., dihydroxybenzoic acid derivatives, such as vanillic acid, syringic acid, and veratric acid resistant bacteria that requires little skill or equipment to complete. An embodiment of the present disclosure includes methods of determining whether a liquid contains a guaiacol-producing bacteria, methods of detecting guaiacol-positive thermophilic acidophilic bacteria, methods of determining whether an ingredient or beverage product with a low pH, is viable for commercial sale and / or consumption by a consumer, methods of determining whether to accept a shipment of an acidic fruit juice (or another susceptible beverage with a low pH), and methods of determining whether to package an acidic fruit juice into personal commercial containers having a size between about 0.1 liter and about 2.0 liters.

[0010] In embodiments, the methods of the present disclosure comprise (a) mixing a media that changes color with a change in pH with a volume of a liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample; (b) placing the sample in a sterile container; (c) incubating the sample in the sterile container at about 30°C to about 60°C; (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c); and (e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d).

[0011] In embodiments, the guaiacol-producing bacteria are Bacillus spp. In additional embodiments, the guaiacol-producing bacteria are Alicyclobacillus spp., Bacillus subtilis, Streptomyces spp., any bacteria that produces guaiacol, or any combination thereof. In specific embodiments, the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilicbacteria (guaiacol -positive TAB). In embodiments, the liquid is an acidic fruit juice, e.g., like orange juice.

[0012] In embodiments, the media comprises a nutrient extract (e.g., yeast extract), a pH indicator (e.g., Bromophenol Blue), a weak acid (e.g., vanillic acid, syringic acid, and / or veratric acid), and water. In certain embodiments the media has a pH of about 3.9 to about 4.4. In certain embodiments, the media is sterilized prior to mixing with a liquid, e.g., by autoclaving the media.

[0013] In embodiments, mixing the media with a volume of liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample comprises mixing the media with the liquid between about a 150: 1 ratio to about a 1 : 10 ratio, or between about a 20: 1 ratio to about a 1 : 1 ratio, or between about 15 : 1 to about a 5 : 1 ratio, or about a 9: 1 ratio, or about a 99: 1 ratio. In embodiments, the sample is about 9 ml of media and about 1 ml of liquid (e.g., acidic fruit juice). In embodiments, membrane filtration may be used, and the liquid media may be filtered in through the membrane.

[0014] In embodiments, the determination of a color change in step (d) is by visual inspection; and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol- producing bacteria. In embodiments, the determination of an odor change in step (d) is by olfactory inspection; and if the odor of the sample has smoky and / or medicinal notes following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol-producing bacteria. In embodiments, the determination of a change in clarity in step (d) is by visual inspection; and if the sample has turbidity following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for guaiacol- producing bacteria. In certain embodiments, (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) and / or (e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d) further comprises subjecting the sample to spectrophotometric and / or fluorometric analysis. In embodiments, spectrophotometric and / or fluorometric analysis is used instead of or before a determination in color change, a determination of an odor change, or a determination of a change in clarity by visual inspection.

[0015] In embodiments, gas chromatography-mass spectrometry may be used for the olfactory inspection and would provide results after as little as 24 hours. In addition to subjecting the sample to spectrophotometric and / or fluorometric analysis, the sample may besubjected to absorbance by plate reader as an alternative. Additional rapid microbial detection systems may be used in embodiments.

[0016] In embodiments, compositions for detecting a guaiacol -producing bacterium in a liquid of the present disclosure comprise a nutrient extract (e.g., yeast extract); a pH indicator (e.g., Bromophenol Blue); a weak acid (e.g., vanillic acid, syringic acid, and / or veratric acid); and water. In embodiments, the composition is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water. In embodiments, the composition is not a solid medium, e.g., an agar dish / plate. In embodiments, the composition the nutrient extract that is about 1.8 g / 1 to about 2.2 g / 1 yeast extract, the pH indicator is about 0.002% Bromophenol Blue, and the weak acid is about 100 mg / 1 of vanillic acid; and the composition has a pH of about 4.1 to about 4.3.

[0017] In embodiments, kits for detecting guaiacol -positive thermophilic acidophilic bacteria of the present disclosure compromise a liquid using any of the disclosed methods, the kit comprising any one of the disclosed compositions; and instructions for using the kit to detect guaiacol-positive thermophilic acidophilic bacteria in a liquid.

[0018] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 illustrates a flowchart of an embodiment of a method used to detect guaiacol-producing bacteria in a liquid sample.

[0020] FIG. 2 illustrates a flowchart of an embodiment of a method used to detect guaiacol-producing bacteria (e.g., guaiacol -positive TAB) in an acidic fruit juice, e.g., orange juice.

[0021] FIG. 3A is an example showing juice contaminated with guaiacol-negative TAB (left), and juice contaminated with guaiacol-positive TAB (right) using media containing vanillic acid. The sample on the left is yellowish brown while the sample on the right is purple.

[0022] FIG. 3B is an example showing dilutions of juice containing guaiacol-positive TAB mixed with media containing vanillic acid. Sample B.l is yellowish brown, Samples B.2- B.4 are all purple. Sample B.5 is also purple but slightly less bright. Sample B.6 is yellowish brown.DETAILED DESCRIPTION

[0023] The present disclosure relates to methods, compositions, and kits for determining whether a liquid contains a guaiacol-producing bacteria. In an embodiment, the media used with the methods, compositions, and kits of the present disclosure contains vanillic acid and a pH indicator for the detection of guaiacol-positive thermophilic acidophilic bacteria (TAB). Vanillin, vanillic acid, syringic acid and / or veratric acid may be toxic for both positive and negative-guaiacol TAB, thus inhibiting growth. Only TAB containing genes that are able to convert vanillic acid into a lesser toxic compound, such as guaiacol, will survive in a media containing vanillic acid.

[0024] Species of bacteria belonging to the Alicyclobacillus genus are characterized for being mostly gram positive, rod-shaped, heat-resistant spore-forming, acidophilic and thermophilic bacteria. Spore germination may be improved by heat shock (of about 10 minutes at 80°C) in order to activate spores. TAB are aerobic, and the conversion of vanillic acid to guaiacol is an aerobic reaction. However, the methods, compositions, and kits disclosed herein provide results without heat shock, as spores may germinate if the growth conditions are adequate.

[0025] The selection medium of the present disclosure may comprise one or more of a compound comprising a derivative of benzoic acid, where benzoic acid is represented by formula I:

[0026] Formula I:

[0027] Benzoic acids are present as basic organic compounds with a benzene ring and a carboxyl group. Benzoic acid derivatives for use with the present disclosure may vary, based on the placement and type of side chain present. Example compounds for use with the present disclosure are illustrated below:wherein Ri is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or - COOH. R2i is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or - COOH. R3is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or - COOH. R4is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or - COOH. R5is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or - COOH.

[0028] In certain embodiments, Ri is -H, R2 is -OCH3, R3 is -OH, R4is -H, and R5 is -H as disclosed in Formula II (vanillic acid (4-hydroxy-3-methoxybenzoic acid))

[0029] Formula II:

[0030] In certain embodiments, R1 is -H, R2 is -OCH3, R3 is -OCH3, R4 is -H, and R5 is - H as disclosed in Formula III (veratric acid (3,4-dimethoxybenzoic acid)):

[0031] Formula III:

[0032] In certain embodiments, R1 is -H, R2 is -OCH3, R3 is -OH, R4 is -OCH3, and R5 is-H as disclosed in Formula IV (syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid)):

[0033] Formula IV:

[0034] In certain embodiments, in reference to Formula V, Ri is -H, -OH, or -OCH3. R2 is -H, -OH, or -OCH3. R3is -H, -OH, or -OCH3. R4is -H, -OH, or -OCH3. R5is -H, -OH, or - OCH3:

[0035] Formula V:Definition and interpretation of selected terms

[0036] As used herein, “weight percent,” “wt%,” “percent by weight,” “% by weight,” and variations thereof refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent,” “%,” and the like are intended to be synonymous with “weight percent,” “wt%,” etc.

[0037] As used herein, “g” represents gram; “L” or “1” represents liter; “mg” represents “milligram (10‘3gram);” “mL” or “cc” or “ml” represents milliliter (10‘3liter). One “pL” equals to one micron liter (10‘6liter). The units “g / lOOg,” “g / lOOmL,” “g / L,” or “g / 1” are units of concentration or content of a component in a composition. One “mg / L” or “mg / 1” equals to one ppm (part per million). The unit of temperature used herein is degree Celsius (°C).

[0038] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial composition. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0039] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes having two or more compounds that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0040] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to 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.

[0041] The term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0042] The term “substantially free” may refer to any component that the composition of the disclosure lacks or mostly lacks. When referring to “substantially free” it is intended that the component is not intentionally added to compositions of the disclosure. Use of the term“substantially free” of a component allows for trace amounts of that component to be included in compositions of the disclosure because they are present in another component. However, it is recognized that only trace or de minimus amounts of a component will be allowed when the composition is said to be “substantially free” of that component. Moreover, if a composition is said to be “substantially free” of a component, if the component is present in trace or de minimus amounts it is understood that it will not affect the effectiveness of the composition. It is understood that if an ingredient is not expressly included herein or its possible inclusion is not stated herein, the disclosure composition may be substantially free of that ingredient. Likewise, the express inclusion of an ingredient allows for its express exclusion thereby allowing a composition to be substantially free of that expressly stated ingredient.

[0043] The term “bacteria” as used herein, includes bacterium (i.e., both clusters of spores, as well as single bacteria spores). Bacteria and bacterium may be used interchangeably in each of the embodiments disclosed herein.

[0044] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0045] As used herein, the transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. Thus, the term “consisting essentially of’ when used in a claim of this disclosure is not intended to be interpreted to be equivalent to “comprising.”Methods of the present disclosure.

[0046] Methods of the present disclose include but are not limited to methods of determining whether a liquid contains a guaiacol-producing bacteria, methods of detecting guaiacol-positive thermophilic acidophilic bacteria, methods of determining whether an acidic fruit juice is viable for commercial sale and / or consumption by a consumer, methods of determining whether to accept a shipment of an acidic fruit juice, and methods of determining whether to package an acidic fruit juice into personal commercial containers having a size between about 0.1 liter and about 2.0 liters.

[0047] In embodiments, a method of determining whether a liquid contains a guaiacol- producing bacteria is disclosed, the method comprising the steps of: (a) mixing a media that changes color with a change in pH with a volume of a liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample, (b) placing the sample in asterile container, (c) incubating the sample in the sterile container at about 30°C to about 60°C, (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c), and (e) determining whether the liquid contains a guaiacol - producing bacteria based upon the determination in step (d). The discloses methods are applicable to the identification of guaiacol-producing bacteria in any liquid, e.g., a beverage, and should not be interpreted as being limited to any specific liquid, e.g., a beverage. For example, methods of the present disclosure are applicable to the beverage industry but also have industrial applicability, e.g., determining whether a hazardous and / or non-edible liquid contains a guaiacol-producing bacteria.

[0048] In embodiments of the present disclosure, the guaiacol-producing bacteria is Bacillus spp. Specific examples may include, but are not limited to, Alicyclobacillus spp., Bacillus sublilis. Streptomyces spp., any guaiacol producing bacteria, or any combination thereof. The disclosed methods may be applied to any liquid that potentially contains a bacteria with genes able to convert vanillic acid, syringic acid and / or veratric acid into a lesser toxic compound, such as guaiacol. In specific embodiments, the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria (guaiacol-positive TAB). Guaiacolpositive TAB is bacteria that commonly spoils edible products in the beverage industry. For example, fruit juices, vegetable juice and juice ingredients, juice related products, sodas, syrups, and other acidic beverages such as tea that are common in the beverage industry. In embodiments, the liquid suspected of being contaminated or potentially contaminated with guaiacol-producing bacteria is an edible liquid. In certain embodiments, the edible liquid is an acidic liquid. In embodiments, the edible liquid is a fruit juice from oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof. In one embodiment, the edible liquid is orange juice, e.g., Minute Maid® Orange Juice, including orange juice not from concentration, orange juice from concentrate, pulp free orange juice, and / or orange juice fortified with vitamins and / or essential nutrients. In another embodiment, the edible liquid is another fruit or vegetable ingredient such as a juice, puree, or concentrate. In another embodiment, the edible liquid is a sugar, syrup, teacontaining ingredient or product, or a flavored water.

[0049] In embodiments of the present disclosure, the media comprises a nutrient extract, a pH indicator, a weak acid, and water. The certain embodiments, the media consists essentially of a nutrient extract, a pH indicator, a weak acid, and water. In embodiments, the media is substantially free of any ingredients other than a nutrient extract, a pH indicator, a weak acid, and water. The nutrient extract provides a source of vitamins and nitrogen necessary to promotegrowth of the vanillic resistant bacteria. In a preferred embodiment the nutrient extract is a yeast extract. For example, a yeast extract from Fischer BioReagents™ Microbiology Media Additives. In embodiments, the media may contain a commercially available nutrient extract, a non-commercially available nutrient extract, and / or a combination of more than one nutrient extract. The extract may be a powder, a liquid, a semi-liquid (e.g., paste), or any combination thereof. In embodiments, nutrient extract (e.g., nutrient extracts typically comprise nucleotides, proteins, amino acids, sugars, etc.) in the media is between about 0.5 g / 1 to about 4.0 g / 1, between about 1 g / 1 to about 3 g / 1, between about 1.5 g / 1 to about 2.5 g / 1, or about 2.0 g / 1. In embodiments, the nutrient extract in the media is greater than about 0.5 g / 1, greater than about 1.0 g / 1, greater than about 1.5 g / 1, greater than about 2.0 g / 1, greater than about 2.5 g / 1, greater than about 3.0 g / 1, greater than about 3.5 g / 1, or greater than about 4.0 g / 1. In embodiments, the nutrient extract is less than about 5.0 g / 1, less than about 4.5 g / 1, less than about 4.0 g / 1, less than bout 3.5 g / 1, less than about 3.0 g / 1, less than about 2.5 g / 1, less than about 2.0 g / 1, or less than about 1.5 g / 1, but still present in the media.

[0050] In embodiments, the pH indicator changes color with an increase in pH. In other words, as the pH of the pH indicator increases the color changes from one color to another color. For example, in an embodiment, the pH indicator is Bromophenol Blue (3',3",5',5"- tetrabromophenolsulfonphthalein). Bromophenol Blue has a yellowish color at a pH of 3.0 but becomes a purple color at about a pH of 4.6. Accordingly, in an embodiment of the present disclosure where the media contains Bromonphenol Blue as the pH indicator, the color of the media will change from a yellowish color to a purple color as the pH increases from about 4.2 to above 4.6. The pH indicator of the present disclosure can be gentian violet, malachite green, thymol blue, methyl yellow, methylene blue, Bromophenol Blue, Congo red, methyl orange, bromocresol green, methyl red, methyl purple, bromocresol purple, bromocresol blue, phenol red, neutral red, cresol red, cresolphthalein, phenolphthalein, thymolphthalein, alizarine yellow R, indigo carmine, or any combination thereof. The pH indicator is chosen, in part, based on the expected change in pH of the sample during the incubation step. In embodiments, the pH indicator in the media is between about 0.0001 % and about 0.01%, between about 0.001% and about 0.005%, between about 0.001% and about 0.004%, or about 0.002%. In embodiments, the pH indicator is present in the media in an amount to effectively change the color of the sample when the pH of the sample increases or decreases with bacteria growth.

[0051] In embodiments, the weak acid is a dihydroxybenzoic acid derivative such as vanillic acid, syringic acid, veratric acid, other dihydroxybenzoic acid derivative, dimethoxybenzoic acid, dimethoxybenzoic acid derivative, or any combination thereof. Theweak acid is selected for its ability to inhibit growth of the suspected bacteria in the liquid that is chosen for testing. Generally, if vanillic acid inhibits growth of a bacteria so will veratric acid. Accordingly, it should be understood that the use of vanillic acid in this disclosure can be exchanged for veratric acid. In embodiments, the total weak acid (e.g., vanillic acid, syringic acid, veratric acid, or a combination thereof) in the media is between about 25 mg / 1 and about 300 mg / 1, or between about 50 mg / 1 and about 250 mg / 1, or between about 75 mg / 1 and about 125 mg / 1, or about 100 mg / 1. In embodiments, the total weak acid in the media is greater than about 25 mg / 1, greater than about 50mg / l, greater than about 75 mg / 1, greater than about 100 mg / 1, greater than about 150 mg / 1, greater than bout 200 mg / 1, greater than about 250 mg / 1, or greater than about 300 mg / 1. In embodiments, the total weak acid in the media is less than about 300 mg / 1, less than about 250 mg / 1, less thana bout 200 mg / 1, less than about 150 mg / 1, less than about 125 mg / 1, less than about 100 mg / 1, less than about 75 mg / 1, or less than about 50 mg / 1. In embodiments where the contribution of weak acid is from both vanillic acid syringic acid, and veratric acid (or any other acid), the contributions can range from 0.01% to 99.99% of the total acid contribution. For example, in embodiments where the total acid contribution in the media is 100 mg / 1, vanillic acid may be 25 mg / 1 and veratric acid may be 75 mg / 1. In other examples, either of vanillic or veratric acid may be replaced with syringic acid. In some embodiments, only one acid may be used rather than a combination of vanillic, syringic, and / or veratric acids.

[0052] In embodiments, a sterile container is a tube, a flask, and / or a beaker. In embodiments, a sterile container must be susceptible to agitation, e.g., shaking on an orbital shaker, during a portion of or all of the incubation step. The sterile container should be selected based on the size of the sample that is the subject of the testing. In a preferred embodiment, the sterile container will be large enough to promote agitation and aerobic growth of a guaiacol- producing bacteria. In embodiments, when the volume of the sample is less than 15 ml then the sterile contain is generally less than 50 ml. In embodiments, when the volume of the sample is greater than or equal to 15 ml then the sterile container is greater than 50 ml. In embodiments, the volume of the sample to the volume of the sterile container is about 1 : 1.5 to about 1 :6, or about 1 : 1.5 to about 1 :5, or about 1 : 1.5 to about 1 :4, or about 1 :2 to about 1 :6 or about 1 :3 to about 1 :6, or about 1 :4 to about 1 :6.

[0053] In embodiments of the present disclosure, methods may further comprise the step of preparing the media before step (a). For example, methods of the present disclosure include mixing media comprising a yeast extract, Bromophenol Blue, and vanillic acid in water to create a media prior to step (a), and adjusting the pH of the media using tartaric acid,hydrochloric acid, or another acid , to between a pH of 3.9 and a pH of 4.5, to between a pH of 4.0 and a pH of 4.4, to between a pH of 4.1 and a pH of 4.3, to about a pH of 4.2 prior to step (a). In embodiments, the mixing step before step (a) comprises about 2 g / 1 of yeast extract, about 0.002% of bromophenol Blue, and about 100 mg / 1 of vanillic acid, and the pH of the media is adjusted to about 4.2. In certain embodiments, the pH of the media is adjusted to a pH below the pH where the pH indicator in the media changes color. In preferred embodiments, the pH in the media is adjusted to a pH close enough to the pH where the pH indicator in the media changes color such that growth of the bacteria during incubation will result in the pH of the sample rising beyond where the pH indicator in the media changes color. In embodiments, the pH of the media is adjusted to between about 2.0 and about 6.0, or between about 2.0 and about 5.0, or between about 3.0 and about 5.0, or between about 3.5 and about 4.5. In embodiments, the pH of the media is adjusted to below about 8.0, or below about 7.0, or below about 6.0, or below about 5.5, or below about 5.0, or below about 4.6, or below about 4.4, or below about 4.3, or below about 4.0, or below about 3.0, or below about 2.5. In embodiments, the pH of the media is adjusted to greater than about 2.0, or greater than about 2.5, or greater than about 3.0, or greater than about 3.5, or greater than about 4.0, or greater than about 4.1, or greater than about 4.2, or greater than about 4.5, or greater than about 5.0.

[0054] In embodiments, the methods of the present disclosure further comprise the step of autoclaving the media for about 5 minutes to about 25 minutes, for about 10 minutes to about 20 minutes, or for about 15 minutes before the creation of a sample. Autoclaving the media is important to remove any potential bacteria contaminants that were introduced during the preparation of the media. In embodiments, the media is autoclaved for greater than about 5 minutes, greater than about 10 minutes, greater than about 15 minutes, greater than about 20 minutes, greater than about 25 minutes, or for the minimal time period necessary to eliminate any potential bacteria contamination in the media. In embodiments, autoclaving the media is at a temperature between about 100°C and about 150°C, between about 110°C and about 140°C, between about 115°C and about 130°C, or about 121°C. In embodiments, autoclaving the media is at a temperature greater than about 100°C, or greater than about 110°C, or greater than about 120°C, or greater than about 130°C. In embodiments, autoclaving the media is at a temperature less than about 150°C but greater than a temperature necessary to eliminate any potential bacteria contamination in the media.

[0055] In embodiments of the present disclosure, the incubation of the sample during the incubation step is for a time long enough to ensure that any bacteria in the sample (contributed by the liquid) is capable of growing enough so that visible or olfactory inspection reveals thepresence or absence of bacteria. For example, in embodiments, incubating the sample in the sterile container is for an amount of time necessary to determine whether the sample has undergone a color change, an odor change, and / or a change in clarity. In embodiments, incubating the sample in the sterile container is for about 1 hour to about 6 days, or for about 5 hours to about 5 days, or for about 12 hours to about 4 days, or for about 12 hours to about 3 days, or for about 24 hours to about 3 days, or for about 2 days to about 3 day, or for about 3 days to about 4 days, or for about 3 days. In embodiments, incubating the sample in the sterile container is for less than about 5 days, or for less than about 4 days, or for less than 3 days, or for less than 2 days, or for less than 24 hours. In embodiments, incubating the sample in the sterile container is for greater than about 2 hours, or for greater than about 4 hours, or for greater than about 6 hours, or for greater than about 8 hours, or for greater than about 10 hours, or for greater than about 12 hours, or for greater than about 24 hours, or for greater than about 2 days, or for greater than about 3 days.

[0056] In embodiments, incubating the sample in a sterile container includes agitation of the sample. Agitation is used broadly and is meant to convey any motion or vibration of the sample, e.g., shaking, vibrating, swirling, vortexing, and / or generally disrupting the sample in the sterile container so as to promote growth of the bacteria in the sample. In embodiments, agitation of the sample is by shaking the sample at between about 75 rpm and about 250 rpm, or between about 100 rpm and about 200 rpm, or about 150 rpm.

[0057] In embodiments, incubating the sample in the sterile container is at a temperature that promotes growth of the bacteria suspected to have contaminated the liquid. In embodiments, incubating a sample in a sterile container in step (b) is at a temperature of between about 35°C and about 55°C, between about 40°C and about 50°C, or about 45°C, or about 37°C. In embodiments, incubating a sample in a sterile container is at a temperature of greater than about 35°C, or greater than about 36°C, about 37°C, greater than about 40°C, greater than about 43°C, about 45°C, or greater than about 47°C. In embodiments, incubating a sample in a sterile container is at less than about 50°C, less than about 46°C, 43 °C, less than about 40°C, or less than about °38°C.

[0058] In embodiments of disclosed methods, mixing a media with a volume of liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample comprises mixing the media with the liquid between about a 150: 1 ratio to about a 1 : 10 ratio, or between about a 20: 1 ratio to about a 1 : 1 ratio, or between about 15: 1 ratio to about a 5:1 ratio, or between about a 8: 1 ratio to about a 10: 1 ratio, or about a 9: 1 ratio, or between about a 90: l ratio to about a 110: 1 ratio, or between about a 95: 1 ratio to about a 105: 1 ratio, or abouta 99: 1 ratio. In embodiment, the ratio of the media to the liquid is between about 7: 1 to about 11 : 1, or between about 8: 1 to about 10: 1, or about 9: 1 when the Brix level of the liquid is less than 20°. In embodiments, the sample is about 9 ml of media and about 1 ml of liquid when the brix value of the liquid is less than 20°. In certain embodiments, a ratio of the media to the liquid is between about a 90: 1 ratio to about a 110: 1 ratio, or between about a 95: 1 ratio to about a 105: 1 ratio, or about a 99: 1 ratio when the Brix level of the liquid is greater than or equal to 20°. In embodiments, the sample is about 99 ml of media and about 1 ml of liquid when the Brix value of the liquid is greater than or equal to 20°.

[0059] In embodiments, determining whether a sample has undergone a color change, an odor change, and / or a change in clarity following step (c) requires determining whether the sample has undergone any two of a color change, an odor change, or a change in clarity. In embodiments, determining whether a sample has undergone a color change, an odor change, and / or a change in clarity following step (c) requires determining whether the sample has undergone all three of a color change, odor change, or change in clarity. While not required, in embodiments of the disclosed methods, the determination of a color change in step (d) is by visual inspection, and if the color of the sample changes (e.g., to purple if the pH indicator is Bromophenol Blue) following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol-producing bacteria. While not required, in embodiments, if the determination of an odor change in step (d) is by olfactory inspection (either identified by human senses or analytically by laboratory instrumentation), and if the odor of the sample has smoky and / or medicinal notes following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol-producing bacteria. While not required, in embodiments, the determination of a change in clarity in step (d) is by visual inspection, and if the sample has turbidity following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for guaiacol- producing bacteria.

[0060] In certain embodiments of the disclosed methods, (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) and / or (e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d) further comprises subjecting the sample to spectrophotometric and / or fluorometric analysis. This may include mass spectrometry, flow cytometry, gas chromatography-mass spectrometry, UV-visible spectrum by plate reader, or another rapid microbial detection system.

[0061] In a specific embodiment of the presently disclosed methods, a method of determining whether a liquid contains a guaiacol-producing bacteria further comprises the step of preparing the media before step (a), comprising mixing between about 1.8 g / 1 and about 2.2 g / 1 yeast extract, between about 0.001% and about 0.003% Bromophenol Blue, and between about 80 mg / 1 and about 120 mg / 1 vanillic acid with water to form the media, adjusting the pH of the media to between a pH of 4.1 and a pH of 4.3, and autoclaving the media for about 10 minutes to about 20 minutes at a temperature of about 115°C to about 125°C, wherein the liquid is a fruit juice (e.g., orange juice) and the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria, wherein the mixing of the media and the fruit juice to form a sample in step (a) is at a ratio of media to fruit juice of about 15: 1 to about 7: 1 if the Brix value of the orange juice is below 20°, or at a ratio of media to orange juice of about 110: 1 to about 80: 1 if the Brix value of the orange juice is greater than or equal to 20°, wherein the incubating the sample in step (c) is for at least 72 hours at a temperature of between about 37°C and about 50°C, wherein the determination of a color change in step (d) is by visual inspection, and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the fruit juice, is positive for a guaiacol-producing bacteria.

[0062] Additional presently disclosed methods include methods of detecting guaiacolpositive thermophilic acidophilic bacteria, the method comprising the steps and compositions disclosed herein. Additional presently disclosed methods include methods of determining whether an acidic fruit juice is viable for commercial sale and / or consumption by a consumer, the method comprising the steps and compositions disclosed herein, where the acidic fruit juice is not viable for commercial sale and / or consumption by a consumer if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria. Additional presently disclosed methods include methods of determining whether to accept a shipment of an acidic fruit juice, the method comprising the steps and compositions disclosed herein, where the shipment of acidic fruit juice is declined and / or returned if the acidic fruit juice contains guaiacol -positive thermophilic acidophilic bacteria. Additional presently disclosed methods include methods of determining whether to package an acidic fruit juice into personal commercial containers having a size between about 0.1 liter and about 2.0 liters, the method comprising the steps and compositions disclosed herein, where the determination is no if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.

[0063] FIG. 1 discloses an embodiment of the present disclosure for detecting a guaiacol- producing bacteria and / or a veratric acid resistant bacteria in a liquid 100. Generally, a nutrientextract 102, a pH indicator 104, a weak acid 106, and water 108, consistent with the disclosures herein, are selected for use in a media. In step 110, the nutrient extract 102 is mixed with a pH indicator 104, a weak acid 106, and water 108 to create a media and the pH of the media is adjusted to a pH appropriate for the pH indicator 104 according to the present disclosure, e.g., a pH that will result in an increase in pH beyond the pH value where the pH indicator changes color. In step 112, the media is autoclaved to remove any bacteria contamination that was introduced during step 110 according to the present disclosure. In step 112, the liquid (e.g., juice) is diluted with media from step 112 in a sterile container to create a sample according to the disclosure herein. In step 116, the sample (media plus liquid) is incubated according to the present disclosure. In step 118, the results of the incubation step are determined according to the present disclosure, e.g., determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) requires determining whether the sample has undergone any one of a color change, odor change, or change in clarity.

[0064] FIG. 2 discloses an embodiment of the present disclosure for detecting a guaiacol- producing and / or a veratric acid resistant bacteria in a liquid 200. Generally, a yeast extract 202, Bromophenol Blue 204, vanillic acid 206, and water 208, consistent with the disclosures herein, are selected for use in a media. In step 210, a yeast extract 202, Bromophenol Blue 204, vanillic acid 206, and water 208 are mixed to create a media and the pH of the media is adjusted to a pH of about 4.2. In step 212, the media is autoclaved for about 15 minutes at about 121°C to remove any bacteria contamination that was introduced during step 210. In step 214, a fruit juice is diluted with media from step 212 in a sterile container to create a sample according to the disclosure herein. In step 216, the sample (media plus fruit juice at a concentration of about 9: 1) is incubated at 45°C for about 3 days in a rotary shaker. In step 218, it is determined whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) by determining whether the sample has undergone any one of a color change, odor change, or change in clarity. In optional step 220, the results of step 218 are confirmed via spectrophotometric and / or fluorometric techniques.

[0065] FIGS. 3A and 3B illustrate results from the method disclosed in FIG. 2. Specifically, FIG. 3 A is an example of the detection of growth of guaiacol-positive TAB from contaminated fruit juice with media containing yeast extract, Bromophenol Blue, vanillic acid, and water, after three days of incubation. The left tube shows 1 ml of a juice contaminated with guaiacol-negative TAB with 9 ml of media (control). The right tube shows 1 ml of fruit juice contaminated with guaiacol-positive TAB mixed with 9 ml of media. Notably, the tube on the left (control) is yellowish brown, clear, and does not have a smoky or medicinal smell, whilethe tube on the right is purple, cloudy, and has a smoky / medicinal smell. FIG. 3B an example of several serial dilutions of fruit juice containing guaiacol-positive TAB mixed in media according to FIG. 2 to roughly calculate the CFUs per ml that can trigger the color change of the media. All tubes contained 1 ml of a fruit juice mixed with 9 ml of media (yeast extract, Bromophenol Blue, vanillic acid, and water). The CFUs / ml were calculated by plating serial dilutions on agar plates and counting the colonies. Results were taken after three days. In the example in FIG. 3B, B.1 is sterile juice used to dilute contaminated juice with guaiacol-positive TAB. No heat shock was carried out. B.2: was 400CFUs / ml (1x10°), B.3: was 40CFUs / ml (IxlO-1), B.4: was 4CFUs / ml (IxlO-2), B.5: was <1 CFUs / ml (IxlO-4), B.6: was <1 CFUs / ml, negative result (IxlO-5). Notably, Sample B. l is yellowish brown, Samples B.2-B.4 are all purple. Sample B.5 is also purple but slightly less bright. Sample B.6 is yellowish brown. Accordingly, methods and compositions of the present disclosure can determine contamination of a liquid with a guaiacol-producing bacteria down to less than 1 CFU / ml.Compositions of the present disclosure.

[0066] Compositions of the present disclosure include the compositions previously discussed and disclosed above and further discussed below. For the sake of brevity, not all embodiments of compositions discussed above are repeated here. In embodiments, compositions for detecting a guaiacol-producing bacteria in a liquid comprise: a nutrient extract, a pH indicator, a weak acid, and water. In embodiments, a guaiacol-producing bacteria is Alicyclobacillus spp., Bacillus sublilis. Streptomyces spp., or any combination thereof. In a specific embodiment, the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria (guaiacol-positive TAB).

[0067] In embodiments, additional reagents may be used including, but not limited to valine or other amino acids. Valine may be used as a reagent to improve the germination of spores.

[0068] In embodiments, compositions of the present disclosure are a liquid, a powder, a semi-dry mixture, or any combination thereof. In specific embodiments, compositions are not solid or semi-solid at room temperature or at an incubation temperature.

[0069] In embodiments, the guaiacol-producing bacteria is an edible liquid. The edible liquid may be acidic, and may be fruit juice including oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof. In certain embodiments, the edible liquid suspected of contamination is an orange juice. In embodiments, a nutrient extract is a yeast extract. The nutrient extract, e.g., yeast extract, provides a source of vitamins and nitrogen to support bacteria growth shouldbacteria be present in the liquid suspected of being contaminated with bacteria. In embodiments, the pH indicator changes color with an increase in pH as previously discussed. In an embodiment, the pH indicator is Bromophenol Blue (3', 3", 5', 5" tetrabromophenolsulfonphthalein). In an embodiment, the weak acid is a dihydroxybenzoic acid derivative such as vanillic acid, syringic acid, veratric acid, other dihydroxybenzoic acid derivative, dimethoxybenzoic acid, dimethoxybenzoic acid derivative, or any combination thereof. In embodiments, the composition is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water. In a preferred embodiment, the composition is not a solid medium, e.g., an agar dish, at room temperature or at an incubation temperature.

[0070] In embodiments, the nutrient extract (e.g., yeast extract) in the composition is between about 0.5 g / 1 to about 4.0 g / 1, between about 1 g / 1 to about 3 g / 1, between about 1.5 g / 1 to about 2.5 g / 1, or about 2.0 g / 1. In embodiments, the pH indicator is Bromophenol Blue and is between about 0.001 % and about 0.003%, or about 0.002% in the composition. In embodiments, the weak acid is vanillic acid, syringic acid and / or veratric acid and is between about 25 mg / 1 and about 300 mg / 1, or between about 50 mg / 1 and about 200 mg / 1, or between about 75 mg / 1 and about 125 mg / 1, or about 100 mg / 1 in the composition. In embodiments, the pH of the composition is between a pH of 3.9 and a pH of 4.5, between a pH of 4.0 and a pH of 4.4, between a pH of 4.1 and a pH of 4.3, or about a pH of 4.2. In a preferred embodiment, the nutrient extract is about 1.8 g / 1 to about 2.2 g / 1 yeast extract, the pH indicator is about 0.002% Bromophenol Blue, and the weak acid is about 100 mg / 1 of vanillic acid, and the composition has a pH of about 4.1 to about 4.3Kits of the present disclosure.

[0071] Kits of the present disclosure include compositions presently disclosed for using any of the methods presently disclosed. In an embodiment, a kit for detecting guaiacol-positive thermophilic acidophilic bacteria in a liquid comprises a presently disclosed composition, and instructions for using the kit to detect guaiacol -positive thermophilic acidophilic bacteria in a liquid according to a presently disclosed method. In embodiments, a kit of the present disclosure may further comprise at least one container to hold a composition of the present disclosure. In embodiments, the container is filled with a composition of the present disclosure and autoclaved prior to inclusion in the kit so a consumer of the kit is provided with ready to use media for use according to a method of the present disclosure.

[0072] The following Examples are given to illustrate the inventive method, compositions, and kits for determining whether a liquid contains a guaiacol-producing bacteria in more detail, although the scope of the invention is not limited thereby in any way.EXAMPLES

[0073] The following examples illustrate embodiments of the present disclosure, but should not be construed as limiting the present disclosure.Example 1 - Detection of Guaiacol-Positive Bacteria Using Vanillic Acid.Background and Objective

[0074] Thermophilic Acidophilic Bacteria (TAB) are a group of non-pathogenic bacteria commonly found to spoil beverages. TAB have the potential to negatively affect the final product due to the production of the off-note guaiacol, a phenolic compound that imparts undesirable smoke-like, medicinal-like or antiseptic-like flavour to the juice. Current methods of testing for the presence of TAB are tedious and time-consuming, thus the present method using vanillic acid and a pH indicator was produced.

[0075] Only TAB containing the genes able to convert vanillic acid into a lesser toxic compound, such as guaiacol, will survive in the media. Furthermore, the media contains a pH indicator, Bromophenol Blue. This pH indicator is yellow at pH 3.0 and purple at pH 4.6. The transformation of vanillic acid to guaiacol generates a change in pH, as vanillic acid is a weak acid and guaiacol is a weak base.Example media to be evaluated for the study

[0076] The media was prepared using 2g / L of yeast extract, 0.002% of Bromophenol blue, and 100 mg / L of vanillic acid in water. The media is prepared in approximately 500 mL of water. The reagents are dissolved in approximately 450 mL of water. After the reagents are dissolved, the volume of the media is adjusted to 500 mL. In embodiments, other volumes (100 mL, 250 mL, 1 L, etc.) may be prepared.Procedure

[0077] The procedure was conducted according to the following steps:

[0078] 1. Prepare media containing vanillic acid (vanillic acid media, VAM).

[0079] The media was prepared by mixing 2 g / L of yeast extract, 0.002% of BromophenolBlue, and 100 mg / L of vanillic acid in water to create a solution. The solution was stirred until all components were completely dissolved and the pH was adjusted using a pH meter to 4.2 using tartaric acid. The final media solution was autoclaved at 121°C for 15 minutes.

[0080] 2. Prepare the sample

[0081] For this study, 1 g of an ingredient with suspected guaiacol presence, or 1 mL of a finished product was used as the sample size. The sample is mixed with 9 mL of the vanillic acid media prepared above. For the purpose of this study, only 1 g or 1 mL of a sample was used, however the example may be scaled accordingly (i.e., 1 :9, 2: 18, 3:27, etc.). For samples with a Brix level of more than 20°, a dilution was used. The dilution was created using 1 g or 1 mL of the sample, with 99 mL of the vanillic acid media.

[0082] 3. Place the media in sterile tubes or containers

[0083] The samples mixed with the vanillic acid media were then placed in a sterile tube or container. Because TAB are aerobic, access to oxygen is required in order to facilitate the conversion of vanillic acid to guaiacol. If the volume of the sample was smaller than 15 mL, a 50-mL Falcon tubes was used. If the volume of the sample was more than 15 mL, a larger flask was used.

[0084] 4. Mix the samples with the liquid media

[0085] The samples were mixed with the vanillic acid media and incubated at 45°C for up to five days. Orbital shaking of 150 rpm was used during the incubation period.

[0086] 5. Reading the results

[0087] The results were read according to three criteria:• Color change: samples that are positive for guaiacol TAB will turn bright purple, in the presence of Bromophenol Blue.• Odor: guaiacol-positive samples will have smoky / medicinal olfactory notes.• Visual growth: turbidity was observed in guaiacol-positive samples.Observations

[0088] The observed results indicate that vanillic acid is toxic for both positive and negative-guaiacol TAB, which results in growth inhibition. Therefore, only guaiacol-positive TAB are able to convert the vanillic acid to a less toxic compound and survive the vanillic acid media.

[0089] The vanillic acid media indicated that the presence of guaiacol-positive TAB produced a color change in the test tubes in shaking, and aerobic conditions in as little as three days. Samples with fewer than 10 CFUs / mL (colony forming units) also showed positive results using the vanillic acid media, indicating that heat is not required even for samples with very little bacteria present.Efficacy

[0090] The study showed that faster methodologies for testing the presence of guaiacolpositive TAB are just as effective and may be tested directly in one test tube rather than using individual agar plates. Further, less medium was successfully used for testing (9 mL of vanillic acid media, compared to 18 mL of solid media for an agar plate). The step of heat shock was not required according to the present methodology, which reduces time and cost when compared to current guaiacol testing methods.

[0091] Positive test results were shown in as little as three days. Further, the media has shown to be selective, and effectively select for guaiacol-producing TAB when vanillic acid is combined with high incubation temperatures, and a low pH.Example 2 — Detection of Guaiacol-Positive Bacteria Using Veratric Acid.Background and Objective

[0092] The background and objective are the same as Example 1.Example media to be evaluated for the study

[0093] The media was prepared using 2g / L of yeast extract, 0.002% of Bromophenol blue, and 100 mg / L of veratric acid in water.Procedure

[0094] The procedure was conducted according to the following steps:

[0095] 1. Prepare the media containing veratric acid (veratric acid media, VAM)

[0096] The media was prepared by mixing 2 g / L of yeast extract, 0.002% of Bromophenol Blue, and 100 mg / L of veratric acid in water to create a solution. The solution was stirred until all components were completely dissolved and the pH was adjusted to 4.2. The final media solution was autoclaved at 121°C for 15 minutes.

[0097] 2. Prepare the sample

[0098] For this study, 1 g of an ingredient with suspected guaiacol presence, or 1 mL of a finished product is used as the sample size. The sample is mixed with 9 mL of the veratric acid media. For the purpose of this study, only 1 g or 1 mL of a sample was used, however the example may be scaled accordingly (i.e., 1 :9, 2: 18, 3:27, etc.). For samples with a Brix level of more than 20°, a dilution was used. The dilution was created using 1 g or 1 mL of the sample, with 99 mL of the veratric acid media.

[0099] 3. Place the media in sterile tubes or containers

[0100] The samples mixed with the veratric acid media were then placed in a sterile tube or container. Because TAB are aerobic, access to oxygen is required in order to facilitate theconversion of veratric acid to guaiacol. If the volume of the sample was smaller than 15 mL, a 50-mL Falcon tubes was used. If the volume of the sample was more than 15 mL, a large flask was used.

[0101] 4. Mix the samples with the liquid media

[0102] The samples were mixed with the veratric acid media and incubated at 45°C for up to five days. Orbital shaking of 150 rpm was used during the incubation period.

[0103] 5. Reading the results

[0104] The results were read according to three criteria:• Color change: samples that are positive for guaiacol TAB will turn bright purple, in the presence of Bromophenol Blue.• Odor: guaiacol-positive samples will have smoky / medicinal olfactory notes.• Visual growth: turbidity was observed in guaiacol-positive samples.Observations

[0105] The observed results indicate that veratric acid is toxic for both positive and negative-guaiacol TAB, which results in growth inhibition. Therefore, only guaiacol-positive TAB are able to convert the veratric acid to a less toxic compound and survive the veratric acid media.

[0106] The veratric acid media indicated that the presence of guaiacol -positive TAB produced a color change in the test tubes in shaking, and aerobic conditions in as little as three days. Samples with fewer than 10 CFUs / mL (colony forming units) also showed positive results using the veratric acid media, indicating that heat is not required even for samples with very little bacteria present.Efficacy

[0107] The study showed that faster methodologies for testing the presence of guaiacol - positive TAB are just as effective and may be tested directly in one test tube rather than using individual agar plates. Further, less medium was successfully used for testing (9 mL of veratric acid media, compared to 18 mL of solid media for an agar plate). The step of heat shock was not required according to the present methodology, which reduces time and cost when compared to current guaiacol testing methods.

[0108] Positive test results were shown in as little as three days. Further, the media has shown to be selective, and effectively select for guaiacol-producing TAB when veratric acid is combined with high incubation temperatures, and a low pH.Example 3 — Detection of Guaiacol-Positive Bacteria Using Syringic Acid.Background and Objective

[0109] The background and objective are the same as Example 1.Example media to be evaluated for the study

[0110] The media was prepared using 2g / L of yeast extract, 0.002% of Bromophenol blue, and syringic acid in water.Procedure

[0111] The procedure was conducted according to the following steps:

[0112] 1. Prepare the media containing syringic acid (syringic acid media)

[0113] The media was prepared by mixing 2 g / L of yeast extract, 0.002% of Bromophenol Blue, and syringic acid in water to create a solution. The solution was stirred until all components were completely dissolved and the pH was adjusted to 4.2. The final media solution was autoclaved at 121°C for 15 minutes.

[0114] 2. Prepare the sample

[0115] For this study, 1 g of an ingredient with suspected guaiacol presence, or 1 mL of a finished product is used as the sample size. The sample is mixed with 9 mL of the syringic acid media. For the purpose of this study, only 1 g or 1 mL of a sample was used, however the example may be scaled accordingly (i.e., 1 :9, 2: 18, 3:27, etc.). For samples with a Brix level of more than 20°, a dilution was used. The dilution was created using 1 g or 1 mL of the sample, with 99 mL of the syringic acid media.

[0116] 3. Place the media in sterile tubes or containers

[0117] The samples mixed with the veratric acid media were then placed in a sterile tube or container. Because TAB are aerobic, access to oxygen is required in order to facilitate the conversion of syringic acid to guaiacol. If the volume of the sample was smaller than 15 mL, a 50-mL Falcon tubes was used. If the volume of the sample was more than 15 mL, a large flask was used.

[0118] 4. Mix the samples with the liquid media

[0119] The samples were mixed with the veratric acid media and incubated at 45°C for up to five days. Orbital shaking of 150 rpm was used during the incubation period.

[0120] 5. Reading the results

[0121] The results were read according to three criteria:• Color change: samples that are positive for guaiacol TAB will turn bright purple, in the presence of Bromophenol Blue.Odor: guaiacol-positive samples will have smoky / medicinal olfactory notes.• Visual growth: turbidity was observed in guaiacol-positive samples.Observations

[0122] The observed results indicate that syringic acid is toxic for both positive and negative-guaiacol TAB, which results in growth inhibition. Therefore, only guaiacol-positive TAB are able to convert the syringic acid to a less toxic compound and survive the syringic acid media.

[0123] The syringic acid media indicated that the presence of guaiacol-positive TAB produced a color change in the test tubes in shaking, and aerobic conditions in as little as three days. Samples with fewer than 10 CFUs / mL (colony forming units) also showed positive results using the syringic acid media, indicating that heat is not required even for samples with very little bacteria present.Efficacy

[0124] The study showed that faster methodologies for testing the presence of guaiacol - positive TAB are just as effective and may be tested directly in one test tube rather than using individual agar plates. Further, less medium was successfully used for testing (9 mL of syringic acid media, compared to 18 mL of solid media for an agar plate). The step of heat shock was not required according to the present methodology, which reduces time and cost when compared to current guaiacol testing methods.

[0125] Positive test results were shown in as little as three days. Further, the media has shown to be selective, and effectively select for guaiacol-producing TAB when syringic acid is combined with high incubation temperatures, and a low pH.Example 4 — Detection of Guaiacol-Positive Bacteria Using Vanillic Acid and Veratric Acid.Background and Objective

[0126] The background and objective is the same as Example 1.Example media to be evaluated for the study

[0127] The media was prepared using 2g / L of yeast extract, 0.002% of Bromophenol blue, and 50 mg / L of vanillic acid, and 50 mg / L of veratric acid in water.Procedure

[0128] The procedure was conducted according to the following steps:

[0129] 1. Prepare the media containing vanillic and veratric acid (vanillic / veratric acid media, VAM)

[0130] The media was prepared by mixing 2 g / L of yeast extract, 0.002% of Bromophenol Blue, 50 mg / L of vanillic acid, and 50 mg / L of veratric acid in water to create a solution. The solution was stirred until all components were completely dissolved and the pH was adjusted to 4.2. The final media solution was autoclaved at 121°C for 15 minutes.

[0131] 2. Prepare the sample

[0132] For this study, 1 g of an ingredient with suspected guaiacol presence, or 1 mL of a finished product is used as the sample size. The sample is mixed with 9 mL of the vanillic / veratric acid media. For the purpose of this study, only 1 g or 1 mL of a sample was used, however the example may be scaled accordingly (i.e., 1 :9, 2: 18, 3:27, etc.). For samples with a Brix level of more than 20°, a dilution was used. The dilution was created using 1 g or 1 mL of the sample, with 99 mL of the vanillic / veratric acid media.

[0133] 3. Place the media in sterile tubes or containers

[0134] The samples mixed with the vanillic / veratric acid media were then placed in a sterile tube or container. Because TAB are aerobic, access to oxygen is required in order to facilitate the conversion of vanillic and veratric acid to guaiacol. If the volume of the sample was smaller than 15 mL, a 50-mL Falcon tubes was used. If the volume of the sample was more than 15 mL, a large flask was used.

[0135] 4. Mix the samples with the liquid media

[0136] The samples were mixed with the vanillic / veratric acid media and incubated at 45°C for up to five days. Orbital shaking of 150 rpm was used during the incubation period.

[0137] 5. Reading the results

[0138] The results were read according to three criteria:• Color change: samples that are positive for guaiacol TAB will turn bright purple, in the presence of Bromophenol Blue.• Odor: guaiacol-positive samples will have smoky / medicinal olfactory notes.• Visual growth: turbidity was observed in guaiacol-positive samples.Observations

[0139] The observed results indicate that vanillic acid and veratric acid are toxic for both positive and negative-guaiacol TAB, which results in growth inhibition. Therefore, onlyguaiacol-positive TAB that are able to convert the vanillic acid and veratric acid to a less toxic compound and survive the vanillic / veratric acid media.

[0140] The vanillic / veratric acid media indicated that the presence of guaiacol-positive TAB produced a color change in the test tubes in shaking, and aerobic conditions in as little as three days. Samples with fewer than 10 CFUs / mL (colony forming units) also showed positive results using the vanillic / veratric acid media, indicating that heat is not required even for samples with very little bacteria present.Efficacy

[0141] The study showed that faster methodologies for testing the presence of guaiacolpositive TAB are just as effective and may be tested directly in one test tube rather than using individual agar plates. Further, less medium was successfully used for testing (9 mL of vanillic / veratric acid media, compared to 18 mL of solid media for an agar plate). The step of heat shock was not required according to the present methodology, which reduces time and cost when compared to current guaiacol testing methods.

[0142] Positive test results were shown in as little as three days. Further, the media has shown to be selective, and effectively select for guaiacol-producing TAB when vanillic acid and veratric acid are combined with high incubation temperatures, and a low pH.

[0143] All of the compositions, methods, and kits disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, methods, and kits of this disclosure have been described in terms of the foregoing illustrative embodiments, it will be apparent to those of skill in the art that variations, changes, modifications, and alterations may be applied to the composition, methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the true concept, spirit, and scope of the disclosure. More specifically, it will be apparent that certain agents, additives, and ingredients that are similar according to their physical, chemical, and / or physiological properties may be substituted for the agents, additives and ingredients described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the hereinafter appended claims.

[0144] The following numbered clauses define further example aspects and features of the present disclosure:1. A method of determining whether a liquid contains a guaiacol-producing bacteria, the method comprising the steps of:(a) mixing a media that changes color with a change in pH with a volume of a liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample;(b) placing the sample in a sterile container;(c) incubating the sample in the sterile container at about 30°C to about 60°C;(d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c); and(e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d).2. The method of clause 1, wherein the guaiacol-producing bacteria is Bacillus spp., Alicyclobacillus spp., Bacillus subtilis, Streptomyces spp., or any combination thereof.3. The method of any one of clauses 1-2, wherein the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria (guaiacol-positive TAB).4. The method of any one of clauses 1-3, wherein the liquid is an edible liquid.5. The method of any one of clauses 1-4, wherein the edible liquid is acidic.6. The method of any one of clauses 1-5, wherein the edible liquid is a fruit juice including oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof, a vegetable juice concentrate or puree, a tea-containing ingredient or product, or a flavored water.7. The method of any one of clauses 1-6, wherein the edible liquid is orange juice.8. The method of any one of clauses 1-7, wherein the media comprises a nutrient extract, a pH indicator, a weak acid, and water.9. The method of any one of clause 1-8, wherein the nutrient extract provides a source of vitamins and nitrogen.10. The method of any one of clauses 1-9, wherein the nutrient extract is a yeast extract.11. The method of any one of clauses 1-10, wherein the pH indicator changes color with an increase in pH.12. The method of any one of clauses 1-11, wherein the pH indicator is Bromophenol Blue (3',3",5',5"-tetrabromophenolsulfonphthalein).13. The method of any one of clauses 1-12, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, - C(O)H, or -COOH, R2i is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, - COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, R3is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, R4 is -H, -CH3, -CH2CH3, -OH, - C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, and R5 is -H, -CH3, - CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH.14. The method of any one of clauses 1-13, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -OH, or -OCH3, R2is -H, -OH, or -OCH3, R3 is -H, -OH, or -OCH3, R4is -H, -OH, or -OCH3, and R5is -H, -OH, or -OCH3.15. The method of any one of clauses 1-14, wherein the weak acid is vanillic acid, syringic acid, veratric acid, or any combination thereof.16. The method of any one of clauses 1-15, wherein the weak acid is vanillic acid.17. The method of any one of clauses 1-16, wherein the media is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water.18. The method of any one of clauses 1-17, wherein the sterile container is a tube, a flask, and / or a beaker.19. The method of any one of clauses 1-18, wherein the sterile container is less than 50 ml if the sample is 15 ml or less, or the sterile container is greater than 50 ml if the sample is greater than 15 ml.20. The method of any one of clauses 1-19, wherein the yeast extract is between about 0.5 g / 1 to about 4.0 g / 1, between about 1 g / 1 to about 3 g / 1, between about 1.5 g / 1 to about 2.5 g / 1, or about 2.0 g / 1.21. The method of any one of clauses 1-20, wherein the Bromophenol Blue is between about 0.001 % and about 0.003%, or about 0.002%.22. The method of any one of clauses 1-21, wherein the vanillic acid is between about 25 mg / 1 and about 300 mg / 1, or between about 50 mg / 1 and about 200 mg / 1, or between about 75 mg / 1 and about 125 mg / 1, or about 100 mg / 1.23. The method of any one of clauses 1-22, further comprising the step of preparing the media before step (a), comprising mixing a yeast extract, Bromophenol Blue, and vanillic acid in water to create a media; and adjusting the pH of the media to between a pH of 3.9 and a pH of 4.5, to between a pH of 4.0 and a pH of 4.4, to between a pH of 4.1 and a pH of 4.3, to about a pH of 4.2.24. The method of any one of clauses 1-23, wherein the mixing step before step (a) comprises about 2 g / 1 of yeast extract, about 0.002% of bromophenol Blue, and about 100 mg / 1 of vanillic acid; and the pH of the media is adjusted to about 4.2.25. The method of any one of clauses 1-24, further comprising the step of autoclaving the media for about 5 minutes to about 25 minutes, for about 10 minutes to about 20 minutes, or for about 15 minutes before the creation of a sample.26. The method of any one of clauses 1-25, wherein the autoclaving the media is at a temperature between about 100°C and about 150°C, between about 110°C and about 140°C, between about 115°C and about 130°C, or about 121°C.27. The method of any one of clauses 1-26, wherein the incubating the sample in the sterile container is for about 1 hour to about 6 days, or for about 5 hours to about 5 days, or for about 12 hours to about 4 days, or for about 12 hours to about 3 days; orfor less than 5 days, or for less than 4 days, or for less than 3 days, or for less than 2 days, or for less than 24 hours; or for greater than 2 hours, or for greater than 4 hours, or for greater than 6 hours, or for greater than 8 hours, or for greater than 10 hours, or for greater than 12 hours; or for the amount of time necessary to determine whether the sample has undergone a color change, an odor change, and / or a change in clarity.28. The method of any one of clauses 1-27, wherein the incubating the sample in the sterile container includes agitation of the sample.29. The method of any one of clauses 1-28, wherein the agitation of the sample is by shaking the sample at between about 75 rpm and about 250 rpm, or between about 100 rpm and about 200 rpm, or about 150 rpm.30. The method of any one of clauses 1-29, wherein the incubating the sample in the sterile container in step (b) is at a temperature of between about 35°C and about 55°C, between about 40°C and about 50°C, or about 45°C.31. The method of any one of clauses 1-20, wherein mixing the media with a volume of liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample comprises mixing the media with the liquid between about a 150: 1 ratio to about a l : 10 ratio, or between about a 20: 1 ratio to about a 1 :1 ratio, or between about 15: 1 to about a 5: 1 ratio, or about a 9: 1 ratio, or about a 99: 1 ratio.32. The method of any one of clauses 1-31, wherein the ratio of the media to the liquid is about 9: 1 when the Brix level of the liquid is less than 20°.33. The method of any one of clauses 1-40, wherein the sample is about 9 ml of media and about 1 ml of liquid.34. The method of any one of clauses 1-33, wherein the ratio of the media to the liquid is about 99: 1 when the Brix level of the liquid is greater than or equal to 20°.35. The method of any one of clauses 1-34, wherein the sample is about 99 ml of media and about 1 ml of liquid.36. The method of any one of clauses 1-35, wherein the determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) requires determining whether the sample has undergone any two of a color change, an odor change, or a change in clarity.37. The method of any one of clauses 1-36, wherein the determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c)requires determining whether the sample has undergone all three of a color change, odor change, or change in clarity.38. The method of any one of clauses 1-37, wherein the determination of a color change in step (d) is by visual inspection; and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol-producing bacteria.39. The method of any one of clauses 1-38, wherein the determination of an odor change in step (d) is by olfactory inspection; and if the odor of the sample has smoky and / or medicinal notes following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol- producing bacteria.40. The method of any one of clauses 1-39, wherein the determination of a change in clarity in step (d) is by visual inspection; and if the sample has turbidity following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for guaiacol-producing bacteria.41. The method of any one of clauses 1-40, wherein (d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) and / or (e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d) further comprises subjecting the sample to spectrophotometric and / or fluorometric analysis.42. The method of any one of clauses 1-41, further comprising the step of preparing the media before step (a), comprising mixing between about 1.8 g / 1 and about 2.2 g / 1 yeast extract, between about 0.001% and about 0.003% Bromophenol Blue, and between about 80 mg / 1 and about 120 mg / 1 vanillic acid with water to form the media; adjusting the pH of the media to between a pH of 4.1 and a pH of 4.3; and autoclaving the media for about 10 minutes to about 20 minutes at a temperature of about 115°C to about 125°C; wherein the liquid is orange juice and the guaiacol-producing bacteria is guaiacol -positive thermophilic acidophilic bacteria, wherein the mixing of the media and the orange juice to form a sample in step (a) is at a ratio of media to orange juice of about 15 : 1 to about 7: 1 if the Brix value of the orange juice is below 20°, or at a ratio of media to orange juice of about 110: 1 to about 80: 1 if the Brix value of the orange juice is greater than or equal to 20°,wherein the incubating the sample in step (c) is for at least 24 hours at a temperature of between about 37°C and about 50°C, wherein the determination of a color change in step (d) is by visual inspection; and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the orange juice, is positive for a guaiacol -producing bacteria.43. A method of detecting guaiacol-positive thermophilic acidophilic bacteria, the method comprising any one of clauses 1-42.44. A method of determining whether an acidic fruit juice is viable for commercial sale and / or consumption by a consumer, the method comprising any one of clauses 1-42, where the acidic fruit juice is not viable for commercial sale and / or consumption by a consumer if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.45. A method of determining whether to accept a shipment of an acidic fruit juice, the method comprising any one of clauses 1-42, where the shipment of acidic fruit juice is declined or returned if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.46. A method of determining whether to package an acidic fruit juice into personal commercial containers having a size between about 0.1 liter and about 2.0 liters, the method comprising any one of clauses 1-42, where the determination is no if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.47. A composition for detecting a guaiacol-producing bacteria in a liquid, the composition comprising: a nutrient extract; a pH indicator; a weak acid; and water.48. The composition of clause 47, wherein the wherein the guaiacol-producing bacteria is Alicyclobacillus spp., Bacillus sublilis. Streptomyces spp., or any combination thereof.49. The composition of any one of clauses 47-48, wherein the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria (guaiacol-positive TAB).50. The composition of any one of clauses 47-49, wherein the composition is a liquid, a powder, a semi-dry mixture, or any combination thereof.51. The composition of any one of clauses 47-50, wherein the liquid is an edible liquid.52. The composition of any one of clauses 47-51, wherein the edible liquid is acidic.53. The composition of any one of clauses 47-52, wherein the edible liquid is a fruit juice including oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof.54. The composition of any one of clauses 47-53, wherein the edible liquid is orange juice.55. The composition of any one of clauses 47-54, wherein the nutrient extract provides a source of vitamins and nitrogen.56. The composition of any one of clauses 47-55, wherein the nutrient extract is a yeast extract.57. The composition of any one of clauses 47-56, wherein the pH indicator changes color with an increase in pH.58. The composition of any one of clauses 47-57, wherein the pH indicator is Bromophenol Blue (3',3",5',5"-tetrabromophenolsulfonphthalein).59. The composition of any one of clauses 47-58, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, - C(O)H, or -COOH, R2i is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, - COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, R3is -H, -CH3, -CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, R4 is -H, -CH3, -CH2CH3, -OH, - C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH, and R5 is -H, -CH3, - CH2CH3, -OH, -C(O)H, -OCH3, -OCH2CH3, -COOH, C1-C3 alkyl or C1-C3 alkenyl, wherein alkyl and alkenyl may optionally be substituted by -OH, -C(O)H, or -COOH.60. The composition of any one of clauses 47-59, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -OH, or -OCH3, R2is -H, -OH, or -OCH3, R3 is -H, -OH, or -OCH3. R4is -H, -OH, or -OCH3, and R5is -H, -OH, or -OCH3.61. The composition of any one of clauses 47-60, wherein the weak acid is vanillic acid, syringic acid, veratric acid, or any combination thereof.62. The composition of any one of clauses 47-61, wherein the weak acid is vanillic acid.63. The composition of any one of clauses 47-62, wherein the composition is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water.64. The composition of any one of clauses 47-63, wherein the composition is not a solid medium, e.g., an agar dish.65. The composition of any one of clauses 47-64, wherein the yeast extract is between about 0.5 g / 1 to about 4.0 g / 1, between about 1 g / 1 to about 3 g / 1, between about 1.5 g / 1 to about 2.5 g / 1, or about 2.0 g / 1.66. The composition of any one of clauses 47-65, wherein the Bromophenol Blue is between about 0.001 % and about 0.003%, or about 0.002%.67. The composition of any one of clauses 47-66, wherein the vanillic acid is between about 25 mg / 1 and about 300 mg / 1, or between about 50 mg / 1 and about 200 mg / 1, or between about 75 mg / 1 and about 125 mg / 1, or about 100 mg / 1.68. The composition of any one of clauses 47-67, wherein the pH of the composition is between a pH of 3.9 and a pH of 4.5, between a pH of 4.0 and a pH of 4.4, between a pH of 4.1 and a pH of 4.3, about a pH of 4.2.69. The composition of any one of clauses 47-68, wherein the nutrient extract is about 1.8 g / 1 to about 2.2 g / 1 yeast extract, the pH indicator is about 0.002% Bromophenol Blue, and the weak acid is about 100 mg / 1 of vanillic acid; and the composition has a pH of about 4.1 to about 4.3.70. A kit for detecting guaiacol-positive thermophilic acidophilic bacteria in a liquid using any one of clauses 1-46, the kit comprising any one of clauses 47-69; and instructions for using the kit to detect guaiacol-positive thermophilic acidophilic bacteria in a liquid.71. The kit of clause 70, further comprising at least one container.

Claims

CLAIMSWhat is claimed is:

1. A method of determining whether a liquid contains a guaiacol -producing bacteria, the method comprising the steps of:(a) mixing a liquid media that changes color with a change in pH with a volume of a liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample;(b) placing the sample in a sterile container;(c) incubating the sample in the sterile container at about 30°C to about 60°C;(d) determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c); and(e) determining whether the liquid contains a guaiacol-producing bacteria based upon the determination in step (d), wherein the guaiacol-producing bacteria is guaiacol-positive thermophilic acidophilic bacteria (guaiacol-positive TAB), wherein the liquid is an acidic edible juice comprising one or more of the following: oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof; a vegetable juice concentrate or puree; a tea-containing ingredient or product; or a flavored water, wherein the liquid media comprises a nutrient extract, a pH indicator, a weak acid, and water, wherein the nutrient extract provides a source of vitamins and nitrogen, wherein the pH indicator changes color with an increase in pH, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -OH, or -OCH3, R2is -H, -OH, or -OCH3, R3 is -H, -OH, or -OCH3, R4is -H, -OH, or -OCH3, and R5is -H, -OH, or -OCH3.

2. The method of claim 1, wherein the guaiacol -producing bacteria is Alicyclobacillus spp., Bacillus sublilis. Streptomyces spp., or any combination thereof.

3. The method of claim 1, wherein the edible liquid is orange juice.

4. The method of claim 1, wherein the nutrient extract is a yeast extract.

5. The method of claim 1, wherein the pH indicator is Bromophenol Blue (3',3",5',5"- tetrabromophenolsulfonphthalein).

6. The method of claim 1, wherein the weak acid is vanillic acid, syringic acid, veratric acid, or any combination thereof.

7. The method of claim 1, wherein the liquid media is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water.

8. The method of claim 6, further comprising the step of preparing the liquid media before step (a), comprising mixing a yeast extract, Bromophenol Blue, and vanillic acid in water to create a media; and adjusting the pH of the media to between a pH of 3.9 and a pH of 4.5, to between a pH of 4.0 and a pH of 4.4, to between a pH of 4.1 and a pH of 4.3, to about a pH of 4.2.

9. The method of claim 8, wherein the mixing step before step (a) comprises about 2 g / 1 of yeast extract, about 0.002% of bromophenol Blue, and about 100 mg / 1 of vanillic acid; and the pH of the media is adjusted to about 4.2.

10. The method of claim 1, wherein the incubating the sample in the sterile container is for about 1 hour to about 6 days, or for about 5 hours to about 5 days, or for about 12 hours to about 4 days, or for about 12 hours to about 3 days; or for less than 5 days, or for less than 4 days, or for less than 3 days, or for less than 2 days, or for less than 24 hours; or for greater than 2 hours, or for greater than 4 hours, or for greater than 6 hours, or for greater than 8 hours, or for greater than 10 hours, or for greater than 12 hours; orfor the amount of time necessary to determine whether the sample has undergone a color change, an odor change, and / or a change in clarity.

11. The method of claim 1, wherein mixing the liquid media with a volume of liquid that is potentially contaminated with one or more guaiacol-producing bacteria to create a sample comprises mixing the media with the liquid between about a 150: 1 ratio to about a 1 : 10 ratio, or between about a 20: 1 ratio to about a 1 : 1 ratio, or between about 15: 1 to about a 5: 1 ratio, or about a 9: 1 ratio, or about a 99: 1 ratio.

12. The method of claim 11, wherein the ratio of the media to the liquid is about 9: 1 when the Brix level of the liquid is less than 20°.

13. The method of claim 12, wherein the sample is about 9 ml of media and about 1 ml of liquid.

14. The method of claim 11, wherein the ratio of the media to the liquid is about 99: 1 when the Brix level of the liquid is greater than or equal to 20°.

15. The method of claim 1, wherein the determining whether the sample has undergone a color change, an odor change, and / or a change in clarity following step (c) requires determining whether the sample has undergone any two of a color change, an odor change, or a change in clarity.

16. The method of claim 1, wherein the determination of a color change in step (d) is by visual inspection; and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol-producing bacteria.

17. The method of claim 1, wherein the determination of an odor change in step (d) is by olfactory inspection; and if the odor of the sample has smoky and / or medicinal notes following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for a guaiacol- producing bacteria.

18. The method of claim 1, wherein the determination of a change in clarity in step (d) is by visual inspection; and if the sample has turbidity following step (c) then the determination in step (e) is that the sample, and therefore the liquid, is positive for guaiacol-producing bacteria.

19. The method of claim 1, further comprising the step of preparing the media before step (a), comprising mixing between about 1.8 g / 1 and about 2.2 g / 1 yeast extract, between about 0.001% and about 0.003% Bromophenol Blue, and between about 80 mg / 1 and about 120 mg / 1 vanillic acid with water to form the liquid media; adjusting the pH of the liquid media to between a pH of 4.1 and a pH of 4.3; and autoclaving the liquid media for about 10 minutes to about 20 minutes at a temperature of about 115°C to about 125°C; wherein the liquid is orange juice and the guaiacol-producing bacteria is guaiacol -positive thermophilic acidophilic bacteria, wherein the mixing of the media and the orange juice to form a sample in step (a) is at a ratio of media to orange juice of about 15 : 1 to about 7: 1 if the Brix value of the orange juice is below 20°, or at a ratio of media to orange juice of about 110: 1 to about 80: 1 if the Brix value of the orange juice is greater than or equal to 20°, wherein the incubating the sample in step (c) is for at least 24 hours at a temperature of between about 37°C and about 50°C, wherein the determination of a color change in step (d) is by visual inspection; and if the color of the sample changes to purple following step (c) then the determination in step (e) is that the sample, and therefore the orange juice, is positive for a guaiacol-producing bacteria.

20. A method of detecting guaiacol-positive thermophilic acidophilic bacteria, the method comprising any one of claims 1-19.

21. A method of determining whether an acidic fruit juice is viable for commercial sale and / or consumption by a consumer, the method comprising claims 1, where the acidic fruit juice is not viable for commercial sale and / or consumption by a consumer if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.

22. A method of determining whether to accept a shipment of an acidic fruit juice, the method comprising claims 1, where the shipment of acidic fruit juice is declined or returned if the acidic fruit juice contains guaiacol-positive thermophilic acidophilic bacteria.

23. A method of determining whether to package an acidic fruit juice into personal commercial containers having a size between about 0.1 liter and about 2.0 liters, the method comprising claims 1, where the determination is no if the acidic fruit juice contains guaiacolpositive thermophilic acidophilic bacteria.

24. A composition for detecting a guaiacol-producing bacteria in a liquid, the composition comprising: a nutrient extract; a pH indicator; a weak acid; and water, wherein the guaiacol-producing bacteria is Alicyclobacillus spp., Bacillus sublilis. Streptomyces spp., or any combination thereof, wherein the liquid is an acidic edible juice comprising one or more of the following: oranges, limes, lemons, cranberries, plums, grapes, pomegranates, grapefruits, blueberries, pineapples, apples, peaches, mangos, or any combination thereof; a vegetable juice concentrate or puree; a tea-containing ingredient or product; or a flavored water, wherein the nutrient extract provides a source of vitamins and nitrogen, wherein the pH indicator changes color with an increase in pH, wherein the weak acid comprises a compound represented by the following formula:wherein Ri is -H, -OH, or -OCH3, R2is -H, -OH, or -OCH3, R3 is -H, -OH, or -OCH3, R4is -H, -OH, or -OCH3, and R5is -H, -OH, or -OCH3.

25. The composition of claim 24, wherein the guaiacol -producing bacteria is guaiacolpositive thermophilic acidophilic bacteria (guaiacol-positive TAB).

26. The composition of claim 24, wherein the composition is a liquid, a powder, a semidry mixture, or any combination thereof.

27. The composition of claim 24, wherein the edible liquid is orange juice.

28. The composition of claim 24, wherein the nutrient extract is a yeast extract.

29. The composition of claim 24, wherein the pH indicator is Bromophenol Blue (3',3",5',5"-tetrabromophenolsulfonphthalein).

30. The composition of claim 24, wherein the weak acid is vanillic acid, syringic acid, veratric acid, or any combination thereof.

31. The composition of claim 24, wherein the composition is substantially free of components other than a nutrient extract, a pH indicator, a weak acid, and water.

32. The composition of claim 24, wherein the nutrient extract is about 1.8 g / 1 to about 2.2 g / 1 yeast extract, the pH indicator is about 0.002% Bromophenol Blue, and the weak acid is about 100 mg / 1 of vanillic acid; and the composition has a pH of about 4.1 to about 4.3.

33. A kit for detecting guaiacol-positive thermophilic acidophilic bacteria in a liquid using any one of method claims 1-23, the kit comprising any one of composition claims 24- 32; and instructions for using the kit to detect guaiacol-positive thermophilic acidophilic bacteria in a liquid.

34. The kit of claim 33, further comprising at least one container.

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