CA3154376A1

CA3154376A1 - Ozone treatment for elimination of pathogens

Info

Publication number: CA3154376A1
Application number: CA3154376A
Authority: CA
Inventors: Jill Lynn Ellsworth; Jason R. Ellsworth
Original assignee: Ellsworth Jason R; Ellsworth Jill Lynn; Willowpure LLC
Current assignee: Willowpure LLC
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2021-03-25
Also published as: AU2019466372A1; EP4030916A1; WO2021054930A1

Abstract

Methods for reducing microbial contamination present in a sample of cannabis plant material by exposure to ozone in an ozone chamber are disclosed herein.

Description

OZONE TREATMENT FOR ELIMINATION OF PATHOGENS
FIELD
The disclosure relates to methods of reducing or eliminating microbial contamination in a sample of cannabis plant material.
BACKGROUND
Cannabis is now legal for medical and/or recreational use in many countries and US states.
During cultivation, harvest, extraction, or even storage, cannabis can become contaminated with pesticides and microbes (e.g., yeast, mold, viruses and/or bacteria). Cannabis testing and compliance with regulations is required to ensure that cannabis has permissible levels of pesticides and acceptable levels of microbial contamination such that it is safe for human consumption.
As regulations are established by regional legislation, testing is typically carried out by approved independent testing laboratories with specific testing requirements dictated by local regulations. Cannabis and cannabis-based products intended for human consumption are generally tested before sale for microbial contaminants, THC levels, CBD levels, residual solvents, and pesticides.
Ozone is an effective oxidizing agent, and in sufficient concentrations, kills bacteria, fungi, viruses and other microorganisms, which contaminate agricultural crops. Ozone has also been shown to oxidize and degrade certain pesticide residues.
Various devices and processes have been developed for disinfecting air and surfaces using ozone gas or ozonated water.
Cannabis cultivators and manufacturers are facing product and revenue loss due to failed compliance with safety regulations. Cannabis growers need to be able to provide hemp and .. marijuana plants to consumers that contain allowable levels of microbial contaminants and pesticides.
A solution is needed to substantially reduce or eliminate microbial contamination of cannabis plant material post-harvest, while maintaining the quality and attributes of the plant material that are important to consumers (e.g., THC, CBD and terpene content).
SUMMARY
This disclosure is directed to methods and devices that safely and effectively reduce the level of microbial contamination in samples of plant material by exposing the plant material to gaseous ozone. The disclosure includes, but is not limited to, the following aspects and examples.

Disclosed herein are methods for reducing microbial contamination, such as contamination with viruses, bacteria, yeast, and/or mold in cannabis plant material (such as a sample of homogenized cannabis flower). In some examples, the contamination includes viable aerobic bacteria (TVAB), coliform bacteria, and/or bile-tolerant gram-negative bacteria (BTGN). In some examples, the contamination includes yeast and mold.
In exemplary embodiments, the method includes (a) providing a pathogen reduction device, including (i) an ozone chamber, (ii) an oxygen concentrator configured to concentrate oxygen from ambient air and produce ozone, (iii) an ozone regulator configured to adjust the ozone concentration in the ozone chamber, and (iv) one or more processors with a memory coupled to the one or more processors, the memory storing instructions that when executed by the one or more processors cause the one or more processors to determine a concentration of gaseous ozone in the ozone chamber and adjust the concentration of gaseous ozone in the ozone chamber to a preset concentration; (b) placing cannabis plant material contaminated with microbes inside the ozone chamber of the pathogen reduction device; (c) setting the ozone concentration in the ozone chamber to between about 200 ppm and about 400 ppm; and (d) treating the cannabis plant material with ozone by leaving it in the ozone chamber for a time period of from 20 minutes to 48 hours. In numerous examples, the method reduced the level (e.g., amount of colony forming units or CFU) of microbial contamination in samples of cannabis plant material.
A variety of conditions and microbes are illustrated herein. For example, the ozone concentration may be between 200 and 400 ppm, between 150 and 300 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm or 300 ppm. In some examples, the treatment time ranges from 20 mm to 2 hours, from 2 hours to 6 hours, from 4 hours to 8 hours, from 4 hours to 18 hours, or from 16 hours to 48 hours, e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 24 hours or 48 hours. In some examples, the methods reduce the microbial contamination level in the cannabis plant material by at least 80%, at least 90%, at least 95%, or at least 99% or 100% relative to the level of microbial contamination of the cannabis plant material prior to treatment.
In all examples where potency was evaluated, the ozone treatment did not impact the potency (e.g., THC level) of the cannabis plant material. In specific examples provided herein, the THC or THCa level prior to and following ozone treatment was substantially the same. The average terpene level prior to and following ozone treatment was also substantially the same in all cases where the terpene level was evaluated. Slight variations in potency and terpene levels may be observed between ozone-treated and untreated (control) sample, however; the observed variations

- 2 -are typical of results using the analytical methods employed for evaluation of potency and terpene levels.
The results presented herein show that the methods can be used to remediate plant material samples with a wide range of contamination levels. For example, in some cases, the initial level of microbial contamination in the cannabis sample was: (1) less than 150,000 CFU
of total yeast and mold, or bacteria; (2) greater than 150,000 CFU of total yeast and mold or bacteria; or (3) greater than 1,000,000 CFU of total yeast and mold (TYM). In some examples, the methods resulted in a reduction in total yeast and mold and/or bacterial contamination in samples cannabis plant material by at least 50,000 CFU, or at least 100,000 CFU. In some examples, the methods herein resulted in a reduction in total yeast and mold and/or bacterial contamination in a cannabis plant material sample to less than 10,000 CFU or less than 1,000 CFU.
In many cases, prior to treatment, samples of cannabis plant material had a level of total yeast and mold and/or bacteria that exceeded an allowable level for compliance with a state regulatory agency, and following treatment using the methods disclosed herein, the contamination level of the cannabis plant material level was reduced to a compliant level.
Methods are disclosed herein for treating cannabis plant material in an ozone chamber of a pathogen reduction device, wherein, prior to treatment, the cannabis plant material has a level of pesticide that exceeds an allowable level for compliance with a regulatory agency or company, and, following treatment, the pesticide level of the cannabis plant material level is reduced to a compliant level.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
Non-limiting examples and non-exhaustive examples are described with reference to the following figures.
FIG. 1 is a graph showing the average microbial counts from multiple trials after treatment of plant pathogens with gaseous ozone.
FIG. 2 is a simplified diagram of a distributed computing system in which aspects of the present invention may be practiced.
FIG. 3 illustrates one example of a suitable operating environment 2000 in which aspects of the present invention may be implemented.
FIGS. 4A-4D show perspectives of an example embodiment of a pathogen reduction device.

- 3 -FIG. 5 shows a tray from an example embodiment of a pathogen reduction device.
FIG. 6 shows a table with the initial and post-treatment levels of Aspergillus (Asp), total viable aerobic bacteria (TVAB), coliform bacteria, TYM, and bile-tolerant gram-negative bacteria (BTGN).
FIGS. 7A-7B show average pass/fail testing for initial (FIG. 7A) and post-ozone treatment (FIG. 7B) for each of Aspergillus (Asp), total viable aerobic bacteria (TVAB), coliform bacteria, TYM, and bile-tolerant gram-negative bacteria (BTGN) in a variety of retail marijuana strains.
FIG. 8 shows both THC and THCa levels before and after treatment with ozone in samples treated for 2 hours.
FIG. 9 shows both THC and THCa levels before and after treatment with ozone in samples treated for 6 hours.
FIG. 10 shows both THC and THCa levels before and after treatment with ozone in samples treated for 24 hours.
FIG. 11 shows CFU counts for two sizes of a Black Afghani cannabis strain before and after treatment with ozone.
FIG. 12 shows CFU counts for two sizes of a Bay Dream strain before and after treatment with ozone.
FIG. 13 shows CFU counts for Mango, Trill OG, and Tangerine Power cannabis strains before and after treatment with ozone.
FIG. 14 shows CFU counts for Bay Dream, Purps, and Mango strains before and after treatment with ozone.
FIG. 15 shows CFU counts for Fluffhead and Trill OG cannabis strains before and after treatment with ozone.
FIG. 16 shows CFU counts for Lemon OG, Tangerine Power, and Purps cannabis strains before and after treatment with ozone.
FIG. 17 shows CFU counts for cannabis strains treated for 360, 600, and 840 minutes before and after treatment with ozone.
FIG. 18 shows CFU counts for cannabis strains treated for 720, 840, and 960 minutes before and after treatment with ozone.
FIG. 19A shows CFU counts for Fall 97 and Strawberry Cough cannabis strains before and after treatment with ozone. FIG. 19B shows percent terpenes for Fall 97 and Strawberry Cough cannabis strains before and after treatment with ozone.
FIG. 20A shows CFU counts for Fall 97 and Blue Dream cannabis strains before and after treatment with ozone for 20 mm and 18 hours, respectively. FIG. 20B shows percent THC for Fall

- 4 -97 and Blue Dream cannabis strains before and after treatment with ozone for 20 min and 18 hours, respectively.
FIG. 21A shows CFU counts for Blue Dream, Strawberry Cough, and Fall 97 cannabis strains before and after treatment with ozone for 18 hours, 20 mm, and 40 mm, respectively. FIG.
.. 21B shows percent THC for Blue Dream, Strawberry Cough, and Fall 97 cannabis strains before and after treatment with ozone for 18 hours, 20 mm, and 40 mm, respectively.
FIG. 22 is a table summarizing the results and conditions for the data in FIGS. 11-21B.
FIG. 23 shows terpene levels for a variety of strains after treatment with ozone ("Willow") for 2 hours compared with an untreated control sample.
FIG. 24 shows terpene levels for a variety of strains after treatment with ozone ("Willow") for 6 hours compared with an untreated control sample.
FIG. 25 shows terpene levels for a variety of strains after treatment with ozone ("Willow") for 24 hours compared with an untreated control sample.
FIG. 26 shows THC and terpene levels for cannabis samples that have been treated with 300 ppm of ozone ("Willow") and untreated control samples.
FIGS. 27A-27B show that treatment of cannabis samples with 300 ppm of ozone resulted in a reduction in pesticide levels. Pesticide levels (ppb) were measured for a series of ozone-treated strains and in no-treatment control samples. FIG. 27A shows a graph of the data, and the values are presented in FIG. 27B.
DETAILED DESCRIPTION
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.
The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a step" includes single or plural steps and is considered equivalent to the phrase "comprising at least one step." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes."
Thus, "comprising yeast, mold, or bacteria," means "including yeast, mold, or bacteria" or "yeast, mold, and bacteria,"
.. without excluding additional elements. All references, including journal articles, patents, and patent publications cited herein are incorporated by reference in their entirety.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be

- 5 -used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.
Applying: Bringing one or more components into nearness or contact with another thing or component. Applying can refer to contacting or administering.
Cannabinoids: A group of secondary metabolites in cannabis. The cannabinoids are synthesized by plants as acid ("a" or "A") forms, and, while some decarboxylation occurs in the plant, it increases significantly at high temperatures. Results of cannabinoids analysis may include both the acidic and decarboxylated versions (for example, THC and THCa). THC
is also referred to as "potency."
Cannabis: A plant from the genus Cannabis. Three species within the genus may be recognized: Cannabis sativa, Cannabis indica, and Cannabis ruderalis. Cannabis includes hemp and marijuana. Cannabis may be used herein to refer to hemp and marijuana or marijuana alone.
Samples of cannabis (such as homogenized flower) may be used as test samples (for example, test samples that have undergone treatment with ozone, such as disclosed herein) or control samples (for example, control samples that have not undergone treatment with ozone).
CBD: The cannabinoid known as cannabidiol.
Colony forming units (CFU): A unit of measuring microbial levels. In some examples, Agar plate counting after an incubation period is used to measure CFU (such as of yeast, mold, or bacteria). Quantitative Polymerase Chain Reaction, or qPCR, can also be used to measure CFU of microbial, by amplifying and detecting a nucleic acid molecule specific for a particular microbe (such as particular genus or species or strain of bacteria, yeast, mold, or virus).
Microbes or microorganisms: include microscopic organisms, such as bacteria, fungi, and viruses. In specific, non-limiting examples, microbes include fungi, such as yeast and mold, and bacteria. In some examples, microbes are present at levels that exceed levels allowed for regulatory compliance or are toxic for consumption (such as by inhalation, topical application, or oral delivery), which is also referred to as microbial contamination. Levels of microbes (such as yeast, mold, and bacteria) can be determined in a variety of ways, such as plating and culturing- or quantitative PCT (qPCR)-based techniques (see, e.g., McKeman et al., F1000Res., 5:2471, 2016, incorporated herein by reference in its entirety).
Ozone (03): It is an allotrope of oxygen and is less stable than dioxygen (02). Ozone occurs naturally at low levels, but can be produced using the corona discharge method, narrow-

- 6 -band UV light, the cold plasma method, and electrolytic ozone generation (EOG). Because ozone cannot be stored, it must be produced on site (for example, in an ozone chamber).
Pathogen: Anything that causes disease or illness, particularly biological organisms such as bacteria, fungi, and viruses that are the source of microbial contamination.
Pesticide: A composition or product that kills or repels plant or seed pests.
Plant Material: Any portion of a plant, including the roots, stems, stalks, leaves, branches, seeds, flowers, fruits, and the like.
Polymerase chain reaction (PCR): Quantitative polymerase chain reaction, or qPCR, is included. qPCR is an analytical technique for evaluating microbial contamination of cannabis samples. qPCR is used to detect and amplify DNA and RNA sequences, comparing them to control sequences for a particular microbial target, calculating the strength of florescent signals. Typically, each test can be done with 0.5g-lg of test material.
Seed: Anything that can be sown to produce a plant. Seed can refer to an unfertilized plant ovule, a fertilized plant ovule, and an embryonic plant.
Terpene: Terpenes are a secondary metabolite found in cannabis and can affect the body in different ways, depending on the type. For example, terpenes can include aromatic properties and functions; communication roles; antibacterial, antifungal, antiviral, antiparasitic, anti-inflammatory, antiallergenic, antihyperglycemic, antispasmodic, antitumoral, and immunomodulatory activities;
cytotoxicity and antimicrobial, antifungal, and antimalarial properties; and insecticide activity.
Examples of terpenes include but are not limited to terpinolene, alpha phellandrene, beta ocimene, carene, limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene, fenchol, camphene, alpha terpineol, alpha humulene, beta caryophyllene, linalool, caryophyllene oxide, and myrcene.
Tetrahydrocannabinol (THC or (¨)-trans-A9-tetrahydrocannabinol): THC is a cannabinoid identified in cannabis and is its principal psychoactive constituent. THCa rapidly converts to THC upon heating. THC may be analyzed. Two analytical techniques have been successfully used for the potency testing of cannabis: Gas chromatography (GC) and High Performance Liquid Chromatography (HPLC). There are advantages and disadvantages to each technique. Specific guidelines are offered by state regulations for suggested analytical technique.
Total THC is of primary interest in cannabis potency testing. High Performance Liquid Chromatography (HPLC) can identify the acid component of THCA before conversion to the corresponding free form of THC. Gas Chromatography (GC) does not detect THCA
directly. The carboxylic acids decarboxylate in the intense heat of smoking, baking, or GC
injector port. GC is generally considered faster and simpler than HPLC so it is often preferred.
GC/FID is preferred for

- 7 -speed of analysis and simplicity in routine identification and quantification of cannabinoid concentrations.
Overview Cannabis is grown under many different conditions, both indoors and outdoors.
As with all agricultural products, it is exposed to a wide range of microorganisms.
Cannabis and cannabis-based products intended for human consumption must comply with state (or other regulatory body) requirements for the level of microbial contaminants and pesticides.
The value of cannabis and cannabis-based products to consumers is dependent upon maintenance of the levels of biologically active compounds including THC, CBD
and terpenes. It follows that any method useful to treat cannabis and cannabis-based products to ensure compliance with regulatory requirements should ideally result in the level of THC, THCa, CBD, CBDa, and terpenes remaining substantially the same.
The inventors have determined that subjecting cannabis plant material to concentrations of ozone between 200 ppm and 400 ppm for an amount of time in the range of 20 minutes to 48 hours results in a significant reduction in or elimination of microbial contamination and pesticides in the cannabis plant material, while maintaining THC, CBD, and/or terpene content that is important to consumers.
Pathogen redaction device Pathogen reduction devices can include a variety of elements, such as an ozone chamber, an oxygen concentrator (for example, configured to concentrate oxygen from ambient air and produce ozone), an ozone regulator (for example, configured to adjust the ozone concentration in the ozone chamber), and one or more processors with a memory coupled to the one or more processors (for example, memory storing instructions that when executed by the one or more processors cause the one or more processors to determine a concentration of gaseous ozone in an ozone chamber; and adjust the concentration of gaseous ozone in the ozone chamber to a preset concentration). In some examples, the device includes an element to add and regulate humidity levels in the ozone chamber. In some examples, the device includes a vacuum pump to reduce the pressure in the ozone chamber, and in some examples reduces water and moisture content of the ozone chamber.
In an exemplary embodiment, FIG. 2 shows a simplified diagram of a distributed computing system 1914 in which aspects of the present invention may be practiced.
According to examples, any of computing devices 1902A (a modem), 1902B (a laptop computer), 1902C (a tablet), 1902D
(a personal computer), 1902E (a smart phone), and 1902F (a server) may be used to send, receive

- 8 -and evaluate signals from pathogen reduction device 1908 via one or more network servers 1906 and a network 1920. Such signals may include data related to ozone concentration, temperature and time of exposure, for example.
According to some aspects, pathogen reduction device 1908 may be a stationary or fixed device. According to other aspects pathogen reduction device 1908 may be a mobile device. For example, pathogen reduction device 1908 may stand on a plurality of wheels for moving the device from one place to another. The wheels may be fixed to the device or they may be readily removed and put back on, by for example, a pop out mechanism. According to an embodiment pathogen reduction device 1908 may have the following dimensions: a length of 4 feet, 4 inches; a width of 2 feet, 0 inches; and a height of 5 feet, 2 inches. With wheels attached the height of the pathogen reduction device may be 5 feet, 5 inches.
According to additional examples pathogen reduction device 1908 may contain a plurality of racks within an ozone chamber. The racks may be positioned suitably for treating plants on each rack level in the pathogen reduction device 1908. For example, the racks may be positioned at 3 inch vertical intervals within the pathogen reduction device 1908. The racks may be made of metal sliders and a metal mesh shelf to effectively ozonate a plant. They may also include a lip around the shelf to prevent loss of a treated plant. For example, the shelves may include a 1 inch lip such that treated plant product is not lost. In accordance with these examples it should be appreciated that such a racking system allows for the processing (e.g., gaseous ozone treatment) of approximately 17-20 pounds of plant product every 20 minutes.
Pathogen reduction device 1908 may comprise one or more of a controller, an ozone system (including an ozone generator and an ozone chamber) and an oxygen concentrator.
Pathogen reduction device 1908 according to certain embodiments may include safety mechanisms including but not limited to a destructor for venting gaseous ozone, providing a mechanism for immediately degrading ozone back to 02, a leak sensor in communicative contact with an alarm display and a safety interlock. According to aspects, one or more of these safety mechanisms may be employed as part of pathogen reduction device 1908 as well as distributed computing system 1914.
A controller as described herein in association with the pathogen reduction device 1908 may control and operate each component within the pathogen reduction device 1908 including the ozone chamber. The controller may comprise one or more processors and a memory coupled to the one or more processors. The memory may store instructions that when executed by the one or more processors cause the one or more processors to implement one or more steps, including:
determining a concentration of gaseous ozone in an ozone chamber; adjusting the concentration of

- 9 -gaseous ozone in the ozone chamber; adjusting the ambient temperature in the ozone chamber;
continuously monitoring the concentration of gaseous ozone and the ambient temperature in the ozone chamber and automatically adjusting the monitored concentration and temperature to a preset concentration and preset temperature.
The controller may also include a graphical user interface for touch screen operation and system interaction. Integrated sensors may be configured to monitor conditions in the pathogen reduction device 1908 so that proper action can be taken to reduce pathogen levels associated with plants being treated in the pathogen reduction device 1908. For example, integrated sensors may provide, via a graphical user interface on the pathogen reduction device or a graphical user interface on computing devices 1902A-F, an indication that an ozone leak has occurred. The controller may be further configured to shut down one or more of the elements described in the pathogen reduction methods and systems described herein to protect the various components of the pathogen reduction device 1908. The controller may also be configured to send a signal to one or more of computing devices 1902A-F if a sensor has failed such that remedial action can be taken.
FIG. 3 illustrates one exemplary embodiment of a suitable operating environment 2000 in which one or more of the present embodiments may be implemented. FIG. 3 provides only one example of a suitable operating environment and is not intended to suggest any limitation as to the scope of use or functionality. Other well-known computing systems, environments, and/or configurations that may be suitable for use include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, programmable consumer electronics such as smart phones, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
In its most basic configuration, operating environment 2000 typically includes at least one processing unit 2002 and memory 2004. Depending on the exact configuration and type of computing device, memory 2004 (storing, among other things, reputation information, category information, cached entries, instructions to perform the methods disclosed herein, etc.) may be volatile (such as RAM), non-volatile (such as ROM, flash memory, etc.), or some combination of the two. This most basic configuration is illustrated in FIG. 3 by dashed line 2006. Further, environment 2000 may also include storage devices (removable, 2008, and/or non-removable, 2010) including, but not limited to, magnetic or optical disks or tape.
Similarly, environment 2000 may also have input device(s) 2014 such as keyboard, mouse, pen, voice input, etc. and/or output device(s) 2016 such as a display, speakers, printer, etc. Also included in the environment may be one or more communication connections, 2012, such as LAN, WAN, point to point, etc.

- 10 -Operating environment 2000 typically includes at least some form of computer readable media. Computer readable media can be any available media that can be accessed by processing unit 2002 or other devices comprising the operating environment.
By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic .. cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other tangible medium which can be used to store the desired information. Computer storage media does not include communication media.
Communication media embodies computer readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term "modulated data signal" means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media.
The operating environment 2000 may be a single computer operating in a networked environment using logical connections to one or more remote computers. The remote computer may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above as well as others .. not so mentioned. The logical connections may include any method supported by available communications media. Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets and the Internet.
Aspects described herein may be employed using software, hardware, or a combination of software and hardware to implement and perform the systems and methods disclosed herein.
Although specific devices have been recited throughout the disclosure as performing specific functions, one of skill in the art will appreciate that these devices are provided for illustrative purposes, and other devices may be employed to perform the functionality disclosed herein without departing from the scope of the disclosure.

- 11-Methods of treating cannabis Disclosed herein are methods for treating cannabis (such as a sample of cannabis plant material). In some embodiments, the methods include reducing microbial contamination (such as contamination with yeast, mold, viruses, and/or bacteria), reducing pesticide content, or both. In some embodiments, the methods include reducing microbial contamination and/or pesticide content to levels allowable by a regulatory agency or company.
A variety of cannabis plant materials can be used in the examples herein.
Various forms of cannabis can be used in the methods herein, including the flower (or bud), leaf, and stem materials or any combination thereof. In some examples, ground or homogenized cannabis plant materials can be used. In some examples, samples of cannabis are homogenized flower (for example, test samples that have undergone treatment with ozone, such as disclosed herein) or control samples (for example, control samples that have not undergone treatment with ozone).
Cannabis plant materials herein can further include a variety of cannabinoids and terpenes.
THC is a cannabinoid found in cannabis and the measure of "potency". The THC
precursor, THCa is an inactive form of THC that rapidly converts to THC upon heating. Other cannabinoids are found in cannabis, such as THC-A, CBD, CBD-A, CBN, CBG, CBG-A, CBC, and CHCV.
In some examples, the methods include providing a pathogen reduction device as disclosed herein. The methods can further include placing a sample of cannabis plant material comprising microbial contamination inside the ozone chamber of the pathogen reduction device. In specific examples, the methods are effective to reduce the level of microbial contamination in the cannabis plant material. For example, the cannabis plant material can have a level of bacteria, yeast, viruses and/or mold that exceeds the allowable level for compliance with a regulatory agency or company prior to treatment, and, following treatment, the pathogen contamination level of the cannabis plant material level is reduced to a compliant level. In further examples, the cannabis plant material has a level of one or more pesticides that exceeds an allowable level for compliance with a regulatory agency or company, and, following treatment, the pesticide level of the cannabis plant material level is reduced to a compliant level.
The methods can further include setting the ozone levels to a variety of ozone concentrations. For example, the methods can include setting the ozone concentration to about 50 ppm to about 400 ppm, about 50 ppm to about 300 ppm, about 200 ppm to about 400 ppm, about 100 ppm to about 300 ppm, about 150 ppm to about 250 ppm, or about 180 ppm to about 220 ppm.
In some embodiments, the concentration of ozone to which the plant material is subjected is greater than 100 ppm, greater than 125 ppm, greater than 150 ppm, greater than 175 ppm, greater than 200 ppm, greater than 225 ppm, greater than 250 ppm, greater than 275 ppm, greater than 300 ppm,

- 12 -greater than 400 ppm, greater than 500 ppm, greater than 600 ppm, greater than 700 ppm, greater than 800 ppm, greater than 900 ppm or greater than 1000 ppm. In some embodiments, the concentration of ozone to which the plant material is subjected is less than 1000 ppm, less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, less than 500 ppm, less than 400 ppm, less than 350 ppm, less than 300 ppm, less than 275 ppm, less than 250 ppm, less than 225 ppm, or less than 200 ppm. In specific, non-limiting examples, the methods can include treating a sample of plant material with an ozone concentration of between about 200 ppm and about 400 ppm, about 200 and about 300 ppm, about 150 ppm and about 250 ppm, about 200 and 225 ppm, e.g., about 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, or 300 ppm.
In some examples, the methods include treating the cannabis plant material with ozone (for example, by leaving it in the ozone chamber) for a specific period of time. In some examples, the period of time can be about 1 minute to about 48 hours, about 2 minutes to about 24 hours, about minutes to about 18 hours, about 30 minutes to about 12 hours, about 45 minutes to about 6 15 hours, about 60 minutes to about 4 hours, about 60 minutes to about 2 hours, about 2 hours to 6 hours, about 4 hours to 8 hours, about 4 hours to 18 hours, about 6 hours to 12 hours, about 10 to 24 hours, and about 16 hours to 48 hours. In some embodiments, the time plant material is exposed to ozone is greater than 20 minutes, greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 2 hours, greater than 6 hours, greater than 12 hours, greater than 18 hours, or 20 greater than 24 hours. In some embodiments, the time plant material is exposed to ozone is less than 48 hours, less than 24 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, or less than 30 minutes. For example, the period of time can be at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, or at least 30 hours, such as about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, or about 48 hours, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 23 hours, 30 hours, or 48 hours.
In some examples, the methods can include treating cannabis plant material at a variety of temperatures. In some cases, the cannabis plant material can be treated at room temperature (such as 59 F to 77 F).
The methods herein can be used to reduce microbial (such as yeast, mold, virus and/or bacteria) contamination. For example, the methods can be used to reduce microbial contamination to less than about 150,000 CFUs, less than about 100,000 CFUs, less than about 50,000 CFUs, less

- 13 -than about 40,000 CFUs, less than about 30,000 CFUs, less than about 20,000 CFUs, less than about 10,000 CFUs, less than about 9,000 CFUs, less than about 8,000 CFUs, less than about 7,000 CFUs, less than about 6,000 CFUs, less than about 5,000 CFUs, less than about 4,000 CFUs, less than about 3,000 CFUs, less than about 2,000 CFUs, less than about 1,000 CFUs, less than about 500 CFUs, or no measurable CFUs. In some examples, the method results in a reduction of microbial contamination (such as yeast, mold, virus, and/or bacteria) in the sample of cannabis plant material sample by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, or by about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%, or by about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
In some examples, the level of THC, THCa, CBD, CBDa, and terpenes in the cannabis plant material prior to and following ozone treatment is substantially the same. The results shown in FIGS 8-10, 19B, 20B, 21B and 23-26, illustrate the fact that the ozone treatment methods disclosed herein do not decrease the level of cannabinoids or terpenes in a manner that impacts the desirability of the treated cannabis to consumers.
Cannabis plant material with a variety of initial microbial (such as yeast, mold, virus and/or bacteria) contamination levels can be used in the methods herein. For example, the initial level of microbial contamination in the cannabis material can be less than about 5,000, less than about 10,000, less than about 50,000, less than about 100,000, less than about 150,000, less than about 200,000, less than about 250,000, less than about 300,000, less than about 350,000, less than about 400,000, less than about 450,000, less than about 500,000, less than about 550,000, less than about 600,000, less than about 650,000, less than about 700,000, less than about 750,000, less than about 800,000, less than about 900,000, less than about 1,000,000, less than about 2,000,000, less than about 3,000,000, less than about 5,000,000, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 150,000, at least about 200,000, at least about 250,000, at least about 300,000, at least about 350,000, at least about 400,000, at least about 450,000, at least about 500,000, at least about 550,000, at least about 600,000, at least about 650,000, at least about 700,000, at least about 750,000, at least about 800,000, at least about 900,000, at least about 1,000,000, at least about 2,000,000, at least about 3,000,000, at least about 5,000,000, about 1,000 to about 5,000, about 5,000 to about 10,000, about 10,000 to about 50,000, about 50,000 to about 100,000, about 100,000 to about 250,000, about 250,000 to about 500,000,

- 14 -about 500,000 to about 1,000,000, about 1,000,000 to about 5,000,000 colony-forming units (CFU) of yeast, mold, virus, and/or bacteria.
In some examples, the methods can include reducing microbial (such as yeast, mold, virus, and/or bacteria) contamination in cannabis plant material to an allowable level for compliance with a regulatory agency or company. For example, prior to treatment (or pre-treatment) with ozone using the methods herein, cannabis plant material can include a level of microbial (such as yeast, mold, virus, and/or bacteria) contamination that exceeds an allowable level.
For example, untreated cannabis material can include a level of microbial contamination greater than or equal to 150,000 CFU microbial contamination/g cannabis.
The methods disclosed herein can be used to reduce the microbial contamination to a level that is compliant with the specific regulatory requirements for given states as shown in Table 1, for example, less than 1,000 or 10,000 CFU/g of cannabis for total yeast and mold, less than 10,000 or 100,000 CFU/g of cannabis for total viable aerobic bacteria, less than 100 or 1,000 CFU /g cannabis for total coliform contamination is allowed.
Table 1. US State Testing Regulations as of August 2019 State Rec or TYM Total Total Bile Tolerant Aspergillus E
Coli/ Mycotoxins Retest Medical (CFU/g) Viable Coliforms Gram Salmonella Allowed Aerobic (CFU/g) Negative Bacteria Bacteria (CFU/g) (CFU/g) Colorado Rec and 10,000 Non-Detect Yes Med MA Rec and 10,000 100,000 1,000 1,000 Non-Detect Non-Detect 201.tg/kg of Yes Med substance California Rec and - Non-Detect Non-Detect 201.tg/kg of With Med substance Approval Michigan Rec and 10,000 100,000 1,000 Non-Detect Non-Detect 20 ug/kg of Yes Med substance Washington Rec and - 10,000 Non-Detect 20 jig/kg of With Med substance Approval Oregon Rec and - 100 CFUsig Yes Med (E Coli only) Nevada Rec and 10,000 1,000 1,000 Non-Detect Non-Detect 20 jig/kg With Med combined Approval Alaska Rec and - Non-Detect 5WMithax Med Approval Delaware Medical Faculties set quality control, testing procedures and thresholds as part of their application.
Only They are subject to random testing by the state at any time.
Illinois Medical 1,000 10,000 100 100 Non-Detect 20 jig/kg of With Only substance Approval Ohio Medical 10,000 100,000 1,000 1,000 Non-Detect Non-Detect 20 jig/kg of Yes Only substance Hawaii Medical 10,000 100,000 1,000 ..
1,000 .. Non-Detect Non-Detect .. Yes Only

- 15 -PA Medical 10,000 10,000 1,000 Non-Detect 20 ug/kg of -Only substance Oklahoma Medical 10,000 Non-Detect Only New Medical 1,000 100,000 1,000 Non-Detect Yes Mexico Only As of August 2019, some states including Washington DC, Maine, Vermont, Alaska, New York, Florida, Connecticut, New Hampshire, Connecticut, New Jersey, Rhode Island, West Virginia, Maryland, Arkansas, Minnesota, North Dakota, Montana, and Arizona, allow medical use only.
In some examples, the methods disclosed herein can reduce pesticides in cannabis plant material to an allowable level for compliance with a regulatory agency or company. In specific, non-limiting examples, the pesticides are Myclobutanil, Bifenazate, Spiromesifen, or Imidacloprid.
For example, prior to treatment with ozone using the methods herein, cannabis plant material can include a level of pesticide that exceeds an allowable level. For example, pre-treatment cannabis material can include a level of pesticide greater than or equal to about 1 ppb, greater than or equal to about 2 ppb, greater than or equal to about 5 ppb, greater than or equal to about 10 ppb, greater than or equal to about 15 ppb, or greater than or equal to about 20 ppb. After treatment, the pesticide in cannabis plant can be reduced, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 45%, at least about 48%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% to a level allowable level for compliance with a regulatory agency or company, such as about 1 ppb, about 2 ppb, about 5 ppb, about 10 ppb, about 15 ppb, or about 20 ppb.
In specific examples, the methods for reducing the microbial contamination in a sample of cannabis plant material include: providing a pathogen reduction device, which includes an ozone chamber, an oxygen concentrator configured to concentrate oxygen from ambient air and produce ozone, an ozone regulator configured to adjust the ozone concentration in the ozone chamber; and one or more processors with a memory coupled to the one or more processors, the memory storing instructions that when executed by the one or more processors cause the one or more processors to determine a concentration of gaseous ozone in an ozone chamber; and adjust the concentration of gaseous ozone in the ozone chamber to a preset concentration; placing a sample of cannabis plant material comprising microbial contamination inside the ozone chamber of the pathogen reduction device; setting the ozone concentration in the ozone chamber to between about 200 ppm and about 400 ppm; and treating the cannabis plant material with ozone by leaving it in the ozone chamber for a time period of at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least

- 16-2 hours, at least 4 hours, at least 12 hours, from 4 to 8 hours, from 6 to 12 hours, from 10 to 24 hours, and from 16 hours to 8 hours, wherein the method reduces the level of microbial contamination in the sample of cannabis plant material.
EXAMPLES
The disclosure is further illustrated by the following examples. The examples are provided for purposes of example only. They are not to be construed as limiting the scope or content of the invention in any way.
Example 1 FIG. 1 is a graph showing average microbial counts from three ozone exposure trials for each of E. coli, Salmonella, Listeria, and Candida inoculated Cannabis plants.
Details regarding that study are as follows.
Cannabis plants were individually contaminated with Escherichia coli, Salmonella enterica .. sv typhimurium, Listeria monocytogenes and Candida albicans then exposed to gaseous ozone for minutes at 100 ppm and 200 ppm. Significant kills of all bacterial pathogens were observed at 200 ppm while a lesser kill rate was seen at 100 ppm except for the E. coli which was observed to be significantly vulnerable to both 100 ppm and 200 ppm ozone concentrations.
The effect of ozone treatment on C. albicans were limited; however, testing at ozone concentrations higher than 20 200 ppm may provide markedly different results, as the results in FIG. 1 from the 200 ppm trials showed an average reduced microbial count for C. albicans when compared with the control group.
Pure cultures of E. coli and L. monocytogenes from the American Type Culture Collection (ATCC) were grown in selective media broths. The C. albicans from ATCC was grown on potato dextrose agar, and after significant growth was achieved, the colonies were transferred and suspended in Butterfield's buffer. After significant turbidity in each broth of pathogens was observed, they, along with Butterfield's buffer with the suspended C.
albicans, were used to inoculate individual Cannabis samples. This process created four individual populations of Cannabis with each population being contaminated with one of the four organisms grown. After inoculating the samples they were allowed to dry overnight by placing them out in the open at room temperature. Each population of Cannabis was then divided into smaller individual 2-4 gram samples which were used in the tests. One test consisted of placing nearly half of the smaller samples into an ozone gas chamber for 20 minutes at an approximate ozone concentration of 100 ppm. The remaining samples were set aside to be used as controls.

- 17 -Three samples for each organism were tested at each of the concentrations. The control samples were placed into the same chamber for 20 minutes with an ozone concentration of 0 ppm.
All samples were then analyzed for their microbial counts by first generating serial dilution using Butterfield's buffer as the diluent for each sample then individually plating the resultant dilutions onto selective media appropriate for the organism being analyzed. After the required incubation temperatures and times were completed, the organisms on the plates were counted. For each set of dilutions of a given sample and starting at the plate representing the lowest dilution ratio, the first plate that was countable was counted and the result was recorded.
Testing occurred over two days. The first day involved testing one sample of Cannabis for each organism at an ozone concentration of 100 ppm and another test of only Listeria at 50 ppm.
Additional testing involving subsequent trials for each organism at ozone concentrations of 100 ppm and 200 ppm were conducted on the second day of testing.
The ozone concentration in the ozone chamber was kept at a range approximating the desired 100 ppm and 200 ppm concentrations. For instance, when the desired concentration was 100 ppm, the actual range of concentrations was 98 ppm to 106 ppm, and when the desired concentration was 200 ppm, the actual range of concentration was 198 ppm to 204 ppm. While the samples were being placed into the chamber ozone gas was lost. It took 3 minutes before the desired concentration of 100 ppm was achieved for tests requiring that ozone concentration and 5 minutes before the desired concentration of 200 ppm was achieved for tests requiring that ozone concentration.
The result of testing is shown in Table 2. At an ozone concentration of 50 ppm, which only Listeria was involved with, there was no statistically significant difference between the control and the treated sample. At an ozone concentration of 100 ppm there was a significant kill of all of the organisms tested.
Table 2: Concentration range of gaseous ozone present during each test, the amount of time the organisms were subjected to the ozone and the microbial counts after the treatments shown for the first day of testing.
Microbe Target Ozone Actual Ozone Treatment Time Microbial Count Concentration Concentration (mm) (cfu/g) (ppm) Range (ppm) E. Coli Control 0 N/A 20 1.2*10"
- -E. Coli Treated 100 95-102 20 2.8*1010 Salmonella Control 0 N/A 20 1.1*10"
Salmonella Treated 100 95-102 20 1.7*1010

- 18 -Listeria Control 0 N/A 20 1.9* 107 Listeria Treated 100 95-102 20 4.8* i05 Listeria Control 0 N/A 20 7.0*102 Listeria Treated 50 46-52 20 2.6*102 Mold Control 0 N/A 20 1.4*10I2 Mold Treated 100 95-102 20 1.9*1011 The results of the second day of testing are shown in Table 3. There was a significant kill of all organisms at an ozone concentration of 100 ppm when compared to the controls and at an ozone concentration of 200 ppm the amount of kills increased when compared to the kills at 100 PPIll=
Table 3: Concentration range of the gaseous ozone present during each test, the amount of time the organisms were subjected to the ozone and the microbial counts after treatments shown for the second day of testing. "BLD" stands for below the limit of detection.
Microbe Target Ozone Actual Ozone Treatment Time Microbial Count Concentration Concentration (mm) (cfu/g) (1)Pm) Range (ppm) E. Coli Control 0 N/A 20 9.0*104 E. Coli Trial 1 100 See Table 2 20 See Table 2 E. Coli Trial 2 100 98-106 20 BLD
E. Coli Trial 3 100 98-106 20 3.6*10I
E. Coli Trial 1 200 198-204 20 BLD
.............. , ....................................................
E. Coli Trial 2 200 198-204 20 2.3*10I
, E. Coli Trial 3 200 198-204 20 2.0*101 .............. , ....................................................
Salmonella 0 N/A 20 4.5*104 Control Salmonella Trial 1 100 98-106 20 See Table 1 .............. , ....................................................
Salmonella Trial 2 100 98-106 20 1.1*104 , Salmonella Trial 3 100 See Table 1 20 1.3*104 Salmonella Trial 1 200 198-204 20 7.0*102 Salmonella Trial 2 200 198-204 20 2.2*103 Salmonella Trial 3 200 198-204 20 8.0*103 -----------------------Listeria Control 0 N/A 20 2.0*104

- 19 -Listeria Trial 1 100 See Table 2 20 See Table 2 Listeria Trial 2 100 98-106 20 2.0*103 Listeria Trial 3 100 98-106 20 1.0*103 Listeria Trial 1 200 198-204 20 3.0*102 Listeria Trial 2 200 198-204 20 3.1*102 Listeria Trial 3 200 198-204 20 1.1*102 Candida Control 0 N/A 20 5.0*105 Candida Trial 1 100 See Table 2 20 See Table 2 Candida Trial 2 100 98-106 20 2.7*105 Candida Trial 3 100 98-106 20 1.5904 Candida Trial 1 200 198-204 20 1.0*105 Candida Trial 2 200 198-204 20 8.3*104 Candida Trial 3 200 198-204 20 9.8*104 This demonstrates that gaseous ozone is an effective antimicrobial step when treating Cannabis samples contaminated with pathogens. It is presumed that plant samples that would be treated for human consumption would not be near the level of contamination that this study generated through the inoculation processes. Therefore the use of gaseous ozone as an antimicrobial step in production would assure that the level of pathogen contamination after this step would be under the tolerance levels set by safety guidelines.
The correlation between the reduction in pathogen levels and ozone exposure observed can be extrapolated to many different plant species. However, these results show that the disclosed methods provide a non-toxic means for reducing harmful pathogen levels to acceptable safety standards in the Cannabis plant.
As shown in FIG. 1, test results for the study described above are provided.
The X-axis of graph 100 is divided into average test results for gaseous ozone treatment of Cannabis inoculated with E. Coli, Salmonella, Listeria and Candida compared against control (i.e., inoculated Cannabis not treated with ozone). The Y-axis of graph 100 depicts averages for the test results, in log values of colony-forming units per gram (CFU/gram), for the study. Log values for average CFU/gram for E. Coli are shown at 102. Log values for average CFU/gram for Salmonella are shown at 104. Log values for average CFU/gram for Listeria are shown at 106 and log values for average CFU/gram for Candida are shown at 108.
Terpene Testing Methods: Headspace Gas-Chromatography with Flame Ionization Detection, or headspace GC-FID was used. This method is used in the environmental and pharmaceutical industries to analyze for product or environmental contamination. For each test, a

- 20 -small sample of cannabis is used. The sample is heated in an airtight vial to vaporize the residual solvents, sample the headspace in the vial and inject the headspace sample into a gas chromatograph for chemical analysis. In analyzing sample headspace, various matrix interferences were screened from the concentrate. The terpene content for the samples may represent lower than expected results as no correction for moisture content was performed. As such, some terpenes may have evaporated upon drying giving lower than expected terpene results.
The ability of ozone to reduce TYM using an embodiment of a disclosed pathogen reduction device was also evaluated. In this series of experiments, homogenized cannabis flower with a TYM bio-burden exceeding 10,000 CFU/g was exposed to ozone using an embodiment of a .. disclosed pathogen reduction device. 20 gram samples of the cannabis flower were separated into two groups (10 grams a group) and placed on different racks within the pathogen reduction device.
One 10 gram sample was placed on a perforated tray with air holes in order to maximize ozone flow and increase the surface area of the flower that would be exposed to ozone.
gram samples as described above were exposed to ozone for 0 minutes (control), 15 .. minutes, 30 minutes, 45 minutes, and 60 minutes at an ozone concentration of 200 ppm. Six 1 gram samples from each treatment time were processed and evaluated using a 3M
Petrifilm Rapid Yeast and Mold Count Plate and accompanying Product Instructions. The ozone treated samples were homogenized, diluted with buffer (distilled water), and then 1 milliliter suspension samples were dispensed onto 3M Petrifilm Rapid Yeast and Mold Count Plates.
The plates 20 were incubated at 27 C for greater than 60 hours in order to quantify the TYM remaining on the cannabis flower following exposure to ozone. Three of the six 1 gram samples were taken from the flower placed on the perforated tray. Two of the 1 gram samples were taken from the flower placed directly on the screen. The final 1 gram sample was collected from the lowest mesh screen in the device where the finest flower collected after falling through the rack screen.
As detailed in the following Table 4 and Table 5, a 92% decrease in TYM bio-burden was observed after treatment with ozone, with Colony Forming Units (CFUs)/gram dropping from an average of 82,000 CFU/g to an average of 7,000 CFU/g after treatment with ozone for 60 minutes.
The most significant reduction in CFU/g was observed in the first 20 minutes of exposure to ozone.
After the first 20 minutes and up until 45 minutes, there was only an additional 1.1% reduction in CFU/g. However, between a 45 minute and 60 minute exposure to ozone, there was another 14.1%
reduction in CFU/g. Notably, there was a reduction in yeast and mold across all 6 samples from roughly 105 CFU/g to below 10,000 CFU/g following treatment with ozone in an embodiment of a disclosed pathogen reduction device.

- 21 -Table 4: Average CFU/g of yeast and mold remaining on the tested cannabis flower following exposure to ozone for varying periods of time.
Sample ( t. =minutes) exposure time Average CFU/g (all samples tested) 0 minutes 68,000 20 minutes 18,000 30 minutes 17,000 45 minutes 16,000 60 minutes 6,000 Table 4 reveals that fungi are strongly inhibited by ozone exposure. Notably, graphing the results of Table 4 (exposure time versus average CFU/g) allows an estimation of treatment time for sample with higher levels of fungal contamination than tested in Table 4. The data to support Table 4 is disclosed in Table 5.
Table 5: Results from experiments described above and presented in Table 4. TO-1 represents sample 1 with an ozone exposure time of 0 minutes. T20-1 represents sample 1 with an ozone exposure time of 20 minutes. T30-1 represents sample 1 with an ozone exposure time of 30 minutes. T45-1 represents sample 1 with an ozone exposure time of 45 minutes.
T60-1 represents sample 1 with an ozone exposure time of 60 minutes.
Sample CFU/g CFU/g (6 samples for (Test 1) (Test 2) each exposure in minutes) TO-1 77,000 61,000 TO-2 30,000 28,000 TO-3 50,000 77,000 TO-4 25,000 53,000 TO-5 60,000 140,000 TO-6 82,000 130,000 T20-1 18,000 24,000 T20-2 15,000 16,000 T20-3 15,000 17,000 T20-4 19,000 23,000 T20-5 12,000 18,000 T20-6 21,000 21,000 T30-1 16,000 20,000 T30-2 18,000 30,000 T30-3 13,000 16,000

- 22 -............................ , ..............
, T30-4 16,000 15,000 T30-5 11,000 .. i .. 14,000 T30-6 20,000 20,000 T45-1 7,100 11,000 ............................ i ..............
45-2 13,000 15,000 145-3 11,000 12,000 T45-4 21,000 48,000 ............................ i ..............
T45-5 16,000 17,000 T45-6 8,000 12,000 , T60-1 7,400 , 9,000 T60-2 6,000 10,000 T60-3 7,800 .. i ... 6,300 T60-4 770 3,100 T60-5 4,300 4,400 ............................ i ..............
T60-6 6,300 7,100 Some additional ozone exposure tests were performed with cannabis flower or trim on a variety of commercially available strains using an embodiment of a disclosed pathogen reduction device. In each experiment (Table 6) a reduction of TYM CFU/g was measured (as disclosed above using 3M Petrifilm Rapid Yeast and Mold Count Plate and Product Instruction) after treating different weights of cannabis with an ozone concentration of 200 ppm for between 20 and 60 minutes.
Table 6: Results of ozone treatment of cannabis flower or trim to reduce mold and yeast. CFUs were measure before treatment (Initial CFU) and after treatment (Ending CUF) with ozone.
Total Weight Ozone Concentration Type of Product Strain Type Initial CFU Ending CFU .. Run (lbs) ..
(PPI11) .. Length of Time (min.) Flower Hybrid / Sativa 100,000 5,400 - 5,500 1 lb 200 20:00 Flower Hybrid / Indica 51,750 30,000-32,000 6 lbs 200 25:00 Trim Hybrid / Indica 17,000 800-11,000 6 lbs 200 25:00 Flower Hybrid/Indica 2,200 220 1 lb 200 45:00 Flower Indica 320,000 91,000 3 lbs 200 60:00 Trim Hybrid / Sativa 50,000 3,000 3 lbs 200 60:00 Flower Hybrid / Indica 110,000 3,000 NA 200 60:00 Flower Hybrid 77,000 4500 5 lbs 200 60:00 While ozone exposure can successfully reduce pesticides and fungus on cannabis flower, it has little to no effect on the terpenes of a cannabis flower.

- 23 -Example 2 Test samples of retail or medical marijuana that are required to be uniform in strain and to be a representative sample of a commercial lot were submitted to a licensed testing facility for testing purposes. Samples that failed the commercial test were provided for remediation together with the TYM level (as colony-forming units, or CFU) at the time of submission. Over 1700 samples were analyzed and remediated using the ozone treatment system disclosed herein. The samples had starting levels of yeast and mold contamination ranging from less than 100 CFU to over 1,000,000 CFU. Samples ranged in size from 0.5 lbs. to over 30 lbs. The samples were placed in the ozone treatment chamber for periods of time ranging from less than 60 minutes to 48 hours (less than 60 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 23 hours, 30 hours, and 48 hours), at ozone concentrations from 200 to 400 ppm.
Tables 7-9 show three sets of results for contaminated samples that were remediated using the ozone system disclosed herein.
Table 7: Results of 240 minute ozone treatment of contaminated cannabis samples. The contamination level was less than 75K CFU prior to testing.
Sample Initial CFU Ending CFU CFU Reduction Pass(1)/Fail(0) after treatment 1 11,000 2,500 77.27% 1 2 11,000 50 99.55% 1 3 11,000 50 99.55% 1 4 11,000 50 99.55% 1 ¨ "- +-5 14,000 2,500 82.14% 1 6 14,000 2,500 82.14% 1 7 15,000 50 99.67% 1 .......... , 8 15,000 50 99.67% 1 9 18,000 2,500 86.11% 1 10 18,000 60 99.67% 1 .......... , ........................................
11 18,000 70 99.61% 1 12 20,000 50 99.75% 1 13 20,000 80 99.60% 1 14 21,000 6,500 69.05% 1 15 21,000 12,000 42.86% 0 ..................................... .: ............ .: ..........
16 22,000 2,500 88.64% 1 17 23,000 530 97.70% 1 ---------- , ------------------------------------------------------18 23,000 220 99.04% 1 19 24,000 250 98.96% 1

24,000 250 98.96% 1 ' 21 30,000 2,500 91.67% 1 ..................................... , ............. ; ...........
22 32,000 540 ' 98.31% 1 ...... , .....................................................
23 32,000 880 97.25% 1 ................................. 4 ............. 4 ..........
24 35,000 2,500 92.86% 1

25 35,000 1,700 95.14% 1 s ..... , ; ;

26 44,000 2,500 94.32% 1 ................................. 4 ............. 4 ..........

27 44,000 6,900 84.32% 1

28 44,000 1,800 95.91% 1

29 48,000 2,500 94.79% 1 --- ,'-- ,`--

30 48,000 29,000 39.58% 0 ................................. 4 ............. 4 ..........

31 48,000 29,000 39.58% 0

32 51,000 9,000 82.35% 1 --- ,'-- ,'--

33 51,000 8,800 82.75% 1 ................................. 4 ............. 4 ..........

34 51,000 8,800 82.75% 1

35 56,000 1,000 98.21% 1 ...... , ........................

36 56,000 11,000 80.36% 0 ................................. 4 ............. 4 ..........

37 56,000 8,100 85.54% 1

38 56,000 4,500 91.96% 1 ...... , ........................

39 56,000 8,100 85.54% 1 ................................. 4 ............. 4 ..........

40 57,000 3,400 94.04% 1

41 57,000 1,300 97.72% 1 Average 88.40% 90.24%
Table 8: Results of 360 minute ozone treatment of contaminated cannabis samples. The contamination level was less than 150K CFU prior to testing.
Sample Initial Ending CFU Pass(1)/Fail(0) CFU CFU Reduction after ..................................... , treatment 1 11,000 50 99.55% 1 2 11,000 150 98.64% 1 3 11,000 50 99.55% 1 ..................................... 4 .........
4 11,000 50 99.55% 1 11,000 50 99.55% 1 6 11,000 270 97.55% 1 ..................................... 4 .........
7 11,000 420 96.18% 1 8 11,000 880 92.00% 1 9 12,000 2,500 79.17% 1 ..................................... 4 .........
13,000 1,100 91.54% 1 11 13,000 840 93.54% 1 12 14,000 50 99.64% 1 ..................................... 4 .........
13 14,000 50 99.64% 1 ..................................... 4 .........
14 14,000 1,700 87.86% 1 14,000 770 94.50% 1 16 16,000 0 100.00% , 1 17 16,000 230 98.56% 1 18 16,000 350 97.81% 1 19 16,000 2,500 84.38% 1 ..................................... 4 .........
16,000 2,500 84.38% 1 - - - -21 20,000 140 99.30% 1 22 20,000 510 97.45% 1 ........ , ..... , .... , ........ 4 .........
23 21,000 2,500 88.10% 1 -- --24 21,000 2,500 88.10% 1 ; .............................................
25 22,000 2,500 88.64% 1 .................................. 4 .........
26 22,000 50 99.77% 1 -- --27 22,000 60 99.73% 1 ; .............................................
28 22,000 2,600 88.18% 1 29 22,000 2,300 89.55% 1 ........ , ..... , .... , ........ 4 .........
30 23,000 4,100 82.17% 1 31 23,000 21,000 8.70% 0 32 24,000 50 99.79% 1 ........ , ..... , .... , ........ 4 .........
33 24,000 50 99.79% 1 34 24,000 180 99.25% 1 .................................. 4 .........
35 24,000 160 99.33% 1 36 24,000 300 98.75% 1 37 24,000 = 330 98.63% 1 .................................. 4 .........
38 25,000 2,500 90.00% 1 .................................. 4 .........
39 27,000 50 99.81% 1 40 27,000 = 50 99.81% 1 ; .............................................
41 27,000 700 97.41% 1

42 27,000 810 97.00% 1 ........ , ..... , .... , ........ 4 .........

43 28,000 680 97.57% 1

44 28,000 3,300 88.21% 1

45 30,000 2,500 91.67% 1 ........ , ..... , .... , ........ 4 .........

46 31,000 1,600 94.84% 1

47 31,000 3,700 88.06% 1 .................................. 4 .........

48 32,000 50 99.84% 1 .................................. 4 .........

49 32,000 50 99.84% 1

50 32,000 1,000 96.88% 1 .................................. 4 .........

51 32,000 620 98.06% 1 .................................. 4 .........

52 33,000 4,800 85.45% 1 ---------------- , ---- , --------------------

53 33,000 5,400 83.64% 1 ; .............................................

54 34,000 1,300 96.18% 1

55 34,000 3,400 90.00% 1 ........ , ..... , .... , ........ 4 .........

56 37,000 4,500 87.84% 1

57 37,000 4,600 87.57% 1

58 38,000 720 98.11% 1 ........ , ..... , .... , ........ 4 .........

59 38,000 730 98.08% 1

60 38,000 250 99.34% 1 .................................. 4 .........

61 38,000 540 98.58% 1 ........ , ..... , .... , ........ 4 .........

62 39,000 50 99.87% 1

63 39,000 = 50 99.87% 1 .................................. 4 .........

64 40,000 2,500 93.75% 1 .................................. 4 .........

65 40,000 2,500 93.75% 1 ........ , ..... , .... , ........ 4 .........

66 41,000 0 100.00% 1 ; .............................................

67 41,000 110 99.73% 1 .................................. 4 .........

68 41,000 190 99.54% 1

69 42,000 0 100.00% 1 ........... , ..................... 4 ........

70 42,000 810 98.07% 1 - - - -

71 42,000 640 98.48% 1 ; ........

72 43,000 730 98.30% 1 ..................................... 4 ........

73 43,000 620 98.56% 1

74 45,000 0 100.00% 1 ; ........

75 47,000 100 99.79% 1 , 4.,

76 47,000 130 99.72% 1 ........... , ..................... 4 ........

77 48,000 0 100.00% 1

78 48,000 2,500 94.79% 1 , õ õ 4.,

79 48,000 2,500 94.79% 1 ........... , ..................... 4 ........

80 53,000 1,200 97.74% 1

81 53,000 520 99.02% 1

82 58,000 0 100.00% 1

83 59,000 120 99.80% 1

84 59,000 220 99.63% 1

85 63,000 11,000 82.54% 0

86 63,000 830 98.68% 1

87 63,000 810 98.71% 1

88 65,000 50 99.92%
, .... .... 4.,

89 65,000 80 99.88% 1 ........... , ..................... 4 ........

90 66,000 620 99.06% 1

91 66,000 620 99.06% 1

92 69,000 0 100.00% 1 ........... , ..................... 4 ........

93 77,000 0 100.00% 1

94 83,000 0 100.00% 1

95 95,000 0 100.00% ' 1

96 100,000 3,400 96.60% 1 - - -

97 100,000 2,500 97.50% 1

98 100,000 2,500 97.50% 1 ..................................... 4 ........

99 100,000 2,500 97.50% 1

100 110,000 21,000 80.91% 0 ; ........

101 120,000 0 100.00% 1

102 130,000 10,000 92.31% 0 ........... , ..................... 4 ........

103 130,000 190 99.85% 1

104 130,000 3,700 97.15% 1 - - - - - ---

105 130,000 3,600 97.23% 1 ........... , ..................... .: .......
Average 95.07% 96.19%
Table 9: Results of 840 minute ozone treatment of highly contaminated cannabis samples. The contamination level was greater than 150K CFU prior to testing.
Sample Initial Ending CFU Pass(1)/Fail(0) CFU CFU Reduction after treatment 1 3,700,000 1,000 99.97% 1 2 3,700,000 1,800 99.95% 1 3 3,000,000 220 99.99% 1 4 3,000,000 390 99.99% 1 3,000,000 430 99.99% 1 6 3,000,000 440 99.99% 1 7 1,500,000 50 100.00% 1 8 1,500,000 90 99.99% 1 9 680,000 800 99.88% 1 680,000 1,000 99.85% 1 11 660,000 4,300 99.35% 1 12 660,000 3,500 99.47% 1 13 640,000 2,300 99.64% 1 14 640,000 3,500 99.45% 1 590,000 4,400 99.25% 1 16 590,000 11,000 98.14% 0 17 470,000 20 100.00% 1 18 370,000 570 99.85% 1 19 370,000 710 99.81% 1 340,000 710 99.79% 1 21 340,000 1,300 99.62% 1 22 330,000 2,400 99.27% 1 23 330,000 2,300 99.30% 1 24 320,000 700 99.78% 1 320,000 810 99.75% 1 26 270,000 2,200 99.19% 1 27 270,000 2,500 99.07% 1 28 270,000 120 99.96% 1 29 270,000 380 99.86% 1 270,000 250 99.91% 1 31 270,000 50 99.98% 1 32 260,000 290 99.89% 1 33 260,000 580 99.78% 1 34 170,000 190 99.89% 1 170,000 610 99.64% 1 Average 99.69% 97.14%
Example 3 In an example, samples of retail marijuana were submitted to a certified testing facility to examine levels of Aspergillus (Asp), total viable aerobic bacteria (TVAB), coliform bacteria, total 5 yeast and molds (TYM), and bile-tolerant gram-negative bacteria (BTGN) (FIGS. 6-7). The TYM
and coliform testing was performed using agar plate counting. Samples that failed the commercial test (one test) were provided for remediation using the ozone treatment system disclosed herein and then re-tested (two tests; FIG. 6, remediated samples denoted with "Willow").
The samples were placed in the ozone treatment chamber for periods of time ranging from 12-24 hours, at ozone 10 concentrations from 200 to 400 ppm.

The samples that failed (FIG. 6, "F") the Asp testing passed after remediation (FIG. 6, "Willow Asp"). The sample TVAB ranged from 3M CFU to 50 CFU, and remediation reduced the CFU of TVAB in all samples to less than 100K CFU. The sample coliform ranged from 670K
CFU to 0 CFU, and remediation reduced the CFU of coliform in all samples to less than 1K CFU.
The sample TYM ranged from 1.5M CFU to 0 CFU, and remediation reduced the CFU
of TYM in all samples to less than 1K CFU. The sample BTGN ranged from 750K CFU to 0 CFU, and remediation reduced the CFU of BTGN in all of samples to less than 1K CFU.
Example 4 To examine the efficacy of the ozone treatment on the fungal load of cannabis flower, previously homogenized cannabis flower was sampled with fungal levels exceeding 10,000 CFU/g. Samples of approximately 3 grams were placed directly onto a rack screen. The samples primarily included larger buds. The effect on chemotype was also examined by extracting and testing for cannabinoid and terpene content before and after ozone exposure.
Methods and materials. Subsamples were removed from the racks after treatment times of 0, 20, 40, and 60 minutes. Three 1-gram samples from each treatment period were aseptically handled for the fungus quantification steps of the cannabis flower, and the samples were incubated at 27 C 2 C for ¨72 hours.
Cannabinoid and terpene content were measured in a blinded sample before ozone treatment and after 20, 40, and 60 minutes. The flower was divided into 2 subsamples.
One subsample was immediately processed for chemotype analysis, while the second subsample was placed in the ozone chamber for 1 hour and then prepared for analysis.
Results. As shown in Table 10, a 96.6% decrease in fungal bioburden was observed with colony forming units (CFUs)/gram dropping from an average of 70,000 CFU/g to an average of 2,700 CFU/g after a treatment period of 60 minutes. Treating the material for 20 and 40 minutes reduced the fungal level to 6,500 CFU/g and 4,800 CFU/g, respectively. Across all samples at multiple dilutions, a reduction in fungi was observed from approximately 104 CFU/g to below 10,000 CFU/g. The high SD at time 0 like reflects the heterogeneity of dense buds. A decrease in SD at a greater rate than the mean at subsequent time points indicates that the ozone successfully permeated all of the buds to the same degree. Table 11 shows the percentage of total potential cannabinoids and total terpenes for a dense bud before and after ozone exposure.

Table 10: Average CFU/g of all dilutions vs. average CFU/g on highest count Time CFU/g SD
Average (all) 0 Minutes 70,000 110,000 20 Minutes 6,500 2,500 40 Minutes 4,800 770 60 Minutes 2,700 970 Table 11:. Percentage of total potential cannabinoids and total terpenes (using GC) for a dense bud before and after ozone exposure.
Trial Run Total potential THC (%) Total terpenes (%) A 11.75 (0 min) 11.26 (20 min) 1.26 (0 mm) 1.38 (20 mm) 14.93 (0 min) 13.59 (40 min) 1.01 (0 mm) 1.00 (40 mm) 13.78 (0 min) 14.79 (60 min) 1.34 (0 mm) 1.4 (60 min) Example 5 In an example, control and test samples were examined for TYM as well as %THCa and THC (potency). Plants produce THCa which is decarboxylated to THC through heat and light.
THC is the active form and is the measure of "potency" of a cannabis sample and may be referred to as such. THC analysis using GC is carried out by extracting dried cannabis plant material with an organic solvent and the supernatant of the extract is injected into a gas chromatograph for separation and detected by either a flame ionization detector or by a mass spectrometer for positive identification. The TYM testing was performed using agar plate counting.
Samples that failed the commercial test (one test) were provided for remediation using the ozone treatment system disclosed herein and then re-tested (two tests; FIG. 6, remediated samples denoted "Willow"). The samples were placed in the ozone treatment chamber for periods of time ranging from 12-24 hours, at ozone concentrations of from 200 to 300 ppm.
The samples that failed the Asp. Test (FIG. 6, column 1, "F") passed after remediation (FIG. 6, "Willow Asp"). The sample TVAB ranged from 3M CFU to 50 CFU, and remediation reduced the CFU of TVAB in 98% of samples to less than 100K CFU. The sample coliform ranged from 670K CFU to 0 CFU, and remediation reduced the CFU of coliform in 98% of samples to less than 1K CFU. The sample TYM ranged from 1.5M CFU to 0 CFU, and remediation reduced the CFU of TYM in all samples to less than 1K CFU. The sample BTGN ranged from 750K CFU to 0 CFU, and remediation reduced the CFU of BTGN in 94% of samples to less than 1K
CFU.

Further, the remediated samples that did not exhibit reduced to the levels of contaminants were located under a leak and were likely higher due to contamination. Data showing the effect of ozone treatment using the methods disclosed herein on TYM, THCa, potency and terpene levels is summarized in Tables 12-14.
Table 12: Percentage of THCa and potency as well as CFU of Total Yeast and Mold before (Control) and after ozone exposure ("Willow").
Treatment time: THCa (%) Potency (%) TYM
16 hours Control 1 15.42 14.30 31,000 CFU
Control 2 14.61 13.44 11,000 CFU
Control 3 14.48 13.37 33,000 CFU
Control Average 14.84 13.70 25,000 CFU
Willow 1 17.32 16.2 120 CFU
Willow 2 18.64 17.1 390 CFU
Willow 3 16.89 15.45 160 CFU
Post Willow 17.62 16.25 223 CFU
Average Table 13: Percentage of terpenes before (Control) ozone exposure.
Control 1 Control 2 Control 3 Control (%) (%) (%) Avg (%) alpha-Pinene 0.130 0.118 0.107 0.118 Camphene 0.000 0.000 0.000 0.000 beta-Myrcene 0.101 0.094 0.096 0.097 beta-Pinene 0.056 0.050 0.046 0.051 delta-3-Carene 0.000 0.000 0.000 0.000 alpha-Terpinene 0.000 0.000 0.000 0.000 Limonene 0.000 0.000 0.000 0.000 p-Cymene 0.000 0.000 0.000 0.000 beta-Ocimene 0.000 0.000 0.000 0.000 Eucalyptol 0.000 0.000 0.000 0.000 gamma-Terpinene 0.000 0.000 0.000 0.000 Terpinolene 0.000 0.000 0.000 0.000 Linalool 0.000 0.000 0.000 0.000 alpha-Ocimene 0.000 0.000 0.000 0.000 Isopulegol 0.000 0.000 0.000 0.000 beta-Carophyllene 0.122 0.128 0.111 0.120 alpha-Humulene 0.050 0.052 0.043 0.048 cis-Nerolidol 0.015 0.012 0.011 0.013 trans-Nerolidol 0.017 0.000 0.000 0.006 Guaiol 0.000 0.000 0.000 0.000 Caryophyllene-oxide 0.000 0.000 0.000 0.000 alpha-Bisobolol 0.000 0.000 0.000 0.000 Total Terpene 0.491 0.454 0.414 0.453 Table 14: Percentage of terpenes after ozone exposure ("Willow").
Willow 1 Willow 2 Willow 3 Willow Avg (go) (go) (%) (%) alpha-Pinene 0.122 0.118 0.117 0.119 Camphene 0.000 0.000 0.000 0.000 beta-Myrcene 0.100 0.091 0.091 0.094 ...................... , ......
beta-Pinene 0.053 0.049 0.050 0.051 delta-3-Carene 0.000 0.000 0.000 0.000 alpha-Terpinene 0.000 0.000 0.000 0.000 Limonene 0.000 0.000 0.000 0.000 ...................... , ..........................
p-Cymene 0.000 0.000 0.000 0.000 beta-Ocimene 0.000 ' 0.000 0.000 0.000 Eucalyptol 0.000 0.000 0.000 0.000 gamma- 0.000 0.000 0.000 0.000 Terpinene Terpinolene 0.000 0.000 0.000 0.000 Linalool 0.000 0.000 0.000 0.000 alpha-Ocimene 0.000 0.000 0.000 0.000 Isopulegol 0.000 0.000 0.000 0.000 beta- 0.127 0.118 0.125 0.123 Carophyllene alpha-Humulene 0.050 0.048 0.050 0.049 cis-Nerolidol 0.015 0.016 0.019 0.017 trans-Nerolidol 0.000 0.026 0.034 0.020 Guaiol 0.000 0.000 0.000 0.000 Caryophyllene- 0.000 0.000 0.000 0.000 oxide alpha-Bisobolol 0.000 0.000 0.000 0.000 Total Terpene 0.467 0.466 0.486 0.473 Example 6 In one study, control and test samples were examined for %THC and %THCa before and after ozone treatment (FIGS. 8-10) The `Willow'-treated samples were treated with 300 ppm ozone for 2, 6, and 24 hrs. The control samples did not receive the ozone treatment.
The %THC and %THCa before and after ozone treatment were substantially the same (i.e., within the expected range of variability of the methods employed).
Example 7 A large number of cannabis strains were evaluated for TYM before and after ozone treatment (FIGS 11-22). Some samples were also evaluated for terpene and THC
content as well (FIGS. 19A-21B). Samples ranged in size from 100 g to 18 lbs. The samples were placed in the ozone treatment chamber for periods of time ranging from 20 mm - 18 hours, at an ozone concentrations of 225 ppm. In the majority of cases, the ozone treatment reduced the microbial burden by about 99%. The terpene and THC percentage was substantially the same before and after ozone treatment for 20 minutes, 40 minutes and 18 hours of ozone treatment (FIGS. 19A-21B).
In a similar study, cannabis samples contaminated with a total yeast and mold content of 13,000 to 100,000 CFU were placed in an ozone treatment chamber for periods of time ranging from 12 hours to 18 hours at an ozone concentrations of 250 ppm. The results in Table 15, below show that in the majority of cases, the ozone treatment significantly reduced the microbial burden in the cannabis samples, such that they were complaint with state regulations.
Table 15. Effect of 250 ppm ozone treatment on total yeast and mold content of cannabis flower.
Initial Ending CFU Length of Strain name CFU CFU Reduction Run(m) Afghani 43,000 380 99.12% 720 Afghani 43,000 630 98.53% 720 SoCal Al 40,000 0 100.00% 720 SoCal Al 40,000 350 99.13% 720 Tangie 13,000 810 93.77% 720 Tangie 13,000 710 94.54% 720 Afghani 43,000 380 99.12% 720 Afghani 43,000 630 98.53% 720 SoCal Al 40,000 0 100.00% 720 SoCal Al 40,000 350 99.13% 720 Tangie 13,000 810 93.77% 720 Tangie 13,000 710 94.54% 720 Glass Slipper 60,000 25 99.96% 960 Glass Slipper 60,000 7,800 87.00% 960 Glass Slipper 60,000 25 99.96% 960 Glass Slipper 60,000 25 99.96% 960 Sueno 14,000 0 100.00% 960 Sueno 14,000 0 100.00% 960 Scott's OG 1/5 100,000 800 99.20% 1080 Scott's OG 1/5 100,000 700 99.30% 1080 Scott's OG 1/7 49,000 300 99.39% 1080 Scott's OG 1/7 49,000 100 99.80% 1080 Example 8 In an example embodiment, a series of strains (Blue Dream, Purps, and Mob Boss) were examined for percent terpene in ozone-treated samples ("Willow") and untreated control samples (FIGS. 23-25). The Blue Dream strain was treated with 200 to 300 ppm ozone for 2 hours, and the Purps and Mob Boss strains were treated for 24 hours each. The percent terpene levels were examined over a variety of terpene types. For most terpene types, the treated and untreated control samples exhibited substantially the same amount of terpenes. The results of the tests shown in FIGS 23-25 demonstrate that terpene levels are not adversely affected by treatment with gaseous ozone according to methods described herein. Rather, total terpene levels for the tested samples for both the control samples and samples treated with gaseous ozone were substantially the same. That is, cannabis samples treated with gaseous ozone were shown to have terpene concentrations similar to cannabis samples that had not been treated with gaseous ozone.

Example 9 In an example embodiment, cannabis plant material was examined for percent terpene in ozone-treated samples ("Willow") and untreated control samples (FIG. 26).
Three samples were treated with 200 ppm to 300 ppm ozone for 18 hours. The individual and average percent THC and total terpene levels were evaluated. On average, the terpene levels increased (by less than one percent) in the treated and untreated control samples, and the THC levels increased (also by less than one percent) compared with the untreated controls.
Example 10 The ability of ozone to reduce pesticide residue using an embodiment of a disclosed pathogen reduction device was evaluated. Myclobutanil and Bifenizate were purchased in liquid formulations (Myclobutanil at 19.7% from Dow AgroSciences as Eagle 20EW and Bifenazate at 22.6% from Chemtura Corporation as Floramite@ SC). The pesticides were sprayed on homogenized hemp flower and air dried. Dried hemp flower with pesticide was then exposed to treatment with 200 ppm of ozone for 20 minutes. Non-pesticide treated hemp, pesticide treated hemp, and pesticide treated hemp that was exposed to ozone were analyzed by spectrometry using AOAC official method 2007.01. The results are disclosed below in Table 16, where Matrix Blank is non-pesticide treated hemp (negative control), Control is pesticide treated hemp that was not exposed to ozone, and Sample 1, Sample 2, and Sample 3 are three samples of pesticide treated hemp that was exposed to ozone at 200 ppm for 20 minutes.
Ozone treatment of hemp contaminated with Myclobutanil or Bifenizate was effective. For example, exposing hemp contaminated with Myclobutanil for 20 minutes at 200 ppm ozone resulted in an average reduction of 0.18 ppm Myclobutanil across the three samples tested.
Similarly, exposing hemp contaminated with Bifenizate for 20 minutes at 200 ppm ozone resulted in an average reduction of 0.1 ppm Bifenizate across the three samples tested.
Table 16: The ability of ozone treatment to effectively reduce Myclobutanil or Bifenizate. "LOD"
stands for below the limit of detection.
Compound Matrix Control Sample 1 Sample 2 Sample 3 Blank (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Myclobutanil LOD 0.5 0.39 0.28 0.28 Bifenazate LOD 0.34 0.28 0.21 0.22 Spiromesifen LOD 0.16 0.14 0.14 0.12 Imidacloprid LOD 0.23 0.22 0.18 0.19 In an example embodiment, cannabis plant material was examined for percent change in pesticides (Imidacloprid) in ozone-treated samples ("Willow") and untreated control samples (FIGS. 27A-27B). Various strains (Afghani, Strawberry Banana Split, Lucky Charms, Skunk Northern Lights, and PapayaDawg) were treated with 200-400 ppm ozone for 24 hours. The amount and percent change in pesticide is reported. The pesticide levels decreased by about 40% to 50% (ppb) in most strains tested, but decreased by about 84% (ppb) in PapayaDawg, compared with the untreated controls.
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting scope.

Claims

We claim:

1. A method for reducing microbial contamination of cannabis plant material, comprising:
(a) providing a pathogen reduction device, comprising:
(i) an ozone chamber;
(ii) an oxygen concentrator configured to concentrate oxygen from ambient air and produce ozone;
(iii) an ozone regulator configured to adjust the ozone concentration in the ozone chamber; and (iv) one or more processors with a memory coupled to the one or more processors, the memory storing instructions that when executed by the one or more processors cause the one or more processors to determine a concentration of gaseous ozone in an ozone chamber and adjust the concentration of gaseous ozone in the ozone chamber to a preset concentration;
(b) placing cannabis plant material contaminated with one or more microbes inside the ozone chamber of the pathogen reduction device;
(c) setting the ozone concentration in the ozone chamber to between about 200 ppm and about 400 ppm; and (d) treating the cannabis plant material with ozone by leaving it in the ozone chamber for a time period of at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 12 hours, from 4 to 8 hours, from 6 to 12 hours, from 10 to 18 hours, from 12 to 24 hours, or from 16 hours to 48 hours, wherein the method reduces microbial contamination in the cannabis plant material.

2. The method of claim 1, wherein the ozone concentration is between 200 ppm and 300 ppm.

3. The method of claim 1 or 2, wherein the ozone concentration is 200 ppm, 225 ppm, or 250 ppm.

4. The method of any one of claims 1-3, wherein the treatment time is from 20 minutes to 2 hours.

5. The method of any one of claims 1-3, wherein the treatment time is from 2 hours to 6 hours.

6. The method of any one of claims 1-3, wherein the treatment time is from 4 hours to 8 hours.

7. The method of any one of claims 1-3, wherein the treatment time is from 6 hours to 18 hours.

8. The method of any one of claims 1-3, wherein the treatment time is from 16 hours to 48 hours.

9. The method of any one of claims 1-8, wherein the method reduces microbial contamination in the cannabis plant material by at least 80% as compared to an amount of microbial contamination prior to the treatment step.

10. The method of any one of claims 1-9, wherein the method reduces microbial contamination in the cannabis plant material by at least 90% as compared to an amount of microbial contamination prior to the treatment step

11. The method of any one of claims 1-10, wherein the method reduces microbial contamination in the cannabis plant material by at least 98% as compared to an amount of microbial contamination prior to the treatment step.

12. The method of any one of claims 1-12, wherein the microbial contamination comprises yeast and mold contamination.

13. The method of any one of claims 1-12, wherein the microbial contamination comprises bacterial contamination.

14. The method of any one of claims 1-13, wherein the tetrahydrocannabinol (THC) or acid form of THC (THCa) level in the cannabis plant material prior to and following ozone treatment is substantially the same as compared to prior to the treatment step.

15. The method of any one of claims 1-14, wherein the average terpene level of the cannabis plant material prior to and following ozone treatment is substantially the same as compared to prior to the treatment step.

16. The method of any one of claims 1-15, wherein the amount of microbial contamination in the cannabis plant material prior to the treatment step is less than 150,000 colony-forming units (CFU) of yeast, mold, and/or bacteria.

17. The method of any one of claims 1-16, the amount of microbial contamination in the cannabis plant material prior to the treatment step is at least 150,000 CFU of yeast, mold, and/or bacteria.

18. The method of any one of claims 1-16, wherein the amount of microbial contamination in the cannabis plant material prior to the treatment step is at least 1,000,000 CFU of yeast, mold, and/or bacteria.

19. The method of any one of claims 1-18, wherein the method reduces yeast, mold, and/or bacteria contamination in the cannabis plant material by at least 50,000 CFU.

20. The method of any one of claims 1-18, wherein the method reduces yeast, mold, and/or bacteria contamination in the cannabis plant material by at least 100,000 CFU.

21. The method of any one of claims 1-18, wherein the method reduces yeast, mold, and/or bacteria contamination in the cannabis plant material to less than 10,000 CFU.

22. The method of any one of claims 1-18, wherein the method reduces yeast, mold, and/or bacteria contamination in the cannabis plant material to less than 1,000 CFU.

23. The method of any one of claims 1-18, wherein the method reduces bacterial contamination in the cannabis plant material by at least 90%.

24. The method of any one of claims 1-23, wherein the cannabis plant material is homogenized cannabis flower.

25. The method of any one of claims 1-24, wherein prior to treatment, the cannabis plant material has a level of bacteria, yeast, and/or mold that exceeds the allowable level for compliance with a regulatory agency or company; and following treatment, the contamination level of the cannabis plant material level is reduced to a compliant level.