Bioluminescent detection kit and method of microbiological contamination of food and surfaces

Description

1 / 38 KIT AND METHOD FOR BIOLUMINESCENT DETECTION OF MICROBIOLOGICAL CONTAMINATION OF FOOD AND SURFACES Certificate of Addition of Invention to invention patent BR 10 2020 000300-3, originally filed on 07 / 01 / 2020.

[001] The present invention relates to a kit and a bioluminescent method for detecting microbiological contamination, intended for quickly and accurately testing the surfaces of food and beverages, for quality control and food safety purposes for manufactured products. DESCRIPTION OF THE STATE OF THE ART

[002] Microorganisms found on the surfaces of industrial foods, especially meats, play an important role in food safety and product quality.

[003] Among the most common microorganisms are pathogenic bacteria such as Salmonella sp., Listeria monocytogenes, and Escherichia coli (E. coli), as well as fungi and yeasts that can cause food spoilage. Contamination by these microorganisms can occur during various stages, from slaughter or harvesting to food processing, handling, and storage. For example, raw meat can be naturally contaminated with bacteria present in the environment, soil, water, or animals. During processing and handling, contact with contaminated equipment, utensils, and surfaces can transfer these microorganisms to the meat, increasing the risk of cross-contamination and food safety problems.

[004] In this sense, it is essential to control and monitor the presence of these microorganisms to guarantee the quality and safety of food. Measures such as regular sanitization of equipment and surfaces, maintaining adequate storage temperatures, and using packaging Petition 870250015693, dated 26 / 02 / 2025, page 5 / 50 2 / 38 appropriate measures help minimize the risk of contamination. Food safety standards are rigorously enforced in the food industry to ensure that products are safe and meet regulatory requirements.

[005] A series of microbiological tests are regularly performed to monitor the presence of microorganisms on surfaces and in food products. Some of the most common tests include: Total Aerobic Microbial Count; Coliform Count; Specific Pathogen Screening; Mold and Yeast Count Tests; Surface Monitoring Tests; Water Sanitization Tests.

[006] Tests for detecting microorganisms based on the bioluminescence of the firefly luciferin-luciferase system in the presence of ATP leaked from live microorganisms offer an effective approach to monitoring the presence of bacteria and other organisms in samples.

[007] Bioluminescence is a natural phenomenon in which living organisms produce light as a result of biochemical reactions. This unique property has been exploited to develop rapid and sensitive methods of microbiological detection.

[008] Bioluminescence tests offer several advantages over traditional methods, most notably, these tests generally do not require lengthy cultivation steps, which significantly reduces the time needed to obtain results. Another important advantage is the ability to promote portable field applications. The ability to perform rapid, real-time testing is particularly valuable in sectors such as food and beverages, where the rapid detection of microbiological contamination is essential to ensure product safety. Petition 870250015693, dated 26 / 02 / 2025, p. 6 / 50 3 / 38

[009] Bioluminescent detection of Adenosine Triphosphate (ATP) utilizes the enzymatic reaction between luciferin and luciferase in fireflies to generate light in the presence of ATP. This reaction occurs naturally in fireflies such as the North American species Photinus pyralis and several other species worldwide, and was used based on the science that all living microorganisms necessarily use ATP to conserve metabolic energy.

[0010] Adenosine triphosphate is the universally found energy-storing molecule in all animals, plants, bacteria, yeast cells and molds, that is, even in food waste contaminated by microorganisms.

[0011] The chemical reaction for ATP detection by bioluminescence is described by the following equation. D — luferlana + O2+ ATP ^ oxyluferlana + CO2+ AMP + PPl + light Where, ATP - Adenosine Triphosphate AMP - Adenosine Monophosphate PPi - Inorganic pyrophosphate

[0012] The light emitted by this reaction is directly related to the amount of ATP present (approximately one photon of light is emitted for each molecule of ATP present). In the presence of molecular oxygen and luciferase, luciferin undergoes oxidative decarboxylation, a process that occurs in several steps, forming oxyluciferin. This process is accompanied by the emission of light. Using devices called luminometers, photometers, or photodetector cameras, it is possible to quantify the relative light intensity (number of photons per Petition 870250015693, dated 26 / 02 / 2025, page 7 / 50 4 / 38 unit of time) which provides the result in Relative Light Units (RLU) or counts per second (cps: count represents an unmetered number of photons).

[0013] In this sense, some studies and devices have been developed to perform rapid tests on surfaces in order to detect the presence of microorganisms.

[0014] US patent 6,266,177 discloses a recombinant mutant luciferase having 70% or more homology with a luciferase from Photinus pyralis (SEQ ID NO: 21), Luciola cruciata (SEQ ID NO: 14), Luciola lateralis (SEQ ID NO: 16), Luciola mingrelica (SEQ ID NO: 16) or Lampyris noctiluca (SEQ ID NO: 20), wherein the amino acid corresponding to amino acid residue number 245 or 318 in the Photinus pyralis luciferase has been substituted relative to the corresponding wild-type amino acid residue, such that the Km for ATP is increased relative to that of the corresponding non-mutated enzyme residue.The invention further discloses a fusion protein comprising said luciferase, a recombinant polynucleotide encoding said luciferase, a replication vector comprising said polynucleotide, a host cell comprising said vector, a process for producing the luciferase comprising said host cell, a method for testing the amount of ATP in a material by contacting said mutant luciferase, and a test kit comprising said mutant luciferase.

[0015] Document CN1908186 discloses a method for rapid detection of the total number of bacteria using a reagent composed of a group of somatic cell adenosine triphosphate-releasing reagents and a group of bacterial cell adenosine triphosphate-releasing reagents and luminescence. Furthermore, the invention discloses a device for determining the total number of bacteria. The device includes a bacterial ATP detection cup and a fluorescent detection device. Petition 870250015693, dated 26 / 02 / 2025, p. 8 / 50 5 / 38

[0016] Document CN105203510 discloses a rapid processing apparatus and a method for detecting ATP in the food field through food pretreatment, membrane filtration, pre-integrated treatment of microbial cell lysis and ATP, as well as the integration of reagent solution for cell lysis and ATP detection.

[0017] Document CN115433757 discloses a set of systems for detecting the level of microbial contamination, including kits, test methods and equipment. The kit comprises an ATP extraction agent, a degradation enzyme, an ATP degradation buffer and / or a microbial cell lysis agent, as well as a luminescence reaction reagent and / or a luminescence reaction buffer. The aforementioned test kit comprises the ATP extraction agent; an ATP degradation enzyme; an ATP degradation buffer and / or a microbial cell lysis agent and a luminescence reaction reagent and / or a luminescence reaction buffer.

[0018] Document EP0126019 discloses a method for measuring ATP concentration across a series of filtering membranes to concentrate organisms on the desired filtering membrane, sizing the various membranes to selectively concentrate a variety of organisms present in a single sample pass. The method includes the separation of microbial ATP, non-microbial ATP, and extracellular ATP. Furthermore, the present invention discloses a method for cell enrichment.

[0019] Document EP0542790 discloses a method for concentrating cells from a sample of liquid milk or other foods and includes the use of a lysis agent or solution and testing for contamination by pre-selected microorganisms. Furthermore, the present invention discloses a microbial test kit for use in testing milk samples comprising nucleic acid, a Petition 870250015693, dated 26 / 02 / 2025, page 9 / 50 6 / 38 carrier, a possible chelating agent if the sample is milk, enzymes, initiators and reagents.

[0020] Document JP2010268792 discloses a method for assessing the presence of bacteria on food surfaces using swabs to collect bacteria from the surface, which are then transferred to a sterile solvent and the number of bacteria is counted under an optical microscope at a predetermined magnification to measure the quantity of bacteria present. Furthermore, a measuring apparatus is disclosed that includes a light source to project light onto a predetermined wavelength region, the intensity of which varies according to the number or quantity of microorganisms. Additionally, the possibility of estimating the quantity of microorganisms from the amount of ATP is mentioned, as well as determining the amount of ATP using spectral characteristics modulated by the ATP present in the microorganisms on the surface, when applying light.

[0021] Document JPH03172194 discloses a simple and quick method for measuring the number of microorganisms, along with a corresponding device. Furthermore, a sample container used in the device is disclosed, which facilitates and speeds up the measurement of the number of bacteria, in particular, general bacteria that cause contamination during quality control in food production or preservation.

[0022] Document US2011076706 discloses methods and compositions for detecting the presence of bacteria in a sample, as well as the quantity and type of bacteria. Furthermore, the invention discloses a kit for detecting the presence of a microorganism in samples comprising a container comprising luciferin and luciferase, a container comprising a bacterial lysis reagent; and a container comprising an agent that selectively lyses a non-bacterial cell. Petition 870250015693, dated 26 / 02 / 2025, page 10 / 50 7 / 38

[0023] Document US2015140583 discloses a method for detecting microorganisms in a sample comprising the steps of concentrating microorganisms, lysing the concentrated microorganisms, mixing the microorganisms and the adenosine diphosphate substrate, generating amplified adenosine triphosphate, mixing a luciferin / luciferase reagent and the adenosine triphosphate generating light emission, and measuring the light emission for microorganism detection. Furthermore, a device and a kit are disclosed, wherein the kit comprises: a device having a single-use branched container, a disposable pre-filter and a pump; and buffers, filters, lysis reagents / extractant, substrate reagents, adenosine diphosphate, detection assay reagents, luciferin / luciferase reagent, filtration containers, concentration or detection assay containers, and instructions for using the devices, methods and detection assays.

[0024] In this sense, it is clear the importance of exploring new technologies that employ more efficient bioluminescent reagents and allow rapid surface testing of food in the industrial environment, using materials and devices that are more precise in detecting microorganisms, without the need for long incubation times and requiring minimal intervention in the tests.

[0025] In this way, the state of the art would benefit from a solution related to faster and more sensitive tests for detecting microorganisms on surfaces, which is practical and has greater sensitivity in detection, to be applied in the control of microorganisms in industry. OBJECTIVES OF THE INVENTION

[0026] In order to solve the problems of the state of the art, the present invention aims to provide a kit for detecting contamination by microorganisms present on surfaces, especially food surfaces in Petition 870250015693, dated 26 / 02 / 2025, page 11 / 50 8 / 38 industrial sector, that is portable, easy to handle and that offers fast and highly accurate results.

[0027] Furthermore, it is an objective of the present invention to provide a method for rapid and more sensitive detection of microorganisms on surfaces, especially food surfaces in the industrial sector, which is simple and can be performed in a few steps. SUMMARY OF THE INVENTION

[0028] In order to achieve the aforementioned objectives, the present invention provides a bioluminescent assay and detection kit for microbiological contamination of food and surfaces based on the use of swabs. Said kit comprises at least one sample collection swab consisting of a plastic shaft comprising cotton or sterile synthetic material at its ends, soaked in ATP extraction solution, a luminometer test tube comprising Amydetes vivianii luciferase solution and firefly luciferin, and a luminometer or photometer device.

[0029] More specifically, the kit in question comprises blue-green light-emitting luciferases for analytical assays and indication of cadmium and mercury comprising the amino acid sequence as defined in SEQ ID NO: 2, the cDNA sequence defined in SEQ ID NO: 1 of the luciferase enzyme from Amydetes viviani, wherein they are used as bright analytical reagents, as bright reporter genes and as cellular and enzymatic biosensors for the metals cadmium and mercury, and for pH at high temperatures (>30°C), further comprising: at least one sample collection swab consisting of a plastic shaft comprising cotton or sterile synthetic material at its ends, Petition 870250015693, dated 26 / 02 / 2025, page 12 / 50 9 / 38 ATP extraction solution, assay solution containing Amydetes vivianii luciferase and firefly D-luciferin; at least one test tube containing said test solution; and a device that measures luminescence intensity.

[0030] The invention further provides a bioluminescent detection method for microbiological contamination of food and surfaces, using said detection kit, and comprising the following steps: sample collection; immersion; luminescence measurement and analysis; and comparison of the luminescent signal with a standard curve.

[0031] More specifically, the method comprises blue-green light-emitting luciferases for analytical assays and indication of cadmium and mercury comprising the amino acid sequence as defined in SEQ ID NO: 2, the cDNA sequence defined in SEQ ID NO: 1 of the luciferase enzyme from Amydetes viviani, wherein they are used as bright analytical reagents, as bright reporter genes and as cellular and enzymatic biosensors for the metals cadmium and mercury, and for pH at high temperatures (>30°C), using a detection kit as defined in claim 1, the method further comprising the steps of: a) Sample collection in which the end of a swab soaked in ATP extraction solution is rubbed over the study surface in a 1cm2 quadrant, in zigzag movements while rotating its shaft, followed by at least 40 minutes of contact time; Petition 870250015693, dated 26 / 02 / 2025, p. 13 / 50 10 / 38 b) Immersion, in which the end of the swab that was in contact with the surface under study is immersed in a test tube containing between 0.5 and 1.5 mL, preferably 1 mL, of assay solution with Amydetes vivianii luciferase and firefly luciferin, followed by an additional 4 to 5 minutes of exposure time; c) Measurement and analysis of luminescence, in which luminescence is measured using a luminometric or photometric instrument that receives the tube containing the sample under study; and d) Comparison of the luminescent signal with a standard curve: comparing the luminescent signal with a standard curve (ruler) of ATP to estimate the degree of microbiological contamination of the food. BRIEF DESCRIPTION OF THE FIGURES

[0032] The subject matter of the present invention will become fully clear in its technical aspects from the detailed description that will be made based on the figures below, in which:

[0033] Figure 1: Illustrates the method for detecting microbiological contamination and the devices and reagents used.

[0034] Figure 2: Demonstrates the linearity curves of standard ATP concentration (ATP), ATP extracted from the dilution culture (Dilution) and from the bacterial swab culture (Swab) at the five concentrations used and their respective results in light intensity (CPS) using Amydetes vivianii luciferase. Petition 870250015693, dated 26 / 02 / 2025, p. 14 / 50 11 / 38

[0035] Figure 3: Refers to the linearity graphs of ATP extracted from the bacterial culture in dilution (Dilution) and from the culture on the swab (Swab) at the five concentrations used and their respective results in CPS, line equations and R2.

[0036] Figure 4: Refers to the graph of the relationship between results in CPS and in CFU / mL of the averages of three different optical densities (OD6oo = 1.08; 1.25 and 1.17) using the luciferase Amydetes vivianii.

[0037] Figure 5: Refers to the graph of the relationship between results in CPS and in CFU / mL of the averages of three different optical densities (OD6oo = 1.08; 1.25 and 1.17) using a commercial swab.

[0038] Figure 6: Refers to the graph of linearity curves constructed for standard ATP (ATP), ATP extracted from cultures on the swab (Swab) and commercial swab (3M) at five different concentrations along with their respective light intensities measured in cps. DETAILED DESCRIPTION OF THE INVENTION

[0039] In accordance with the aforementioned objectives, the present invention relates to an assay and kit and a method for bioluminescent detection of microbiological contamination of foods such as meats and beverages. The method applies the technique of immersing a swab soaked in ATP extraction solution in a luminometer tube containing 0.5 to 1.5 mL, preferably 1 mL, of assay solution with Amydetes vivianii luciferase and firefly luciferin, followed by a luminometric assay of the emitted luminescence.

[0040] Thus, the kit and method can be applied for the following purposes: (1) assays for the presence of ATP in biological samples; (2) rapid and sensitive assays for microbiological contamination of food using Petition 870250015693, dated 26 / 02 / 2025, page 15 / 50 12 / 38 swab; (3) rapid and sensitive microbiological contamination tests of beverages using swabs; and (4) adaptation for microbiological contamination testing of surfaces such as countertops and solid objects. The invention provides more sensitive analyses to ATP and therefore more accurate results regarding microbiological contamination. This is because it uses the enzyme luciferase Amydetes vivianii, which is a more efficient and stable enzyme when used as a bioanalytical reagent.

[0041] The bioluminescent detection kit for detecting microbiological contamination of food and surfaces comprises: (1) Sample collection swab consisting of a plastic shaft comprising cotton or sterile synthetic material at its ends, soaked in ATP extraction solution, (2) ATP extraction solution: The cell lysis buffer is essential for the accurate quantification of bacterial contamination. This buffer has been optimized to ensure maximum efficiency in the release of ATP from bacterial cells, without compromising the integrity of the released ATP or other components of the bioluminescent reaction. The combination of components results in cell lysis, allowing intracellular ATP to be quantified under ideal conditions for constructing an accurate response curve that correlates bacterial concentration with the measured bioluminescence intensity. The buffer composition includes: - Tris-HCl 0.10 M, pH 8.0: used as a buffer to maintain the solution pH between 7.5 and 8.5, preferably 8.0, ideal for preserving the integrity of ATP after cell lysis and the enzymatic activity of luciferase, preventing its degradation and ensuring that the measured ATP levels accurately reflect the amount present in the cells before lysis; Petition 870250015693, dated 26 / 02 / 2025, page 16 / 50 13 / 38 - TCA (Trichloroacetic Acid) 1%: used to denature proteins and precipitate biomolecules that may interfere with ATP measurement. A concentration of 0.5 to 1.5%, preferably 1%, was chosen to be sufficiently effective in precipitation without causing significant ATP degradation; and - Triton X-100 2%: a non-ionic detergent that facilitates the lysis of cell membranes, releasing intracellular contents, including ATP. A concentration of 1.5 to 2.5%, preferably 2%, is used to ensure efficient lysis of bacterial cells and other microorganisms, releasing as much ATP as possible for subsequent detection, (3) a test tube where the swab should be immersed in the presence of an assay solution containing Amydetes vivianii luciferase and firefly D-luciferin, (4) Assay solution containing Amydetes vivianii luciferase and firefly D-luciferin: The composition of the assay solution was developed to maximize the efficiency of the bioluminescent reaction and ensure accuracy in the quantification of ATP, which is a direct indicator of the presence of biological material and consists of: - Tris-HCl 0.10 M pH 8.0, ideal for the bioluminescent activity of luciferase; - MgSO4 4 mM: an essential cofactor for luciferase activity at a concentration that ensures maximum enzyme activity without being inhibited by excess magnesium; - BSA (Bovine Serum Albumin) 1 mg / mL: protein stabilizer in solution, preventing luciferase denaturation. Petition 870250015693, dated 26 / 02 / 2025, page 17 / 50 14 / 38 during the detection process, and may be between 1 mg / mL and 1.5 mg / mL, preferably 1; - DTT (Dithiothreitol) 5 mM: reducing agent used to prevent the oxidation of thiol groups in luciferase, ensuring the maintenance of its enzymatic activity over time, and can be between 4.5 mM and 15.5 mM, preferably 5 mM. - Glycerol 10%: included in the solution to increase viscosity, which helps stabilize the mixture and prevent rapid diffusion of components, ensuring a controlled and consistent reaction; - Amydetes vivenii luciferase (0.025 to 0.05 mg / mL); and - luciferin 0.5 mM: The assay solution for dipping the swab must have at least 65 pL to 80 pL, preferably 75 pL, and (5) A light detection device which may be a luminometer or photometer or photodetection camera or any other light detection equipment.

[0042] The bioluminescent method for detecting microbiological contamination of food and surfaces comprises the following steps: a) Sample collection: the sample is collected by sliding the end of a swab soaked in ATP extraction solution over the study surface in a 1cm2 quadrant, in zigzag movements while rotating its shaft, followed by 40 minutes of contact time; b) Immersion: consists of immersing the end of the swab that was in contact with the surface under study in a test tube containing 75 pL. Petition 870250015693, dated 26 / 02 / 2025, p. 18 / 50 15 / 38 of an assay solution containing Amydetes vivianii luciferase and firefly luciferin, followed by at least another 4 minutes of exposure time; c) Luminescence analysis: luminescence analysis is performed using a luminometer, photometer, or photodetector camera coupled to a CCD camera that receives the ELISA tube or plate containing the study sample(s); and d) Comparison of the luminescent signal with a standard curve: in this step, a comparison is made between the luminescent signal and a standard curve (ruler) of ATP to estimate the degree of microbiological contamination of the food.

[0043] The methodology was developed for specific microbiological testing in food, focusing on beef and chicken. Among the advantages of the invention, the suitability of the methodology for specific use in food stands out, given the lack of commercial products and prior art documents for this purpose. The commercially available products are geared towards microbiological detection on surfaces, water, dairy products, as well as protein and allergen detection on surfaces, not directly addressing the need for microbiological evaluation in meat. Furthermore, the method of the present invention provides the use of technology for the detection of high microbiological loads in meat, overcoming limitations observed with the use of commercial swabs, which do not produce results consistent enough to differentiate between foods suitable and unsuitable for consumption.

[0044] Furthermore, unlike commercially available products, which have additional components in the swab and lysis solution and an immediate analysis time, the method described in the present invention requires a longer contact time in the collection phase (40 minutes), followed by a 4-minute immersion phase. This extended time was tested and adjusted to provide greater reproducibility and accuracy in the analyses, considering that the swab is a fibrous matrix with a high capacity for absorption and progressive distribution of the liquid. Petition 870250015693, dated 26 / 02 / 2025, page 19 / 50 The 16 / 38 approach avoids immediate saturation and ensures controlled and uniform absorption, resulting in greater consistency in testing, especially in situations that demand high precision.

[0045] The method was tested with different types of swabs, produced with various absorbent materials, and showed similar results in terms of efficiency, reinforcing its applicability and reliability for microbiological detection in meat.

[0046] For its development, the Adenosine Triphosphate (ATP) detection method by bioluminescence is based on the detection of ATP from viable microorganisms, performed with test microorganisms to ensure that it is possible to meet validation criteria. Tests and standard definitions

[0047] In order to evaluate the proposed method for use in microbiological quality control, E. coli Xl1-Blue cultures were used, prepared on the day of each experiment, and five replicates were used for each analysis to allow the calculation of means, standard deviations, and precision according to parameters that will be described below. Therefore, the development and validation of the rapid microbiological method employing the ATP bioluminescence technique was first standardized using dilutions of commercial ATP at the following concentrations: 5.0E10-3, 5.0E10-4, 5.0E10-5, 5.0E10-6, and 5.0E10-7 mM, followed by standardization with cell culture and with a swab to allow comparable ATP quantifications between the sources.

[0048] The analyses were performed on an AB2200 luminometer (ATTO, Tokyo, Japan) using 75 μL of the analysis solution, 5 μL of Amydetes vivianii luciferase, and 5 μL of 10 mM luciferin. The analysis solution was previously prepared with 45 μL of 0.10 M Tris-HCl, 5 μL of 80 mM MgSO4, and 10 μL of BSA (Bovine Petition 870250015693, dated 26 / 02 / 2025, page 20 / 50 17 / 38 serum albumin) 1 mg / mL, 5 μL DTT (Dithiothreitol) 5 mM, and 15 μL of glycerol. Values ​​from luminometer analyses are given in light intensity - counts per second (CPS).

[0049] Curves were performed to standardize the culture to be used throughout the assay. For this purpose, E. coli Xl1-Blue cells were cultured in liquid LB medium containing tetracycline at 37°C until an optical density (OD600) of 1.0. From the culture, the following 1 mL dilutions in phosphate buffer were prepared: 1000, 100, 10, 1, 0.1, and 0 μL / mL. Subsequently, the dilutions were further diluted in equal parts of extraction buffer so that all ATP inside the cells would be available for quantification. The extraction buffer consisted of 0.1M Tris HCl pH 8.0, 1% TCA, and 2% Triton. After 10 minutes, 10 μL of the dilutions in extraction buffer were analyzed in a luminometer using 75 μL of the analysis solution, 5 μL of luciferase, and 5 μL of 10 mM luciferin after 4 minutes of contact between the dilution and the analysis solution.

[0050] The final standardization consisted of verifying the possibility of using swabs to collect microorganisms from diverse matrices, therefore first during the method validation and subsequently in food or on workbenches and locations that require it. Therefore, considering the 120 μL capacity of each swab, 60 μL of extraction buffer was added to the swab followed by 60 μL of the culture dilutions previously prepared in phosphate buffer. The dilution was seeded on a previously sterilized plate so that it was possible to verify the swab's ability to adsorb all the seeded content. The swabs were left to rest for 40 minutes, then the swab was inserted into a luminometer tube and after 4 minutes of waiting the swab was placed in contact with the analysis solution. Validation of a bioluminescent method for detecting ATP extracted from E. coli XL1-Blue cultures. Petition 870250015693, dated 26 / 02 / 2025, page 21 / 50 18 / 38

[0051] The Brazilian regulatory acts that guide the validation of analytical methods are Resolution RE No. 899 of May 29, 2003, with the Guide for the validation of analytical and bioanalytical methods, and Resolution RDC No. 166 of July 24, 2017, which establishes criteria for the validation of analytical methods, admitting alternative approaches for the validation of analytical methods applied to biological products and the use of other official compendia recognized by ANVISA. Therefore, the validation of the proposed method followed the validation protocols in microbiology described in the European Pharmacopoeia (Ph. Eur.) chapter 5.1.6 (Alternative methods for control of microbiological quality) and the United States Pharmacopeia (USP) chapter 5.1.6 (Alternative methods for control of microbiological quality). <1223> .

[0052] The validation procedures for the bioanalytical assay using standard commercial ATP and ATP extracted from E. coli Xl1-Blue cultures as analytes were performed in accordance with regulatory guidelines and comprise measures and requirements that will be explained below: - Linearity: Linearity aims to demonstrate the method's ability to provide results directly proportional to the concentration of the substance being analyzed, within a specified range. This parameter can be demonstrated by the coefficient of determination of the graph (R²), which should not be statistically different from 1, observing that the slope of the line (angular coefficient or slope) is different from zero. The linearity of the calibration curve was validated over three days at five different concentrations. The means, standard deviations, and precision of the three days were calculated and compared in linear curves between standard ATP, ATP extracted from the culture only, and ATP from the culture in a swab. The quadratic regression function was also calculated, as well as the coefficient of determination (R²). The acceptance criterion was > 0.99 for the correlation coefficient.For reproducibility and accuracy, based on the average of each point on the calibration curve, the acceptance criteria were up to 35%. Petition 870250015693, dated 26 / 02 / 2025, page 22 / 50 19 / 38 - Limit of detection: The limit of detection (LOD) is the smallest amount of an analyte that can be detected in a matrix. For this definition, the calculation was performed using the average of the values ​​of the analytical solution without the standard ATP (blank - LOB), the blank, the standard deviation of the blank, and the standard deviation of the lowest concentration sample from the linearity curve, according to the following formulas: LOB mediumwhite + 1.645(Dpwhite) LOD = LOB + 1.645 (DPlow concentrations sample) - Limit of quantification: The limit of quantification is the lowest value at which an analyte can be quantified in a matrix. This parameter was defined from a curve with successive dilutions of standard ATP, where the lowest reliably quantifiable value was defined as the limit of quantification and the lowest point on the linearity curve. - Inter-day and intra-day precision: Precision expresses the closeness of results obtained within the degree of dispersion of all measurements of an analyte in a sample, where analytical variability must be within established quantitative limits. Therefore, precision aims to assess whether the results obtained are repeated on different days, analyses, analysts, and laboratories, to verify their reliability. To this end, the method was reproduced on the same day (intra-day) and on different days (inter-day). Inter-day precision was verified over three days with the culture in dilution and on a swab, at the highest, medium, and lowest concentrations, while the intra-day evaluation was performed on only one day at the same three concentrations described. The final precision should be up to 35%. - Accuracy: Accuracy aims to determine if there is a significant difference between the mean results of two groups, or if the mean of the sample being analyzed presents the value observed in another group. The groups of Petition 870250015693, dated 26 / 02 / 2025, page 23 / 50 20 / 38 comparisons can be independent, or the same group in a different situation. In this validation, the accuracy of the method aims to evaluate the proximity between the data obtained in a given study and data obtained by a standard analyte. Therefore, it will be determined how close the extracted ATP will be to the standard ATP curve in the proposed method as follows: the results of the standard ATP curve and the results of ATP extracted from the diluted culture were correlated, and the results of the standard ATP curve and the results of ATP extracted from the diluted culture on the swab were compared. The comparison was analyzed by Student's t-test where the allowed recovery criterion should be between 70-100%. - Matrix effect: The matrix effect is evaluated to ensure that components present in the sample matrix will not interfere with the analysis and quantification of the analyte of interest. The interference of the response on the sample matrix was evaluated at the lower and upper concentrations of the ATP linearity curve. For the dilution, the culture was used without extracting ATP from the cells, and standard ATP was added to verify if the result would be close to the standard ATP curve. For the swab, 110 μL of sterile water was used as a diluent in the swab and 10 μL of standard ATP at the lower and upper concentrations of the linearity curve. The accuracy criterion for the Matrix Effect was established by Student's t-test between the standard ATP and the ATP evaluated under the described conditions, and should be between 70-100%. - Stability: The stability of the analytical solution is being evaluated at the highest, average, and lowest concentrations of the linearity curve over time. The stock analytical solution was tested with and without the presence of luciferin, under storage conditions of -20°C (freezer) and 5°C (refrigerator) after 15 days, 1, 3, and 6 months of storage, comparing it with an analysis performed at time zero (fresh solution) using Student's t-test. The acceptance criterion will be a maximum deviation of 30% from the fresh stock. Petition 870250015693, dated 26 / 02 / 2025, page 24 / 50 21 / 38 Relationship between CFU and bioluminescence

[0053] After method validation, it is necessary to correlate the light intensity results obtained with real values ​​used as an evaluation standard. Therefore, in order to estimate the number of colony-forming units (CFU), used as a standard unit in current regulations to define acceptable microorganism limits in food, tests were performed in triplicate with three different optical densities (OD600) in five dilutions of E. coli Xl1-Blue culture in phosphate buffer pH 8.0 (1000, 100, 10, 1 and 0.1 μL / mL). Subsequently, a graph was created with the averages of the three optical densities, obtained in CFU / mL and in CPS, after adjusting the dilution factor and defining the noise. The equation of the line generated by the graph of the relationship between CFU / mL and cps is used in subsequent calculations to estimate the number of CFU / mL based on the cps results obtained by the luminometer.

[0054] The same correlation was made with a commercial swab from the 3M brand, aiming to compare the final results in food, to weigh the advantages and disadvantages between a known and commercially available luciferase, and the Amydetes luciferase used in this invention.

[0055] The proposed method, using the luciferase Amydetes vivianii, demonstrated high bioluminescent activity and excellent stability, exhibiting reproducible and precise properties. There was a significant increase of approximately 22 times in luminescence intensity at the highest measured ATP concentration and 12 times at the most concentrated optical dilution. These results indicate superior performance when compared to traditional methods.

[0056] Figure 6 presents a comparison between measurements performed with standard ATP, a swab with Amydetes vivianii luciferase, and a commercial swab, highlighting the advantages of the proposed method. It allowed for a clear distinction between... Petition 870250015693, dated 26 / 02 / 2025, page 25 / 50 22 / 38 efficient, contaminated and uncontaminated beef and chicken in less than an hour. In terms of sensitivity, reproducibility, and precision, the method demonstrated superior performance to the commercial 3M kit, as shown in Table 18, in the Forced Degradation Assay section, described later in this document. In this table, the results of ATP quantification in foods subjected to forced degradation allowed differentiation between foods suitable and unsuitable for consumption, in accordance with current legislation – a distinction that would not be possible using the commercial methodology and swab. Food trials

[0057] After developing the method using transformed E. coli BL21-DE3 cell culture, the same method was applied to real samples to verify whether the established parameters were maintained. The objective was to estimate whether a food sample was contaminated or not, according to current laws. For this purpose, meat products such as raw beef, raw chicken, and dairy products such as type A whole milk were chosen. Food matrix effect

[0058] The interference of the food matrix in the quantification of ATP was evaluated in each food by adding the standard ATP solution at a known concentration to the surface or solution of the food - 120 μL in solid foods and dilution of ATP in milk. For this purpose, the lower and upper concentrations of the linearity curve (5.00E-03 and 5.00E-07 mM) were used instead of extracting ATP from microorganisms present in the food. The acceptance criterion was evaluated by Student's t-test between the standard ATP and the ATP evaluated under the described conditions, and should be between 70-100%. Application of the method to food Petition 870250015693, dated 26 / 02 / 2025, page 26 / 50 23 / 38

[0059] For each solid food, both the commercial swab and the common swab were rubbed in a 1 cm2 quadrant, and for the liquid food, the swab was immersed in 1 mL. The assay was performed on three different days. The means, standard deviations, and precision were calculated for each day and for the three days, using the respective equations of the lines obtained from the correlation graphs between light intensity and CFU / mL, in the topic "Relationship between CFU and bioluminescence", in order to verify compliance with current legislation. Forced degradation test

[0060] In order to verify whether the method could be applied in cases of contaminated food or food with limits above those specified by legislation, the food was subjected to processes in which it was possible to accelerate its degradation and, therefore, increase the contamination.

[0061] To this end, the foods were incubated at 36°C for 12 hours, followed by analysis and calculation of means, standard deviations and precision using the luciferase A. vivianii. Then the equation of the line obtained from the correlation graph between Light Intensity and CFU / mL, in the topic “Relationship between CFU and bioluminescence”, was used. Bioluminescence Imaging (BLI)

[0062] Cell and tissue bioimaging was performed using a NightOwl cooled CCD photodetector system (Berthold), a multi-user instrument available in our laboratory. Results Development and validation of a method for detecting microbiological contamination by E. coli XL1-Blue.

[0063] Aiming at the development of a method for detecting ATP from microorganisms present in food or on surfaces, the detection of ATP Petition 870250015693, dated 26 / 02 / 2025, page 27 / 50 24 / 38 of microorganisms from diluted and standardized cultures of E. coli Xl1-Blue were compared with the same cultures adsorbed onto swabs and with pre-established concentrations of standard ATP. The results obtained in the validation of the bioanalytical assay using ATP extracted from E. coli Xl1-Blue cultures are presented below. Linearity

[0064] According to the established criteria for verifying the linearity of the concentrations chosen for method validation, obtained through ATP extracted from microorganisms in diluted cultures (Dilution) and from microorganisms in culture on the swab (Swab), the curves demonstrate linearity. The means, standard deviation, and precision were calculated for the five dilutions with extracted ATP in five replicates and three different days, and are presented in Tables 1 and 2. The equations of the lines of the graphs generated by the curves of the means of the five dilutions with extracted ATP obtained on the three days of study are presented in Table 3. Table 1 - Mean, SD and precision of light intensity values ​​in counts per second (CPS) for each concentration tested over three days for the E. coli Xl1-Blue culture in dilution. Average Dilution of Light Intensity Value (cps) 1 1.70E+06 2.10E+05 3.89E+04 1.53E+04 9.22E+03 2 1.95E+06 1.79E+05 3.09E+04 1.34E+04 1.01E+04 3 1.18E+06 2.25E+05 2.98E+04 1.23E+04 1.07E+04 Average 1.61E+06 2.05E+05 3.32E+04 1.36E+04 1.00E+04 SD 3.89E+05 2.37E+04 4.97E+03 1.54E+03 7.60E+02 Accuracy % 24.2 11.6 15.0 11.3 7.6 Petition 870250015693, dated 26 / 02 / 2025, page 28 / 50 25 / 38 Table 2 - Mean, SD, and precision of light intensity values ​​in CPS for each concentration tested over three days for E. coli Xl1-Blue culture on swab. Swab Average Day Light Intensity Value Found (cps) 1 4.90E+05 1.01E+05 2.88E+04 1.47E+04 9.03E+03 2 5.93E+05 1.07E+05 1.92E+04 1.06E+04 7.75E+03 3 5.97E+05 7.17E+04 2.14E+04 8.76E+03 5.81E+03 Average 5.60E+05 9.32E+04 2.32E+04 1.13E+04 7.53E+03 SD 6.05E+04 1.89E+04 5.02E+03 3.04E+03 1.62E+03 Accuracy % 10.8 20.2 21.7 26.8 21.5

[0065] Using the data from the standard ATP curves, the dilution culture, and the swab culture, a graph was created, as seen in Fig. 2, aiming at a visual comparison between the standard ATP concentrations and the dilution and swab methods that are being established in the laboratory. Thus, the graph in Fig. 2 shows the linearity curves of the standard ATP concentration (ATP), ATP extracted from the dilution culture (Dilution) and from the swab culture (Swab) at the five concentrations used and their respective results in light intensity (CPS).

[0066] The average values ​​found in the linearity assessment of the diluted culture and the swab culture were also shown in a graph and in Table 3 with the data from the equation of the line, as well as the respective averages and standard deviations, demonstrating that on three different days there is repeatability of the linearity curves at the chosen concentrations.

[0067] In summary, Fig. 3 presents linearity graphs of ATP extracted from the bacterial culture in dilution (Dilution) and from the culture on the swab (Swab) at the five concentrations used and their respective results in CPS, equations of the line and R2. Petition 870250015693, dated 26 / 02 / 2025, p. 29 / 50 26 / 38 Table 3 - Values ​​of the equations of the line of averages of three days of linearity evaluation Dilution Swab Day Intercept Slope R2 Intercept Slope R2 1 58.00 1701.10 0.9997 58.00 495.63 0.9926 2 58.00 1945.20 0.9998 58.00 598.02 0.9955 3 58.00 1194.50 0.9931 58.00 597.91 0.9998 N 3 3 3 3 3 3 Average 58.00 1613.60 0.9975 58.00 563.85 0.9960 SD 0 382.92 0.00 0 59.08 0.00 Precision % 0 23.73 0.38 0 10.48 0.36 Detection limit and quantification limit

[0068] The lowest amount at which ATP could be detected in the cultures was defined from the values ​​of the solution without the presence of ATP (blank), as shown in Table 4. From these values ​​and the standard deviation of the sample with the lowest ATP concentration from the linearity curve, it was possible to calculate the limit of detection (LOD) and use it as a noise value in the other parameters to be analyzed.

[0069] The limit of quantification was defined as the average value of the lowest quantified standard ATP concentration from the linearity curve, therefore being 1.00E+04 cps for the dilution and 7.53E+03 cps for the swab, according to the “Linearity” parameter. Table 4 - Mean, SD and precision of light intensity values ​​in counts per second (CPS) for solutions without the presence of the analyte (standard ATP). Petition 870250015693, dated 26 / 02 / 2025, page 30 / 50 27 / 38 Average Swab Dilution 58.2 45.2 SD 5.54E+00 9.93E+00 Precision% 10% 22% LOD for the swab: - LOB = 6.15E+01 LOD = 2.73E+03 LOD for dilution: - LOB = 6.73E+01 LOD = 1.32E+03 Interday and intraday accuracy

[0070] According to the pre-established criteria for verifying the accuracy of the extracted ATP concentrations chosen for the method, and tested in five replicates on three different days (interday), and on the same day (intraday), all acceptance criteria were met for the culture in dilution mode and on the swab (Tables 5 to 8). In the interday evaluation, the accuracy ranged from 15.4% to 27.4%, and in the intraday evaluation from 4.9% to 16.5%, within the established criteria of up to 35%. The accuracy values ​​indicate that the method has repeatability if reproduced on the same day (intraday) or on different days (interday) at the tested concentrations. Table 5 - Results of light intensity in CPS of the mean, SD and precision in the interday evaluation of the E. coli Xl1-Blue culture in dilution mode. Petition 870250015693, dated 26 / 02 / 2025, page 31 / 50 28 / 38 Dilution Day Standard Value* Average of the highest value found* Standard Value* Average of the average value found* Standard Value* Average of the lowest value found* 1 1.43E+06 2.54E+04 1.89E+04 2 2.02E+06 1.56E+06 3.21E+04 2.69E+04 3.87E+03 1.24E+04 3 2.18E+06 3.27E+04 1.10E+04 N 15 15 15 Average 1.72E+06 2.83E+04 1.41E+04 SD 3.86E+03 4.37E+03 3.88E+05 Precision % 27.4 15.4 22.5 Table 6 - Results of light intensity in CPS of the mean, SD and precision in the interdia evaluation of E. coli Xl1-Blue culture in swab. Swab Day Standard Value* Average of the highest value found* Standard Value* Average of the average value found* Standard Value* Average of the lowest value found* 1 3.86E+05 1.60E+04 6.48E+03 2 2.02E+06 4.29E+05 3.21E+04 2.00E+04 3.87E+03 7.77E+03 3 4.51E+05 1.88E+04 6.27E+03 N 15 15 15 Average 4.22E+05 1.83E+04 6.84E+03 SD 9.56E+04 3.21E+03 1.57E+03 Precision% 22.6 17.6 22.9 Table 7 - Results of light intensity in CPS of the mean, SD and precision in the intraday evaluation of the E. coli Xl1-Blue culture in dilution. Dilution Standard Value* Average of the high value found* Standard Value* Average of the average value found* Standard Value* Average of the low value found* 2.02E+0.6 1.43E+0.6 3.21E+0.4 2.54E+0.4 3.87E+0.3 1.89E+0.4 Petition 870250015693, dated 26 / 02 / 2025, page 32 / 50 29 / 38 N 5 5 5 DP 2.13E+05 1.24E+03 1.32E+03 Precision % 14.9 4.9 7.0 Table 8 - Results of light intensity in CPS of the mean, SD and precision in the intraday evaluation of E. coli Xl1-Blue culture in swab. Swab Standard Value* Average of the highest value found* Standard Value* Average of the average value found* Standard Value* Average of the lowest value found* 1.92E+06 3.86E+05 3.21E+04 1.60E+04 3.87E+03 7.25E+03 N 5 5 5 SD 6.38E+04 9.87E+02 4.77E+02 Precision% 16.5 6.2 6.6 Accuracy

[0071] According to the established criteria for verifying the accuracy of the ATP concentrations extracted in dilution and in swab, and compared with a standard ATP curve by Student's t-test, all acceptance criteria were met: the dilution and the swab achieved 72% and 71% accuracy respectively, both within the permitted recovery criterion (between 70-100%). Matrix effect

[0072] The results of the matrix effect accuracy assessment, shown in Table 9, were 71% and 70% for the dilution and swab methods, respectively. The results are within the established criteria (70-100%), demonstrating that the method was not affected by endogenous components in the matrix or by the swab. Considering that the method accuracy calculated for the culture in dilution mode and with the swab was 72% and 71% respectively, the accuracy calculated by the matrix effect is very close to the values ​​established for culture. Petition 870250015693, dated 26 / 02 / 2025, page 33 / 50 30 / 38

[0073] Table 9 below presents results of light intensity in CPS of mean, SD, precision and accuracy in evaluating the matrix effect of the culture and swab at two standard ATP concentrations (5.00E-03 and 5.00E-07 mM). Table 9 - Results of light intensity in CPS of mean, SD, precision and accuracy in evaluating the matrix effect of the culture and swab at two standard ATP concentrations (5.00E-03 and 5.00E-07 mM). Culture with standard ATP without swab, with water and standard ATP buffer extraction, and without buffer extraction. ATP Concentration (mM) 5.00E-03 5.00E-07 5.00E-03 5.00E-07 Average (CPS) 3.76E+06 4.55E+04 3.95E+06 5.57E+04 SD 3.64E+05 8.11E+03 3.87E+05 1.05E+04 Precision% 10% 18% 10% 19% Accuracy% 71% 70% Stability of the analysis solution

[0074] The results of the stability assessment of the analytical solution are presented in Table 10. According to the results obtained, the solution with luciferin stored at 5°C showed light intensity results below the results at time zero, demonstrating degradation after 3 months of storage under the aforementioned conditions. Table 10 - Mean, SD and Precision of light intensity values ​​in counts per second (CPS) for four conditions in which the solution was analyzed for the stability of the analysis solution in three different periods. With luciferin at -20°C Without luciferin at 20°C With luciferin at 5°C Without luciferin at 5°C Concentration 5.00E03 5.00 E-05 5.00E-07 5.00 E-03 5.00E-05 5.00 E-07 5.00E-03 5.00E05 5.00E-07 5.00 E-03 5.00 E-05 5.00 E-07 15-day averages 1.46E+07 1.52E+05 6.01E+03 1.15E+07 1.39E+05 6.65E+03 1.01E+07 1.29E+05 7.22E+03 1.32 E+07 1.44 E+05 1.60 E+05 DP 1.83E+ 06 1.49 E+04 2.55E +02 1.27 E+06 1.14E +04 7.01 E+02 3.62E +05 6.85 E+03 7.29E +02 5.40 E+05 3.80 E+04 4.88 E+05 Petition 870250015693, dated 26 / 02 / 2025, page 34 / 50 31 / 38 Precision % 12% 10% 4% 11% 8% 11% 4% 5% 10% 4% 26% 30% Averages 1 1.89E+ 1.92 2.04E 2.04 2.14E 2.68 3.33E 4.77 4.89E 1.89 2.66 2.82 month 07 E+05 +04 E+07 +05 E+04 +06 E+04 +03 E+07 E+05 E+04 SD 1.88E+ 1.52 1.47E 1.35 3.19E 2.22 2.86E 4.47 7.28E 1.38 5.76 4.07 06 E+04 +03 E+06 +04 E+03 +05 E+03 +02 E+06 E+04 E+03 Precision % 10% 8% 7% 7% 15% 8% 9% 9% 15% 7% 22% 14% Averages 3 1.58E+ 1.98 1.13E 2.98 2.58E 1.68 4.54E 1.58 2.74E 2.37 2.74 2.81 months 07 E+05 +04 E+07 +05 E+04 +03 E+02 +01 E+06 E+04 E+03 SD 1.79E+ 1.92 2.20E 3.87 2.47E 1.68 4.91E 3.08 5.37E 7.02 8.29 7.81 06 E+04 +03 E+06 +04 E+03 +02 E+01 +00 E+05 E+03 E+02 Precision % 11% 10% 19% 13% 10% 10% 11% 19% 20% 30% 36% 28% 6-month averages 9.38E+06 2.59E+05 1.01E+04 1.91E+07 2.69E+05 1.17E+04 3.01E+03 2.43E+03 2.46E+03 6.76E+04 1.62E+03 2.51E+02 SD 6.69E+05 6.77E+03 1.26 E+03 9.57 E+0 5 1.36E +04 2.14 E+0 3 9.46E +02 1.05 E+0 3 1.46 E+03 1.19 E+0 4 3.42 E+0 2 4,50 E+0 1 Accuracy % 7% 3% 12% 5% 5% 18% 31% 43% 59% 18% 21% 18%,

[0075] Table 11 was created for better visualization of the data from the comparison by Student's t-test between the analysis solution at time zero and the other conditions. The accepted data are above 70%, therefore only solutions maintained at -20°C are preserved over time. Table 11 - Accuracy Table demonstrating the results of the Student's t-test comparison of each condition analyzed - analysis solution with or without luciferin stored at -20°C or 5°C - with the solution from time zero. Incubation time: With Luciferin 20°C; Without Luciferin 20°C; With Luciferin 5°C; Without Luciferin 5°C; 15 days; 84%; 96%; 97%; 88%; 1 month; 72%; 69%; 56%; 72%; 3 months; 80%; 70%; 41%; 51%; 6 months; 94%; 72%; 41%; 41% Relationship between CFU and bioluminescence Petition 870250015693, dated 26 / 02 / 2025, page 35 / 50 32 / 38

[0076] In order to estimate the amount of CFU / mL, used as a standard unit in current regulations defining acceptable microorganism limits in food, tests were carried out to relate the results obtained by light intensity, in CPS, and the results obtained in CFU / mL. The averages of the analyses performed in triplicate for each optical density (OD600 = 1.08; 1.25 and 1.17) are shown in Tables 12 and 13.

[0077] Next, a graph (Fig. 5) was created with the averages of the three optical densities, obtained in CFU / mL and in CPS, after adjusting the dilution factor and including noise as the intersection point on the graph's y-axis. The equation of the generated line (y=5.1952x + 2726) will be used in subsequent food analysis tests using the swab to capture microorganisms.

[0078] Table 12 below presents mean, SD and precision results of light intensity values ​​in counts per second (CPS) for four conditions in which the solution was analyzed for the stability of the analysis solution in three different periods. Table 12 - Results in CFU / mL of the averages of the triplicates for each optical density evaluated, at five different dilutions. CFU / mL Average Dilution Average Average Average (μL) OD600=1.08 OD600=1.25 OD600=1.17 1000 3.10E+08 1.49E+08 4.10E+08 2.90E+08 100 5.30E+07 2.34E+07 6.30E+07 4.65E+07 10 3.70E+06 1.42E+06 2.60E+06 2.57E+06 1 6.80E+05 1.76E+04 5.30E+05 4.09E+05 0.1 5.30E+04 1.21E+03 2.50E+04 2.64E+04 Table 13 - Light intensity results in counts per second (CPS) of the averages of the triplicates for each optical density evaluated, in five different dilutions using Amydetes viviani luciferase. Petition 870250015693, dated 26 / 02 / 2025, page 36 / 50 33 / 38 Light Intensity (CPS) Average Dilution Average Average Average (μL) OD600=1.08 OD600=1.25 OD600=1.17 1000 1.23E+07 1.46E+07 1.87E+07 1.52E+07 100 1.47E+06 1.24E+06 1.42E+06 1.37E+06 10 2.02E+05 1.86E+05 1.40E+05 1.76E+05 1 3.04E+04 5.50E+04 3.85E+04 4.13E+04 0.1 3.71E+04 3.66E+04 3.89E+04 3.75E+04

[0079] The same assay was performed with a commercial swab, in order to compare the results obtained from a local luciferase with a commercially available luciferase. To this end, assays were performed on an optical density at five different concentrations, aiming to relate the results obtained by light intensity, in CPS, and the results obtained in CFU / mL from the previous assay. The average of the analysis performed in triplicate (OD6oo = 1.17) is shown in Table 14. Subsequently, a graph was created, as seen in Fig. 5, with the averages of the optical density, obtained in CFU / mL and in CPS, after adjusting the dilution factor and including noise as the point of intersection on the graph's y-axis. The equation of the generated line (y = 0.4231x + 2726) will be used in subsequent food analysis assays using the commercial swab. Table 14 - Light intensity results in counts per second (CPS) of the averages of the triplicates of each optical density evaluated, in five different dilutions using a commercially available swab (3M). Light Intensity (CPS) Average Dilution Average Average Average (μL) OD600=1.08 OD600=1.25 OD600=1.17 1000 1.25E+08 9.53E+07 1.43E+08 1.21E+08 100 2.83E+07 2.47E+07 3.55E+07 2.95E+07 10 2.71E+06 2.07E+06 5.62E+06 2.47E+06 1 5.07E+05 4.10E+05 1.00E+06 5.07E+05 0.1 2.71E+05 2.56E+05 6.51E+05 2.93E+05 Petition 870250015693, dated 26 / 02 / 2025, p. 37 / 50 34 / 38 Essays using food

[0080] Based on the method developed and validated in cell cultures, the same method was used in food, in order to verify its repeatability and final reproducibility. For this purpose, meat, chicken and milk were used in the presence of the Amydetes vivianii luciferase and a commercial swab, thus verifying the advantage of the luciferase developed in our laboratory compared to a commercially available luciferase. Food matrix effect

[0081] The accuracy was 73% for meat, 80% for chicken, and 70% for milk, all within the established criteria (70-100%), demonstrating that the method was not affected by food matrix components or the swab (Table 10). Considering that the matrix effect of the method calculated for the culture in dilution mode and on the swab was 71% and 70% respectively, the matrix effect on the food is very close to the values ​​found previously.

[0082] Table 15 below refers to the results of light intensity in CPS of the mean, SD, precision and accuracy in the evaluation of the matrix effect in foods at two standard ATP concentrations (5.00E-03 and 5.00E-07 mM). Table 15 - Results of light intensity in CPS of mean, SD, precision and accuracy in the evaluation of the matrix effect in foods at two standard ATP concentrations (5.00E-03 and 5.00E-07 mM). Sample Meat Chicken Milk ATP Concentration (mM) 5.00E-03 5.00E-07 5.00E-03 5.00E-07 5.00E-03 5.00E-07 Average (CPS) 1.07E+06 4.80E+04 1.21E+06 1.58E+05 3.64E+06 1.83E+05 SD 6.12E+04 9.00E+03 3.68E+05 3.78E+04 3.00E+05 3.47E+04 Precision% 6% 19% 30% 24% 8% 19% Accuracy% 73% 80% 70% Petition 870250015693, dated 26 / 02 / 2025, page 38 / 50 35 / 38 Application of the method to food

[0083] To verify the viability of the possible commercial use of the swab containing the luciferase Amydetes vivianii, the developed method was applied to real food samples, with results shown in Table 16. The same analysis was performed with the commercial 3M swab, shown in Table 17. The results in CFU / mL were obtained from the equations of the line in the correlation graphs in Fig. 4 and Fig. 5 so that it was possible to compare them with the limits available from regulatory bodies.

[0084] Therefore, based on the limits available from IN 60 / 2019, in which meat and chicken have acceptable limits of 3.00E+06 CFU / mL and 3.00E+05 CFU / mL, the results obtained with Amydetes vivianii remained within the established limits, while the results obtained with the 3M commercial swab were outside the permitted limits. Probably, the difference between the results is due to the ability of A. vivianii luciferase to reach higher limits on the correlation curve in Fig. 4, making it possible to accommodate the results within the trend line, while the commercial swab has a lower acceptance limit, as also evidenced by Fig. 2. Table 16 - Results of ATP quantification in light intensity in CPS of mean, SD, precision and calculations in CFU of three different foods based on the correlation line equation between CPS and CFU / mL using Amydetes viviani luciferase. Food Day 1 Day 2 Day 3 Average CFU Meat Average (CPS) SD Precision % 4.68E+06 8.19E+05 18% 1.42E+06 2.44E+05 17% 7.00E+06 1.73E+06 25% 4.37E+06 7.51E+05 17% 8.40E+05 Chicken Average (CPS) SD 4.43E+06 1.09E+06 4.06E+06 8.03E+05 6.49E+05 1.93E+05 3.04E+06 457606.822 5.85E+05 Petition 870250015693, dated 26 / 02 / 2025, page 39 / 50 36 / 38 Accuracy % 25% 20% 30% 15% Average (CPS) 1.09E+04 5.17E+04 4.76E+04 3.67E+04 6.55E+03 Milk DP Accuracy % 9.58E+02 8.79% 4.76E+03 9.22% 9.38E+03 19.68% 5.03E+03 13% Table 17 - Results of ATP quantification in light intensity in CPS of mean, SD, precision and calculations in CFU of three different foods on three different days based on the equation of the correlation line between CPS and CFU / mL of the commercial swab (3M). Food Day 1 Day 2 Day 3 Average CFU Average (CPS) 2.07E+06 1.10E+06 1.71E+06 1.63E+06 Meat DP 3.94E+05 1.83E+05 3.33E+05 494505.677 3.84E+06 Accuracy % 19% 17% 19% 30% Average (CPS) 7.72E+05 5.51E+05 4.34E+05 5.85E+05 Chicken DP 2.02E+05 1.30E+05 1.28E+05 171791.257 1.38E+06 Accuracy % 26% 24% 30% 29% Average (CPS) 9.23E+04 1.68E+05 1.51E+05 1.37E+05 Milk DP 1.26E+04 1.68E+04 6.62E+03 39822.0597 3.18E+05 Precision % 14% 10% 4% 29% Forced degradation test

[0085] The results of the forced degradation assay allowed for ATP quantification in such a way that it was possible to correlate it with the permitted limits of CFU / g, thus confirming that the respective food was contaminated. Therefore, it is possible to state that the luciferase A. vivianii is suitable for use in ATP quantification in food because it possesses an intense glow, with Petition 870250015693, dated 26 / 02 / 2025, pages 40 / 50 37 / 38 CPS results are more intense than commercial swab luciferase and therefore it is possible to detect contamination above permitted limits.

[0086] Table 18 refers to results of ATP quantification in light intensity in CPS of mean, SD, precision and calculations in CFU of forced degradation of three different foods, in CPS and CFU, based on the results of the correlation line equation between CPS and CFU / mL of the luciferase Amydetes vivianii. Table 18 - Results of ATP quantification in light intensity in CPS of mean, SD, precision and calculations in CFU of forced degradation of three different foods, in CPS and CFU, based on the results of the correlation line equation between CPS and CFU / mL of the luciferase Amydetes viviani. Food CPS UFC Average Meat (CPS) SD Accuracy % 2.29E+07 6.44E+06 28% 4.41E+06 Average Chicken (CPS) SD Accuracy % 2.00E+07 5.81E+06 29% 3.85E+06 Average Milk (CPS) SD Accuracy % 2.62E+06 8.30E+05 30% 5.03E+05

[0087] Based on the results, it is possible to differentiate between food samples that are suitable and unsuitable for consumption due to high levels of microbiological contamination. It is also possible to enable studies using Amydetes vivianii luciferase to quantify ATP from microorganisms in food. Therefore, a patent application is being filed to protect the use of Amydetes vivianii luciferase in detecting microbiological contamination in food. These studies do not exclude the Petition 870250015693, dated 26 / 02 / 2025, pp. 41 / 50 38 / 38 need for further tests to validate the method for the commercialization of a screening kit to determine the suitability for consumption of food of animal origin.

[0088] The luciferase from Amydetes vivianii is the great differentiator of the technique, which allows the detection of ATP from a wide range of light intensity, with a brighter brightness than the luciferases from commercially available kits. This fact makes it possible to detect high levels of ATP above the acceptable limits for microorganisms in food. When the same assay is performed using a commercially available luciferase kit, the limit is lower and, consequently, the smaller range of light intensity does not allow the extrapolation of ATP detection levels, making it impossible to differentiate between contaminated food and food viable for consumption, according to the limits stipulated by ANVISA. Microbiological contamination tests of surfaces

[0089] In further tests using this methodology, the swab was tested on contaminated surfaces and objects in a research laboratory. Luminometric analyses revealed considerable contamination inside a centrifuge and adjacent workbench. Furthermore, the level of contamination resulted in a bioluminescent signal high enough for photographic detection.

[0090] It should be understood that the present description does not limit the application to the details described herein and that the invention is capable of other embodiments and of being practiced or performed in a variety of ways, within the scope of the claims. Although specific terms have been used, such terms should be interpreted in a generic and descriptive sense, and not for the purpose of limitation. Petition 870250015693, dated 26 / 02 / 2025, pp. 42 / 50

Claims

1 / 2 CLAIMS 1. BIOLUMINESCENT DETECTION KIT FOR MICROBIOLOGICAL CONTAMINATION OF FOOD AND SURFACES USING AMYDETES VIVIANII LUCIFERASE, comprising blue-green light-emitting luciferases for analytical assays and indication of cadmium and mercury comprising the amino acid sequence as defined in SEQ ID NO: 2, the cDNA sequence defined in SEQ ID NO: 1 of the Amydetes viviani luciferase enzyme, wherein they are used as bright analytical reagents, as bright reporter genes and as cellular and enzymatic biosensors for the metals cadmium and mercury, and for pH at high temperatures (>30°C), characterized by comprising at least one sample collection swab formed by a plastic shaft comprising cotton or sterile synthetic material at its ends; ATP extraction solution; assay solution containing luciferase from Amydetes vivianii and D-luciferin from firefly;at least one test tube containing said test solution; and a device for measuring luminescence intensity.

2. BIOLUMINESCENT DETECTION METHOD FOR MICROBIOLOGICAL CONTAMINATION OF FOOD AND SURFACES, comprising blue-green light-emitting luciferases for analytical assays and indication of cadmium and mercury comprising the amino acid sequence as defined in SEQ ID NO: 2, the cDNA sequence defined in SEQ ID NO: 1 of the luciferase enzyme from Amydetes viviani, wherein they are used as bright analytical reagents, as bright reporter genes and as cellular and enzymatic biosensors for the metals cadmium and mercury, and for pH. Petition 870250015693, dated 26 / 02 / 2025, p. 43 / 50 2 / 2 at high temperatures (>30°C), using a detection kit as defined in claim 1, characterized by comprising the steps of: a) sample collection in which the end of a swab soaked in ATP extraction solution is rubbed over the study surface in a 1cm2 quadrant, in zigzag movements while rotating its shaft,followed by at least 40 minutes of contact time; b) Immersion, in which the end of the swab that was in contact with the study surface is immersed in a test tube containing between 0.5 and 1.5 mL, preferably 1 mL of test solution with Amydetes vivianii luciferase and firefly luciferin, followed by another 4 to 5 minutes of exposure time; c) Measurement and analysis of luminescence, in which luminescence is measured using a luminometric or photometric instrument that receives the tube containing the study sample; d) Comparison of the luminescent signal with a standard curve, comparing the luminescent signal with a standard curve (ruler) of ATP to estimate the degree of microbiological contamination of the food. Petition 870250015693, dated 02 / 26 / 2025, pp. 44 / 50.

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