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WO2025137725A2 - Procédé de culture, de préparation et d'utilisation d'une biomasse de blanc de matrice - Google Patents

Procédé de culture, de préparation et d'utilisation d'une biomasse de blanc de matrice Download PDF

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Publication number
WO2025137725A2
WO2025137725A2 PCT/US2024/061807 US2024061807W WO2025137725A2 WO 2025137725 A2 WO2025137725 A2 WO 2025137725A2 US 2024061807 W US2024061807 W US 2024061807W WO 2025137725 A2 WO2025137725 A2 WO 2025137725A2
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panaeolus
matrix
blank
cannabis
reference material
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WO2025137725A3 (fr
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Christopher Stephen Pauli
Caleb Daniel KING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/28—Cannabaceae, e.g. cannabis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H15/00—Fungi; Lichens
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells

Definitions

  • novel matrix-blank organisms relate to novel matrix-blank organisms, the methods used to grow and produce these organisms, and the processes to create a novel bioactive-free organism to be used to generate an analytical matrix blank. Methods to produce extracts free of an active compound or group of compounds obtained therefrom and their uses are also disclosed.
  • the novel matrix-blank organism is a Cannabis plant devoid of one or more cannabinoids to produce a representative Cannabis matrix blank.
  • GC gas chromatography
  • LC liquid chromatography
  • Leghissa et al. (2018b) discuss the use of GC with triple quadrupole mass spectrometry for analyzing cannabinoids, highlighting the method’s applicability to plant materials and Cannabis products.
  • Leghissa et al. (2018c) explore the use of gas chromatography with vacuum ultraviolet spectroscopy (GC-VUV) for the rapid detection of cannabinoids.
  • GC-VUV vacuum ultraviolet spectroscopy
  • Aizpurua-Olaizola et al. (2016) and Dalliige et al. (2003) demonstrate the efficacy of two-dimensional gas chromatography (GC x GC) for analyzing complex cannabinoid mixtures.
  • Cannabis industry has established a unique standard for analytical testing to ensure product safety. This is particularly notable given the Cannabis plant’s ability to produce over 500 bioactive compounds, with cannabinoids being the primary focus.
  • cannabinoids being the primary focus.
  • a cannabinoid- free version of Cannabis is crucial for precise measurement of bioactive molecules, while avoiding matrix-induced artifacts as Cannabis is known to produce hundreds of metabolites other than cannabinoids.
  • Cannabis Plants of the genus Cannabis have been used as a drug for centuries as a botanical drug, although the precise basis for the plant’ s activity is not known. Both tetrahydrocannabinol (THC) and cannabidiol (CBD), two of the plant’s most abundant cannabinoids, are known to have distinct pharmacological activities. It is noted that botanists disagree regarding whether the genus Cannabis includes only one species or multiple species. For purposes of this disclosure, reference to Cannabis is intended to refer to any species or subspecies of Cannabis, whether the species is sativa, indica, ruderalis or is unspecified.
  • GPP mevalonate pathway
  • DOX deoxyxylulose pathway
  • cannabigerolic acid acts as a direct precursor for other cannabinoids like tetrahydrocannabinolic acid (THC A), cannabidiolic acid (CBD A), and cannabichromenic acid (CBCA).
  • THC A tetrahydrocannabinolic acid
  • CBDA cannabidiolic acid
  • CBCA cannabichromenic acid
  • the specific phenotype observed could be due to a malfunction in the enzyme geranyl diphosphate:olivetolate geranyltransferase (GOT), which is vital for combining resorcinolic acids (OA and DA) with GPP to produce cannabigerolic acid.
  • GOT geranyl diphosphate:olivetolate geranyltransferase
  • An ineffective GOT enzyme might lead to an accumulation of OA and/or DA.
  • cannabinoid content which would have detected these acids in both their carboxylated and decarboxylated forms, as per De Meijer et al., 2003.
  • this exemplary botanical genotype referred to herein as the “knock-out organism,” is characterized by its absence of conventional organism-specific compounds due to a mutation in the biosynthetic pathway of that compound, as exemplified by the production of cannabinoid-free Cannabis in the case of cannabis, through selectively breeding non-functional cannabinoid biosynthetic genes.
  • Such a specimen is instrumental in corroborating accurate quantification of various bioactive compounds intrinsic to the Cannabis plant and its affiliated extracts.
  • Embodiments of the invention disclosed herein relate to biological matrix-blank reference materials derived from a selectively-bred organism, the selectively-bred organism being characterized by an absence of one or more bioactive compounds expressed in the comparator organism.
  • the selectively, bred Cannabis is characterized by the absence one or more of the following cannabinoids: tetrahydrocannabiphorol (9-THCP), tetrahydrocannabivarin (9-THCV), delta-8- tetrahydrocannabinol (8-THC), delta-9-tetrahydrocannabinol (9-THC), tetrahydrocannabinolic acid (9-THCA), tetrahydrocannabiphorolic acid (9-THCP A), tetrahydrocannabivarinic acid (9- THCVA), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabichromeorcin (CBC
  • the selectively-bred Cannabis is further characterized by the presence of trichomes of clear, white, and/or amber coloration.
  • the selectively-bred Cannabis is the Cannabis variety designated as ‘ZCP.’
  • the matrix -blank organisms or reference materials disclosed herein include flavonoids including cannflavin A, cannflavin B, isocannflavin B, cannflavin C, or other Cannabis-s ⁇ Qc c, flavonoids.
  • the matrix-blank organisms or reference materials disclosed herein include terpenes.
  • matrix-blank organisms or reference materials disclosed herein are further characterized by one or more of the presence of both monoterpenes and sesquiterpenes; the absence of either monoterpenes or sesquiterpenes; the absence of both monoterpenes and sesquiterpenes, the presence of di terpenes, the absence of di terpenes, the presence of tri terpenes, the absence of tri terpenes, the presence of tetraterpenes, and/or the absence of tetraterpenes.
  • the matrix-blank reference materials disclosed herein comprise a homogenized bulk biomass lot devoid of a profile of entourage or contaminant compounds or organisms.
  • the matrix-blank organisms or reference materials disclosed herein are further characterized by at least one of the presence of a known compound or organism spiked in at a known concentration for analytical reference; and/or the addition of one or more compounds selected from the group consisting of flavonoids, alkaloids, terpenoids, carotenoids, cannabinoids, alcohols, ketones, ethers, amines, amides, esters, aldehydes, sterols, pesticides, herbicides, fungicides, hydrocarbons, synthetic cannabinoids, synthetic psychedelics, synthetic opioids, isoxazoles, halogenated compounds, radioactive elements, heavy metals, Aspergillus, Salmonella, Shiga-Toxin Producing Escherichia coli, non-pathogenic Es
  • the spiked amount of the compound is at a regulatory body’s limit of detection and/or limit quantification for that analyte. In some embodiments, the spiked amount of compound is greater than the regulatory body’s limit detection and/or limit of quantification for that given analyte. In some embodiments the spiked amount of compound is less than the regulatory body limit detection and/or limit of quantification for that given analyte.
  • the matrix-blank reference materials disclosed herein for use in biological consumption to create a bioactive-free matrix-blank material from biological tissue or excrement further characterized by the addition of one or more compounds selected from the group consisting of flavonoids, terpenoids, carotenoids, cannabinoids, alcohols, ketones, pesticides, herbicides, fungicides, solvents, synthetic cannabinoids, synthetic psychedelics, synthetic opioids, isoxazoles, halogenated compounds, radioactive elements, Aspergillus, Salmonella, Escherichia coli, and/or heavy metals.
  • cannabinoid-free Cannabis is sprayed with certain pesticides, fungicides, or herbicides specific to the contaminated matrix blank material desired, such as spraying pyrethrins as a pest management strategy when creating a pesticide- contaminated mixture of biomass to use in determining a laboratories ability to detect that given pesticide.
  • a method for cultivating Cannabis to produce pesticide-, herbicide-, fungicide-, heavy metal-, mycotoxin-, endotoxin-, and microbial-free Cannabis biomass as described in Example 1 is provided.
  • a method for sterile post-harvest processing of Cannabis to prevent environmental contamination as described in Example 2 is provided.
  • the tissues of the matrix-blank organisms disclosed herein are used for comparison with unknown or known bioactive-containing samples of the same organism.
  • the organism is selected from one of the following organisms: Lion’ s Mane mushroom species such as Hericium erinaceus. Hericium coralloides, and Hericium americanum: Reishi mushroom species including Ganoderma lucidiim. Ganoderma tsugae, and Ganoderma applanalum Chaga mushroom, specifically Inonotus obliquus,' Cordycep mushrooms such as Cordyceps militaris, Cordyceps gunni, and Cordyceps dipterigena; Amanita mushroom species such as Amanita citrina, Amanita muscaria, Amanita gemmata, Amanita.
  • Lion s Mane mushroom species such as Hericium erinaceus. Hericium coralloides, and Hericium americanum: Reishi mushroom species including Ganoderma lucidiim. Ganoderma tsugae, and Ganoderma applanalum Chaga mushroom, specifically Inonotus obliquus,' Cordycep mushrooms
  • the selectively-bred organism is selected using a genetic marker complementary to one or more biosynthetic pathway genes that select against the biosynthesis of an active component.
  • the bioactive compound is selected based on the compound being unique to the organism, for example, cannabinoids in Cannabis, Cordycepin in Cordyceps, and Psilocybin in Psilocybe, and the like.
  • the matrix-blank reference material comprises biomass or an extract of the organism.
  • the matrix-blank organism is used to create an extract utilizing either a solventless method or a solvent selected from a range including, but not limited to, ethanol, methanol, carbon dioxide, propane, butane, heptane, hexane, naphtha, limonene, pinene, or analogous polar or non-polar solvent system.
  • the matrix-blank reference material is combined with one or more molecules selected from one or more of the following bioactive constituents including, but not limited to, alkaloids, monoterpenes, diterpenes, triterpenes, tetraterpenes, sterols, amino acids; contaminant constituents such as but not limited to lead, mercury, cadmium, arsenic, chromium, nickel, copper, zinc, gold, uranium, radon, silver, synthetic drugs of abuse, myclobutanil, bifenthrin, avermectin, imazalil, permethrin, spiromesifen, spirotetramat, chlorfenapyr, pyrethrins, imidacloprid, carbaryl, malathion, chlorothalonil, mancozeb, iprodione, propi conazole, pyraclostrobin, vinclozolin, copper-based fungicides,
  • bioactive constituents including,
  • the matrix-blank reference material is combined with one or more molecules selected from the group consisting of bioactive constituents, contaminant constituents, biological contaminants, and extracted or synthesized DNA/RNA of the aforementioned species.
  • the tissues of the matrix blank organisms disclosed herein are utilized for analytical chemistry analyses such as, matrix -matched calibrations, validations, and quality controls as a bioactive-free matrix blank.
  • the tissues of the matrix blank organisms disclosed herein are used for medical studies to determine the efficacy, toxicity, or potential benefits of a given organism or compound produced by that organism.
  • the tissues of the matrix blank organisms disclosed herein are used for proficiency testing of analytical labs to determine the accuracy, precision, and ability to detect and quantify bioactive or contaminant compounds.
  • the matrix-blank reference materials disclosed herein are utilized as control samples in scientific research, including but not limited to pharmacological, toxicological, and botanical studies. In some embodiments, the matrix-blank reference materials disclosed herein are employed in the development and calibration of analytical instruments and techniques for detecting and quantifying bioactive compounds in an organism’s biomass. In some embodiments, the matrix-blank reference materials disclosed herein are used in educational settings for demonstration and training purposes in fields related to botany, chemistry, and pharmacology.
  • a method for producing animal feed using a biological matrix-blank from any selectively bred, bioactive-free organism is provided, ensuring safety and compliance with animal health regulations due to the absence of specific bioactive compounds.
  • a method for creating non-psychoactive, bioactive compound-free cosmetic products, such as creams, lotions, and ointments, using a bioactive- free matrix derived from any selectively bred, bioactive-free organism is provided.
  • a method is provided for developing new varieties of bioactive-free organisms using genetic markers associated with a bioactive-free phenotype in any selectively bred, bioactive-free organism.
  • a process for extracting non-bioactive compounds, such as terpenes, flavonoids, and fatty acids, from the bioactive-free matrix of any selectively bred, bioactive-free organism is provided for use in various industries.
  • a method for utilizing the bioactive-free biomass of any selectively bred, bioactive-free organism in environmental remediation and phytoremediation processes is provided, leveraging its ability to absorb and break down pollutants in soil or water.
  • a method for producing biofuels using a bioactive-free matrix from any selectively bred, bioactive-free organism is provided where the absence of specific bioactive compounds facilitates a more efficient conversion process.
  • a method for using a bioactive-free, selectively bred organism as a research tool in botanical and agricultural studies is provided, providing a unique model for understanding plant biology without the interference of specific bioactive pathways.
  • a seed from a Cannabis plant designated ‘ZCP’ wherein a representative sample of seed of said plant has been deposited under .
  • a Cannabis plant, or plant part, tissue, or cell thereof produced by growing the seed from the Cannabis plant designated ‘ZCP’, or a descendant thereof is provided; wherein flower produced from the plant, or plant part, tissue, or cell thereof comprises a biochemical profile as described herein.
  • the Cannabis plant part is selected from the group consisting of: a stem, a trichome, a leaf, and a flower.
  • the Cannabis plant descended from the plant, or plant part, tissue, cell, or seed above is provided, wherein the plant is a clonal descendent.
  • a method of breeding a Cannabis plant, or plant part, tissue, or cell thereof wherein the plant, plant part, tissue, or cell is produced by growing a seed or clone from: a Cannabis plant designated wherein a representative sample of seed of said plant has been deposited under ; or a descendant of the Cannabis plant designated ‘ZCP,’ wherein the plant comprises a cannabinoid profile and/or a terpene profile as described herein, and wherein the method comprises providing the plant as at least one parent in a breeding program and selecting progeny displaying a cannabinoid profile as described herein and/or a terpene profile as described herein.
  • Figure 1 shows chromatogram analysis of ‘ZCP’ via HPLC-PDA.
  • Figure 2 shows chromatogram analysis of reference materials spiked in to an extract of ‘ZCP’ via HPLC-PDA.
  • Embodiments of the invention disclosed herein provide biological matrix-blank organisms (also referred to herein as “matrix-blank organism” or “bioactive-free organism”) and compositions derived therefrom (each such composition being referred to herein as a “biological matrix-blank reference material” or “matrix-blank material” or “matrix-blank biomass” or “matrix-blank extract”).
  • Organisms are provided in which expression and/or accumulation of one or more key biochemical components (the “deleted analyte” or “deleted analytes”) has been modified, while substantially preserving the expression and accumulation of the remaining biochemical components of the organism (the “background analytes”).
  • a matrix-blank organism has a substantially normal biochemical profile as compared with the organism from which it was derived (the “reference organism” or “comparator organism”), while having a finite and limited number of deleted analytes that are present in the reference organism.
  • the matrix-blank organism provides a source of preparation of matrixblank reference materials in any desired form useful for any of the analyses and/or comparisons described herein, such as, but not limited to, biomass and extracts, as well as others that would be evident to those of skill in the art.
  • Embodiments of the invention can be any organism including but not limited to plants, fungi, microorganisms, and research animals. While the invention is primarily described in terms of plants, embodiments of the invention are not limited to plants. Likewise, while a specific matrix-blank Cannabis plant is described herein, the invention specifically contemplates other matrix-blank Cannabis varieties as well as varieties of other matrix-blank plants, fungi, microorganisms, and the like.
  • a matrix-blank reference material can be used to provide a baseline, a reference, or a control for a number of different analyses. Since the biochemical profile of the matrix-blank reference material is substantially the same as the biochemical profile of the same type of reference material from the reference organism, except for the deleted analytes, use of the matrix-blank reference material in any of these different analyses permits more accurate and informative results of such analyses.
  • the two matrices being substantially the same, except for the deleted analytes, permits a level of fidelity and quality control on any analytical test that could not be achieved in any other way. This is partly because the tremendous number of variants of some classes of compounds, including but not limited to cannabinoids, for example, can lead to undesirable imprecision in many kinds of testing or analyses.
  • THC For example, if a specific test is intended to detect and quantify the presence of THC, other cannabinoids or terpenes can be mistakenly classified as THC if the quality or precision of the testing is not suitable for precise differentiation between THC and other compounds.
  • Use of a matrix-reference material to establish a baseline of non-THC components in the reference plant permits a vastly more precise detection of actual THC by avoiding false positive identification of background analytes as being THC.
  • the term “comparator plant” or “reference plant” refers to a plant variety expressing at least one medicinally active compound used to develop a new plant that has been selectively bred to not express the at least one medicinally active compound.
  • the terms “biological matrix-blank organism” or “matrix-blank organism” or “bioactive-free organism” refer to an organism that has been selectively bred to not express at least one medicinally active or bioactive compound but to express at least one other compound expressed in a comparator organism.
  • biological matrix-blank reference material or “matrixblank reference material” and “biological matrix-blank extract,” (or “matrix-blank extract”) and “biological matrix-blank biomass” (or “matrix-blank biomass”) are used to refer to a reference material, such as an extract or biomass from an organism that expresses all or substantially all of the compounds of a comparator organism but does not express at least one medicinally active compound.
  • the term “comparative analytical chemistry matrix” refers to the use of a bioactive-free organism to create a non-organism matrix material.
  • “comparative analytical chemistry matrix” refers to the use of cannabinoid-free Cannabis to create a non-plant matrix material. In some embodiments, this includes infusing cannabinoid-free Cannabis, or an extract thereof, into a finished product such as, but not limited to a chocolate, cooking oil, tincture, sublingual spray, tablet, topical, candy, or transdermal patch, in order to accurately distinguish cannabinoids from the other metabolites of Cannabis present in cannabinoid-free Cannabis and the matrix material itself.
  • the cannabinoid-free Cannabis is consumed by a human parti cipant/animal subj ect or used to create one or more biological tissue or excreted samples that contains the other metabolites of Cannabis without having cannabinoids to create a biological material specific Cannabis matrix blank.
  • biological samples include but are not limited to blood, hair, salvia, urine, fecal matter, and other biological tissues, fluids, and excrement.
  • the term “medicinally active” refers to a compound that has demonstrated biological effects beneficial for health, such as, for treating, preventing, or managing diseases or medical conditions.
  • “medicinally active” compounds have pharmacological effects on the body, such as reducing pain, inflammation, or symptoms of diseases.
  • Medicinally active compounds are sometimes referred to as “active” compounds or ingredients, which are responsible for the therapeutic effects of a plant.
  • a “medicinal plant” refers to a plant used or consumed primarily for the purpose of treating or managing health conditions and alleviate symptoms of diseases.
  • medicinal Cannabis plants typically contain cannabinoids and terpenes, such as cannabidiol (CBD) and/or beta caryophyllene, which have potential therapeutic benefits.
  • CBD cannabidiol
  • beta caryophyllene cannabidiol
  • a “recreational plant” refers to a plant used or consumed primarily for the purpose of enjoyment, relaxation, or social interaction.
  • recreational Cannabis is typically consumed for its psychoactive effects primarily from THC. Consumers experience euphoric effects, altered perception, and/pr mood enhancement.
  • contaminant chemical and “contaminant organism” refer to a material incorporated into a matrix blank organism to mimic a sample that would fail testing, for example, regulatory testing for a given set of regulations.
  • contaminant chemicals and organisms include, but are not limited to: mycotoxins, neurotoxins, drugs of abuse, tryptamines, hericenones, erinacines, ganoderic acids, lucidenic acid, inotodiol, lanosterol, betulinic acid, betulin, mescaline, hordenine, anhalonidine, and tyramine; contaminant constituents including but not limited to Per- and polyfluoroalkyl substances (PF AS), lead, mercury, cadmium, arsenic, chromium, nickel, copper, zinc, gold, uranium, radon, silver, myclobutanil, bifenthrin, avermectin, imazalil, permeth
  • PF AS Per- and polyfluor
  • Coli Listeria monocytogenes, Staphylococcus aureus, Botritiys spp, Aspergillus spp, Clostridium botulinum, Bacillus cereus, Campylobacter spp, yeasts, viroids, molds, as well as extracted or synthesized DNA/RNA of these species.
  • spikeked refers to the process of adding a known quantity of a substance, for example, a specific compound or contaminant, to a sample or a matrix for the purpose of analytical testing, calibration, verification, and/or validation.
  • biomass refers to the total mass of organic material produced by an organism during its growth cycle. In terms of a plant, for example, this can include leaves, stems, flowers, preflowers, trichomes, and roots. Biomass can be measured in terms of dry weight or fresh weight.
  • extract refers to a composition derived from the processing of plant or fungal biomass, including but not limited to roots, stems, leaves, flowers, fruiting bodies, spores, or mycelium, through physical, chemical, or biological methodologies to isolate, concentrate, or purify one or more constituents including but not limited to primary or secondary metabolites, lipids, proteins, tryptamines, cannabinoids, alkaloids, phenolic compounds, flavonoids, carbohydrates, nucleic acids, or derivatives thereof.
  • the term encompasses products obtained via solvent extraction (e.g., ethanol, methanol, acetone, supercritical CO2), mechanical processing (e.g., pressing, grinding, maceration), chromatographic fractionation, distillation, enzymatic or microbial biotransformation, or advanced technologies such as cold plasma or microwave-assisted extraction.
  • solvent extraction e.g., ethanol, methanol, acetone, supercritical CO2
  • mechanical processing e.g., pressing, grinding, maceration
  • chromatographic fractionation e.g., distillation, enzymatic or microbial biotransformation
  • advanced technologies such as cold plasma or microwave-assisted extraction.
  • Specific examples include cannabinoid-rich, flavonoid-rich or terpene-rich compositions derived from Cannabis sativa I.., Cannabis indica.
  • extracts may exist in crude, semi-purified, or purified forms and are suitable for applications as intermediates, active pharmaceutical ingredients, or final formulations.
  • a biological matrix-blank plant which has been selected to not express at least one medicinally active or bioactive compound, but to express at least one other compound expressed in a comparator plant.
  • the at least one other compound is one or more non-medicinally active compounds.
  • the amount of at least one non-medicinally active compound expressed by the biological matrix-blank plant is at least greater than 50% (weight/weight) of the total compounds in the plant.
  • the amount of at least one non-medicinally active compound expressed by the biological matrix-blank plant is greater than 60% (weight/weight) non-medicinally active compounds, or greater than 70% (weight/weight) non-medicinally active compounds, or greater than 80% (weight/weight) non-medicinally active compounds, or greater than 90% (w/w) non- medicinally active compounds, or the greater than 95% (weight/weight) non-medicinally active compounds.
  • the biological matrix-blank plant is a Cannabis plant.
  • Cannabis plants synthesize a diverse spectrum of compounds including cannabinoids, which are identified as primary active components, as well as “entourage” compounds, molecular entities that exhibit minimal interaction with cannabinoid receptors.
  • entourage compounds modulate cannabinoid activity, potentially amplifying their pharmacological effectiveness. It is therefore advantageous to engineer a botanical specimen devoid of primary cannabinoids but containing these entourage compounds.
  • the biological matrix-blank plant retains other significant compounds in proportions that mirror, both qualitatively and quantitatively, the comparator plant.
  • the biological matrix-blank plant is one that chemotypically aligns with medicinal plants employed in the formulation of pharmaceuticals, nutraceuticals, or functional foods.
  • the biological matrix-blank plant is a Cannabis plant and the at least one medicinally active compound is at least one cannabinoid, such as tetrahydrocannabinol (THC) or cannabidiol (CBD), and the like.
  • the biological matrix-blank plant is a THC-free Cannabis plant.
  • the biological matrix-blank plant is a CBD-free Cannabis plant.
  • the biological matrix-blank plant is a THC-free and CBD-free Cannabis plant.
  • the biological matrix-blank plant is a cannabinoid-free Cannabis plant.
  • the at least one other compound present in the comparator plant includes one or more entourage compounds selected from terpenes, lignans, sterols, and flavonoids.
  • the biological matrix-blank plant does not express at least one of CBG, THC, or CBD but expresses one or more terpenes, lignans, sterols, or flavonoids.
  • the biological matrix-blank plant expresses one or more compounds selected from monoterpenes, diterpenes, carotenoids, alkaloids, triterpenes, flavonoids, sterols, and lignans. In some embodiments, the biological matrix-blank plant lacks expression of one or more compounds selected from monoterpenes, diterpenes, tetraterpenes, alkaloids, triterpenes, flavonoids, sterols, and/or lignans.
  • Cannabis species encompassing both wild and cultivated varieties.
  • These cultivated varieties are distinct in their purposes and genetic makeup. They include fiber and grain-producing plants characterized by low THC content, varieties bred for recreational and medicinal applications with high THC levels, and medicinal plants specifically selected for their cannabinoid profile. The latter category may exhibit a predominance of one or more cannabinoids and, optionally, a specific profile of accompanying compounds, commonly referred to as “entourage compounds.”
  • a biological matrix-blank plant with targeted characteristics is developed using a selective breeding approach.
  • the selective breeding approach focuses on reducing the bioaccumulation of one or more cannabinoids with and without adversely impacting the synthesis and presence of compounds present in medicinal and recreational organisms. The methodology employed in achieving this selective breeding and its implications are detailed in the subsequent sections of this application.
  • the biological matrix-blank plant is a Cannabis plant containing a monogenic or multigenic mutation that blocks the biosynthesis of one or more cannabinoids.
  • the biological matrix-blank plant comprises a cannabinoid knockout factor governing a reaction in the pathways towards the phenolic moieties olivetolic and divarinic acid.
  • a method for producing a biological matrixblank plant that does not express at least one medicinally active compound yet expresses at least substantially qualitatively, most other non-medicinally active compounds present in a bioactive-containing organism wherein the method includes: a) selecting an organism that does not express at least one medicinally active compound; b) selecting a bioactive-containing organism; and c) crossing the organism which does not express at least one medicinally active compound with the bioactive-containing plant to obtain an Fl progeny and self-crossing the F 1 progeny to obtain an F2 progeny which is selected for the characteristics sought.
  • Ukrainian plant breeders reported the existence of cannabinoid-free breeding materials multiple times. Pacifico et al. analyzed individual plants from the Ukrainian cultivar USO 31, revealing that approximately one third contained no cannabinoids. Similarly, they found that a minority of plants ( ⁇ 10%) in a French fiber cultivar, Epsilon 68, were cannabinoid-free.
  • cannabinoid-free plants differ phenotypically and chemotypically from those engineered artificially and those isolated in nature. They potentially manifest because of two distinct physiological conditions: (1) a disruption in the formation of glandular trichomes, which are pivotal for cannabinoid synthesis, as suggested by Sirikantaramas et al. and (2) blockage of biochemical pathways crucial for the formation of biochemical precursors preceding CBGA.
  • a disruption in the formation of glandular trichomes which are pivotal for cannabinoid synthesis, as suggested by Sirikantaramas et al.
  • blockage of biochemical pathways crucial for the formation of biochemical precursors preceding CBGA [0088]
  • field-grown cannabinoid-free plants resulting from the Gorshkova et al. program exhibited a lack of glandular trichomes on their bracts and bracteoles.
  • Cannabis fragrance did not emit the typical Cannabis fragrance, indicating a potential absence of volatile mono- and sesquiterpenes. Consequently, these cannabinoid-free plants might have been deemed unsuitable for breeding a typical entourage compound-bearing, cannabinoid-free plant.
  • the second condition affecting metabolites beyond cannabinoids, might hinder the common precursor basic pathways for various end products. For instance, the synthesis of cannabinoids and certain terpenes, sterols, and triterpenes are distinct, occurring in different cellular compartments.
  • the matrix-blank Cannabis plants disclosed herein include stalked glandular trichomes.
  • the trichomes are present at a density comparable to those present in comparator medicinal and recreational cannabinoid-producing plants.
  • the biological matrix-blank plants have small, grey, dull trichomes of various shapes.
  • the trichomes can be headless; pinhead and/or shriveled trichomes, which may be flat, convex or concave, while others are comparable to drug-type cannabinoid producing plants.
  • the matrix-blank Cannabis plants are selectively bred for capitate stalked glandular trichomes or bulbous stalked trichomes. In some embodiments, other trichome types are present.
  • the biological matrix-blank plants exhibit branching characteristic of a drug producing phenotype as opposed to a fiber producing phenotype. In some embodiments, the biological matrix-blank plant exhibits vigor, characterized in that the total above ground dry weight is comparable to drug producing phenotypes.
  • the matrix-blank plants disclosed herein are selectively bred to be dioecious to increase biomass yield and eliminate undesired seed production.
  • the plants autoflower or photoperiod flowering versions of cannabinoid-free Cannabis. Photoperiod insensitivity and dioecy introduction provides a competitive advantage over the plant known in the art.
  • the biological matrix-blank organism disclosed herein is used to generate a biological matrix-blank reference material having a chemical profile that resembles that of the comparator organism less the at least one medicinally active compound.
  • the biological matrix-blank reference material exhibits a profile of entourage compounds that is quantitatively similar to that of the biological matrix-blank plant as shown in FIG. 1 and FIG. 2.
  • This similarity is much greater than prior-art attempts to create control materials for testing.
  • Such attempts typically created the control materials by attempting to wash out (or otherwise remove) the analytes to be tested.
  • Such approaches also typically wash out other biochemical components of the material, resulting in an analytical “blank” that lacked the substantial similarity to the comparator material.
  • prior-art attempts to create control materials would involve assembling the major components of a “blank” minus the analytes to be tested, without any effective attempt to replicate the presence of minor components. Since the minor components can interact in various ways and can be, in some testing formats, mistaken for the analytes to be tested, the absence of such minor components had the effect of the “blank” not being an accurate control material.
  • the biological matrix-blank reference material is used for comparison with unknown or known bioactive-containing samples of the same organism.
  • the biological matrix-blank reference material is used as a standard reference material to be used as a matrix subtraction for analytical chemistry method development and/or method validation.
  • a biological matrix-blank reference material comprising a biomass or an extract from a biological matrix-blank organism, such as a plant or fungus, is provided for use as a reference material.
  • the biomass is a homogenized bulk biomass.
  • the homogenized bulk biomass includes a homogenized biomass of stems, leaves, seeds, and/or buds.
  • the homogenized bulk biomass includes a homogenized biomass of mycelium, spores, and/or fruiting bodies.
  • the biological matrix-blank reference material mirrors the biological matrix -blank plant or fungus in that it lacks at least one medicinally active or bioactive compound but includes at least one other compound expressed in a comparator plant.
  • the biological matrix-blank reference material disclosed herein being derived from a biological matrix-blank organism does not require removal of medicinally active or other organism-produced compounds through means such as extraction, chemical conversion, genetic modification or genetic manipulation.
  • the matrix-blank reference materials disclosed herein represent a significant improvement over current material used for matrix blank subtraction, as the removal of noncannabinoid metabolites in efforts to create current cannabinoid-free Cannabis has led to nonrepresentative matrix blanks being used leading to inaccurate quantification due to the inability to properly subtract the background.
  • the matrix-blank reference materials and associated methods disclosed herein provide the first chemically representative matrix-blank reference material that contains all primary and secondary metabolites produced by the comparator organism without containing the bioactive compound of interest, such as, in the case of cannabinoids being absent in ‘ZCP.’
  • many previous attempts at using a cannabinoid-free matrix for Cannabis has led researchers to use other plant biomass; however, due to the lack of other non-cannabinoid Cannabis-specific metabolites, it is limited in use due to not being comparable to the unprocessed Cannabis material being tested.
  • the embodiments herein allow for the first full spectrum representative matrix-blank reference material in Cannabis that is comparable to the chemical compositions of recreational marijuana or hemp plants.
  • a method of producing a biological matrix-blank reference material includes selecting a biological matrix-blank organism that does not express at least one medicinally active compound but express at least one non- medicinally active compound present in a comparator plant, generating a reference material therefrom, wherein the reference material has a chemical profile that resembles the chemical profile of the comparator organism minus the at least one medicinally active compound not expressed.
  • Producing organisms, such as plants, and mixtures of biomass derived from the organisms in this embodiment provides additional avenues of using the biomass or products derived from the matrix-blank organism including but not limited to enabling studies to identify bioactive effects of individual components of a given botanical drug substance, as well as providing specialized Cannabis with non-naturally occurring ratios of metabolites that provide medicinal effects.
  • the biological matrix-blank reference materials disclosed herein are prepared by any method generally known in the art, for example, but not limited to, by maceration, percolation, vaporization, chromatography, distillation, recrystallisation and extraction with organic polar and non-polar solvents such as, but not limited to, methanol, ethanol, propanol, butanol, pentanol, norflurane, butane, propane, acetone, acetonitrile, benzene, toluene, chloroform, ethyl ether, xylenes, pyridines, methylene chloride, dimethyl sulfoxide, isobutanol, nitrobenzene, cyclohexane, chlorobenezene, hexanes, heptanes, pentanes, and other alcohols.
  • organic polar and non-polar solvents such as, but not limited to, methanol, ethanol, propanol, butanol, pen
  • Such extracts may be prepared also by utilizing polar solvents such as water and supercritical or subcritical liquid carbon dioxide.
  • the extracts can be obtained by the methods and processes described in international patent application numbers WO02/089945 and WO 2004/016277, each of which is hereby incorporated by reference in its entirety.
  • the biomass and extract derived therefrom are handled as disclosed in sterile conditions to eliminate the possibility of post-harvest or post-processing contamination so that the material is suitable to be used as an analytical matrix blank material.
  • the biological matrix-blank reference materials disclosed herein include an extract or a mixture of plant biomass from a selectively-bred Cannabis plant characterized by the absence of at least one of the following cannabinoids: Tetrahydrocannabiphorol (9-THCP), Tetrahydrocannabivarin (9-THCV), Delta-8- Tetrahydrocannabinol (8-THC), Delta-9-Tetrahydrocannabinol (9-THC), Tetrahydrocannabinolic Acid (9-THCA), Tetrahydrocannabiphorolic Acid (9-THCPA), Tetrahydrocannabivarinic Acid (9-THC VA), Cannabichromene (CBC), Cannabichromenic Acid (CBCA), Cannabichromeorcin (CBCO), Cannabichromevarin (CBCV), Cannabichromevarinic Acid (CBCVA), Cannabidiol (CBD), Cannabidiolic Acid (CBDA), Cannabidi
  • cannabinoids Te
  • the selectively-bred Cannabis plant is characterized by trichomes of clear, white, and/or amber coloration.
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘LCP.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘ZCP.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘ZCSB.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘ZCCC.’
  • the selectively -bred Cannabis plant is derived from the Cannabis variety, ‘ZCBC.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘CK15.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘CP21.’
  • the selectively-bred Cannabis plant is derived from the Cannabis variety, ‘ZCO.’
  • the plant material selected as the source of a matrix-blank reference material is derived through breeding.
  • the presence of cannabinoids is selected against over a period of five or more generations of inbreeding and chemical selection.
  • the selection is further bred to introduce various subsets of entourage compounds to be more similar and comparable to commercially available Cannabis varieties. This allows for a more accurate comparison in analytical testing to have a more representative matrix material to compare against, which currently does not exist on the market.
  • the biological matrix-blank reference materials disclosed herein naturally include flavonoids such as, cannflavin A, cannflavin B, cannflavin C, or other Q//wa/v.s-specific flavonoids expressed by the biological matrix-blank plant.
  • the biological matrix-blank reference materials naturally include terpenes expressed by the biological matrix-blank plant.
  • the biological matrixblank reference materials disclosed herein are characterized by the presence of one or more of monoterpenes and sesquiterpenes; the absence of either monoterpenes or sesquiterpenes; the absence of both monoterpenes and sesquiterpenes; the presence diterpenes; the absence of diterpenes; the presence of triterpenes; and/or the absence of triterpenes.
  • a method of obtaining a certified biological matrix-blank reference material includes cultivating a biological matrixblank organism under optimized conditions to produce a pesticide-, herbicide-, fungicide-, heavy metal-, mycotoxin-, endotoxin-, and microbial-free biological matrix-blank organism, as described in Example 1, from which a biomass or extract is obtained.
  • a method for sterile post-harvest processing of the biological matrix-blank organism is provided, wherein the method includes post-harvest processing that has been optimized to prevent environmental contamination, as described in Example 2.
  • a method for packaging a reference material from a biological matrix-blank organism in sterile containers meeting or exceeding EPA standards in a clean environment, ensuring no contamination during packaging, transport, or storage is provided, wherein the packaging is performed under ISO 17034 accreditation as a certified reference material.
  • the biological matrix-blank reference material is devoid of a profile of entourage or contaminant compounds or organisms, further characterized by at least one of: the presence of a known compound or organism spiked in at a known concentration for analytical reference; and/or the addition of one or more compounds selected from the group consisting of flavonoids, alkaloids, terpenoids, carotenoids, cannabinoids, alcohols, ketones, ethers, amines, amides, esters, aldehydes, sterols, pesticides, herbicides, fungicides, hydrocarbons, synthetic cannabinoids, synthetic psychedelics, synthetic opioids, isoxazoles, halogenated compounds, radioactive elements, heavy metals, Aspergillus spp., Salmonella spp., Escherichia coli.
  • Pseudomonas spp. Listeria, hepatitis, norovirus, Clostridium, Campylobacter, Taxoplasma, Psilocybe spp., Cryptosporidium, Saccharomyces spp., hops-latent viroid, beat-curly top virus, tobacco mosaic virus, and radiolabeled nucleic acids.
  • Some embodiments provide a selectively bred matrix-blank organism wherein the tissues of the organism are used for comparison with unknown or known bioactive-containing samples of the same organism.
  • the matrix-blank organism can be selected from one of the following organisms: Lion's Mane mushroom species such as Hericium erinaceus, Hericium coralloides, and Hericium americanunr, Reishi mushroom species including Ganoderma hicidum. Ganoderma tsugae, and Ganoderma applanalum Chaga mushroom, specifically Inonotus obliquus,' Cordycep mushrooms such as Cordyceps militaris, Cordyceps gunni, and Cordyceps dipterigena; Amanita mushroom species such as Amanita citrina, Amanita muscaria, Amanita gemmata, Amanita.
  • Lion's Mane mushroom species such as Hericium erinaceus, Hericium coralloides, and Hericium americanunr, Reishi mushroom species including Ganoderma hicidum. Ganoderma tsugae, and Ganoderma applanalum Chaga mushroom, specifically Inonotus obliquus,' Cordy
  • Medicinal cactus species such as Lophophora williamsii, Lophophora diffusa, Lophophora fricii, Trichocereus pachanoi, and Trichocereus peruvianus and other medicinal plant species such as Mimosa hostilis/tenuiflora, Acacia confusa, Banisteriopsis caapi, Passiflora incarnata, and Peganum harmala.
  • the matrix-blank organism can be selected using a genetic marker complementary to one or more biosynthetic pathway genes that select against the biosynthesis of the active component.
  • the matrix-blank reference material is used to create an extract utilizing either a solventless extraction method including but not limited to ice water extraction, heat and pressure based extraction, or size based extraction or a solvent based extraction method selected from a one or more solvents including ethanol, methanol, carbon dioxide, propane, butane, heptane, hexane, naphtha, limonene, pinene, or analogous polar or non-polar solvent system.
  • a solventless extraction method including but not limited to ice water extraction, heat and pressure based extraction, or size based extraction
  • a solvent based extraction method selected from a one or more solvents including ethanol, methanol, carbon dioxide, propane, butane, heptane, hexane, naphtha, limonene, pinene, or analogous polar or non-polar solvent system.
  • the matrix-blank or reference material includes one or more molecules spiked in at a known concentration selected from the following classes: pesticides, herbicides, Per- and polyfluoroalkyl substances (PF AS), fungicides, mycotoxins, elements, isotopically labels, bioactive molecules, biologicals, and hydrocarbons.
  • PF AS Per- and polyfluoroalkyl substances
  • the matrix-blank or reference material spiked with one or more compounds from the previously mentioned classes of compounds allows for proficiency testing of laboratories using this spiked material, as well as producing known bioactive concentration materials specific to treating a specific condition.
  • the matrix-blank reference material biomass or extract is combined with one or more molecules selected from one or more of the following bioactive constituents including but not limited to tryptamines, hericenones, erinacines, ganoderic acids, lucidenic acid, inotodiol, lanosterol, betulinic acid, betulin, mescaline, hordenine, anhalonidine, and tyramine; contaminant constituents including but not limited to Per- and polyfluoroalkyl substances (PFAS), lead, mercury, cadmium, arsenic, chromium, nickel, copper, zinc, gold, uranium, radon, silver, myclobutanil, bifenthrin, avermectin, imazalil, permethrin, spiromesifen, spirotetramat, chlorfenapyr, pyrethrins, imidacloprid, carbaryl, mal
  • bioactive constituents including but
  • Coli Listeria monocytogenes, Staphylococcus aureus, Botritiys spp, Aspergillus spp, Clostridium botulinum, Bacillus cereus, Campylobacter spp, yeasts, viroids, molds, as well as extracted or synthesized DNA/RNA of these species.
  • the matrix-blank reference material is combined with one or more molecules selected from the group consisting of bioactive constituents, contaminant constituents, biological contaminants, and extracted or synthesized DNA/RNA of the aforementioned species.
  • the biological matrix-blank reference material is devoid of a profile of entourage compounds, for use in biological consumption to create a matrix-blank material from biological tissue or excrement, further characterized by one or more of: the addition of one or more compounds selected from the group consisting of flavonoids, terpenoids, tetraterpenes, triterpenes, diterpenes, alcohols, ketones, pesticides, herbicides, fungicides, solvents, Aspergillus, Salmonella, Escherichia coli, microplastics, and heavy metals; the use of the spiked plant biomass for biological consumption by humans or animals to create a comparative analytical chemistry matrix.
  • Some embodiments include the addition of alkaloids, phenolic compounds, isoxazoles, sterols, vitamins, and/or minerals.
  • the biological matrix-blank reference materials disclosed herein are used for proficiency testing of analytical labs to determine the accuracy, precision, and ability to detect and quantify bioactive or contaminant compounds.
  • the biological matrix-blank reference materials disclosed herein are spiked with one or more contaminants, such as a microbe or a pesticide, and used for proficiency testing or regulatory testing enforcement to evaluate the ability of a testing facility or laboratory to detect and quantify pathogens or adulterants in a test organism, such as Cannabis.
  • the biological matrix-blank reference materials disclosed herein are used for analytical chemistry analyses such as, matrix-matched calibrations, validations, and quality controls as a bioactive-free matrix blank.
  • the biological matrix-blank reference materials disclosed herein are employed for analytical chemistry purposes to compare to unknown organism samples, such as Cannabis samples.
  • the biological matrix-blank reference materials disclosed herein are used for medical studies to determine the efficacy, toxicity, or potential benefits of a given organism or compound produced by that organism.
  • the biological matrix-blank reference materials disclosed herein are used as control samples in scientific research, including but not limited to pharmacological, toxicological, and botanical studies. In some embodiments, the biological matrix-blank reference materials disclosed herein are used in the development and calibration of analytical instruments and techniques for detecting and quantifying bioactive compounds in an organism’s biomass. In some embodiments, the biological matrix-blank reference materials disclosed herein are used to test the hypothesis that at least one active compound is responsible for an extract’s perceived medicinal value. In some embodiments, the biological matrix-blank reference materials disclosed herein are used as a placebo.
  • a method of testing a hypothesis that one or more compounds present in a reference material are responsible for the pharmacological activity of that reference material includes: i) selecting a biological matrix-blank organism as described herein; ii) obtaining a reference material therefrom; and iii) running comparative tests against a reference material obtained from a comparator organism.
  • the biological matrix-blank reference materials disclosed herein are used in educational settings for demonstration and training purposes in fields related to botany, chemistry, and pharmacology.
  • the biological matrix-blank reference materials disclosed herein are used in the development of new varieties of organisms through genetic modification or selective breeding to introduce or enhance desirable traits while maintaining the absence of one or more bioactive compounds.
  • a method of producing a designer biological organism extract used for analytical testing includes: i) selecting an extract obtainable from a biological matrix-blank organism as taught herein; and ii) combining the extract of (i) with one or more medicinally active components.
  • designer organism extract refers to an organism extract which includes one or more medicinally active components which do not naturally occur in the biological matrix-blank organism of part i. While sometimes exemplified by botanical extracts herein, this invention can additionally be further characterized by designer fungal extracts.
  • the medicinally active components are purified naturally occurring compounds, synthetic compounds or a combination thereof.
  • the medicinally active components are present in a plant extract.
  • the plant extract is an extract from a “drug producing” plant of the same species as the biological matrix-blank plant of part i). Typically, this drug producing plant will not be the comparator plant to the matrix-blank plant of part i).
  • the organisms and reference materials described herein are used to create other matrices that allow for research into the metabolite profiles of these organisms when exposed to the organism without one or more of the bioactive-components.
  • a method of making such a matrix includes administering to a subject who has not previously ingested the bioactive compounds, a specific amount of a matrix-blank organism or a matrix-blank reference material; and collecting blood, hair, saliva, urine, serum, or other bodily excretions or tissues from the subject after the consumption of the matrix-blank material.
  • the subject is a human.
  • the subject is an animal.
  • the new matrix is used to avoid false positives in analyte testing.
  • the new matrix material is created through infusing metabolites or metabolite-derived compounds from a cannabinoid-free Cannabis into a human or animal sample or a synthetic matrix created to represent a human or animal tissue or excrement.
  • this method enables researchers and drug testing laboratories to more accurately analyze these matrices in cases where there is a desire to identify and quantify the presence of a given bioactive molecule or metabolite of that molecule in human or animal subjects.
  • urine analysis for 11-HO-THC is commonly performed by employers, courts, and police departments. Having a true matrix-blank sample of urine that contains cannabinoid-free Cannabis metabolites allows for a more accurate determination and detection of the metabolites produced by cannabinoid-containing Cannabis through comparison against the cannabinoid-free infused urine matrix blank.
  • Cannflavins are a group of flavonoids found in Cannabis that have been studied for their potential therapeutic properties.
  • cannflavin A is known for antiinflammatory properties and may have analgesic effects.
  • Cannflavin B also exhibits antiinflammatory effects and may have neuroprotective properties.
  • the biological matrix blank plants still produce flavonoids and are selected for upregulation of flavonoids, including, but not limited to, cannflavin A, cannflavin B, isocannflavin B and cannflavin C.
  • the resulting biological matrix-blank reference materials having high levels of cannflavins have medicinal applications such as anti-inflammatory properties through their proposed role of inhibiting the production of pro-inflammatory prostanoids and leukotrienes.
  • the resulting products resembling cannabinoid-containing Cannabis provide potential health benefits associated with terpenes and other non-cannabinoid compounds including but not limited flavonoids, alcohols, esters, ketones, and other secondary metabolites.
  • the resulting biomass produced would contain elevated flavonoids, including but not limited to cannflavin A, B, and/or C that would be extracted and used as an active pharmaceutical ingredient in a formulated Cannabis-derived product, or the elevated non-cannabinoid bioactive compound containing plant parts itself are used as a botanical drug substance within clinical trials or medical applications.
  • the biological matrix-blank reference materials described herein are used as substrates or mediums in agricultural research. In some embodiments, the biological matrix-blank reference materials are used for studying plant growth, nutrient uptake, or disease resistance in the absence of bioactive compounds. In some embodiments, the biological matrix-blank reference materials described herein are used as research tools in botanical and agricultural studies, providing a unique model for understanding plant biology without the interference of cannabinoid pathways. In some embodiments, a method for using a biological matrix-blank reference material as described herein as a research tool in botanical and agricultural studies is provided. In some embodiments, the method provides a unique model for understanding plant biology without the interference of specific bioactive pathways.
  • the biological matrix blank organisms are used as a comparator plant to understand the biological effects of cannabinoids, terpenoids, or flavonoids as a pest deterrent against specific species of herbivores to identify unique chemical profiles to repel environmental-specific pests.
  • the biological matrix blank organisms are used as a comparator plant to understand the biological effects of cannabinoids, terpenoids, or flavonoids as anti-microbial compounds against specific species of fungi or bacteria to identify unique chemical profiles to gain resistance to environmental-specific microbes.
  • the biological matrix blank reference materials are spiked with one or more cannabinoids to determine the level of the one or more cannabinoids necessary to produce anti-fungal, anti-viral, anti-inflammatory, anti-biotic, anti-microbial, antianxiety or other medicinal or agricultural properties.
  • the one or more compound at those amounts are then bred into susceptible Cannabis varieties.
  • the biological matrix-blank organisms and reference materials described herein are used in environmental studies to assess the impact of bioactive compounds on ecosystems, particularly in soil and water analyses.
  • the biological matrix-blank organisms and reference materials described herein are used in environmental remediation and phytoremediation processes, exploiting its ability to absorb and break down pollutants in soil or water without the risk of cannabinoid accumulation.
  • a method for using a biological matrix-blank plant in environmental remediation and phytoremediation processes is provided. In some embodiments, the method leverages the ability of the biological matrix-blank plant to absorb and break down pollutants in soil or water.
  • Cannabis is known to perform bioremediation; however, this is limited in application due to the presence of controlled substances, namely cannabinoids, produced by the plant.
  • Cannabinoid-free Cannabis used for bioremediation or environmental remediation is not limited by local, state or federal laws that cannabinoid-containing Cannabis is regulated under as the plant contains no illicit or prohibited compounds.
  • the cannabinoid-free Cannabis shows an increased bioremediation capacity compared to cannabinoid-containing Cannabis due to increased biosynthetic capacity of other pathways useful for remediating environmental contaminants due to the absence of cannabinoid biosynthesis.
  • the environmental contaminants being bioremediated include petrochemicals such as oil and gas spills.
  • the environmental contaminants being bioremediated include radioactive materials such as those leaked from nuclear power plant disasters.
  • the environmental contaminants being bioremediated include microplastics and PFAS chemicals.
  • the environmental contaminants being bioremediated include metals and minerals from the soil.
  • the environmental contaminants being bioremediated include chemical spills of toxic manufacturing compounds such as vinyl chloride, benzene, styrene or other environmentally detrimental compounds.
  • the biological matrix-blank organisms or reference materials described herein are used in the production of biofuels, where the absence of cannabinoids facilitates a more efficient conversion process.
  • the biological matrixblank reference materials described herein are used in the production of non-psychoactive, environmentally friendly biofuels or bioplastics.
  • a method for producing biofuels using a biological matrix-blank organism as described herein is provided, wherein the absence of specific bioactive compounds facilitates a more efficient conversion process.
  • the biological matrix-blank reference materials described herein are used in the production of biofuels and bioplastics wherein the absence of cannabinoids allows for a greater yield of biofuels or bioplastics per gram of biomass processed. In some embodiments, the biological matrix-blank reference materials described herein are used in the production of biofuels and bioplastics wherein the absence of cannabinoids produces higher quality biofuels and/or stronger tensile strength bioplastics.
  • the biological matrix -blank reference materials described herein are used in the production of biofuels and bioplastics wherein the absence of cannabinoids allows for a more environmentally sustainable manufacturing process through eliminating the processes used to remove or remediate the cannabinoids from cannabinoid-containing biomass.
  • the biological matrix-blank organisms or reference materials described herein are used in horticulture as a grafting stock for other plant species, leveraging its unique genetic attributes.
  • cannabinoid-free Cannabis due to the agronomic properties and diverse growing conditions cannabinoid-free Cannabis can tolerate, cannabinoid-free Cannabis is used to cultivate grafts of other plant species and varieties that are unable to grow in those conditions naturally.
  • the cannabinoid-free Cannabis is used as root stock in high salinity environments where certain ornamental or crops are unable to survive due to environmental or pest-related selective pressures.
  • the cannabinoid-free Cannabis is planted as a biological method to prevent erosive forces where it provides an advantage to other cover crops due to its larger and deeper root mass than traditional cover crops.
  • cannabinoid-free Cannabis is used as a companion crop to provide pest resistance to a crop that is pest susceptible.
  • the biological matrix-blank material provides economical and technical advantages compared to cannabinoid-containing Cannabis in the manufacturing of raw materials, biofuels, building materials, paper products, animal feed, cosmetic products, and food products, wherein the cannabinoids cause complications in machinery due to the physical properties of the resin.
  • current best practices for producing derived products from hemp or Cannabis are applied to ‘ZCP,’ which enables the production of these materials without being limited to certain countries’ laws against the scheduled compounds Cannabis is known to produce.
  • the biological matrix-blank materials described herein are used in the manufacturing of textiles and fibers, wherein the absence of cannabinoids does not affect the physical properties of the fibers and provide advantages of limiting or removing cannabinoid resin content in the raw material that is known to pose issues for industrial manufacturing.
  • a method for manufacturing textiles and fibers using a biological matrix-blank material as described herein is provided, wherein the absence of specific bioactive compounds improves the physical properties of the fibers as well as allows for easier processing of the raw material due to the lack of adhesive resin found in cannabinoid-containing plants.
  • the biological matrixblank materials described herein are used in the production of paper and cardboard materials, where the lack of cannabinoids contributes to a more environmentally sustainable processing due to the lack of necessity to remove cannabinoids that is required for processing all cannabinoid-containing hemp, which involves potentially toxic solvents and CO2-generating processes.
  • a method for producing paper and cardboard materials using a biological matrix-blank material as described herein is provided, wherein the lack of specific bioactive compounds contributes to more environmentally sustainable processing due to the lack of necessity to remove cannabinoids that is required for processing all cannabinoid- containing hemp, which involves potentially toxic solvents and CO2-generating processes.
  • a method for producing paper and cardboard materials using a biological matrix-blank material as described herein wherein the lack of specific bioactive compounds decreases the cost of production of these materials through eliminating the need of removing these regulated bioactive compounds.
  • cannabinoid-free Cannabis provides economic and ergonomic advantages over cannabinoid-containing Cannabis in the manufacturing process through limiting time cleaning cannabinoid-resin from equipment, as well eliminating costs associated with extraction and disposal of cannabinoid waste products produced in the production of paper, cardboard, or other hemp-derived materials.
  • cannabinoid-content has posed issues for automating industrial Cannabis processing due to the properties of the cannabinoid resin causing it to accumulate on machinery that limits the throughput of these processing facilities. Due to the unique lack of cannabinoid resins in ‘ZCP,’ it provides ergonomical advantages compared to commercially available industrial hemp cultivars that contain cannabinoid resin. Additionally, with the absence of cannabinoids, all input ingredients and intermediary products would be allowed within the law to be processed anywhere; which provides a solution to regulatory and legal challenges currently faced in the industrial hemp industry and production of industrial hemp derived products.
  • the biological matrix-blank materials described herein are used in animal feed, wherein the absence of cannabinoids ensures safety and compliance with animal health regulations and best practices for animal safety and pharmacology.
  • a method for producing animal feed using a biological matrix-blank material as described herein is provided.
  • the method ensures safety and compliance with animal health regulations due to the absence of specific bioactive compounds, such as THC.
  • the absence of certain bioactive compounds in the biological matrix-blank material prevents adverse reactions for animals known to negatively interact with cannabinoid-containing products.
  • the biological matrix-blank material is used to provide necessary nutritional content as well as optionally including selected bioactive compounds such as terpenes or flavonoids that are beneficial for a given condition or environment that the animal resides.
  • bioactive compounds such as terpenes or flavonoids that are beneficial for a given condition or environment that the animal resides.
  • the incorporation ‘ZCP’ material containing cannflavins in animal feed produces anti-inflammatory effects in inflamed animals without risking adverse reactions to the presence of cannabinoids in the animal feed.
  • the ‘ZCP’ material is selected based on a combination of terpenes known to cause calming effects in animals, that enables specialty purpose cannabinoid-free Cannabis derived animal feed products.
  • the biological matrix-blank materials described herein are used as a base for non-psychoactive, cannabinoid-free cosmetic products, including but not limited to creams, lotions, and ointments.
  • a method for creating nonpsychoactive, bioactive compound-free cosmetic products, such as creams, lotions, and ointments, using a biological matrix-blank material as described herein is provided.
  • the biological matrixblank materials described herein are used as a base for non-psychoactive, cannabinoid-free food products, including but not limited to chocolates, tinctures, gummies, pills, transdermal patches, topicals, drink mixes, and other finished products.
  • a method for creating non-psychoactive, bioactive compound-free food products including but not limited to chocolates, tinctures, gummies, pills, drink mixes, topicals, and other finished products is provided.
  • the non-psychoactive, bioactive compound-free products derived from cannabinoid-free Cannabis are used as product-type specific analytical matrix blank materials, as a placebo formulation for research into the effects of cannabinoids or other non-cannabinoid bioactive metabolites, as a drug delivery system for non-cannabinoid bioactive compounds, or as a method of consuming other cannabis-derived compounds without consuming cannabinoids.
  • these cannabinoid-free Cannabis products are spiked with a known contaminant compound or organism to allow for the proficiency testing of analytical laboratories.
  • the biological matrix blank material is incorporated into a kit containing spiked and non-spiked matrix blank material that evaluate the ability of an analytical method to detect and quantify concentrations of target analytes at or below a regulatory body’s specifications for that compound.
  • a process for the extraction of non-cannabinoid compounds including but not limited to hydrocarbon for primarily terpene extractions; water for primarily flavonoid extraction, and/or chloroform for fatty acid targeted extraction, from the biological matrix-blank materials described herein is provided for use in various industries.
  • crude extracts are produced via extraction methods described here or known in the art, which are then processed and packaged to be a crude extract matrix-blank, or the extract is further refined to produce high purity isolated compounds that are used as active pharmaceutical or nutraceutical ingredients, or used for the production of reference standards of isolated compounds in the analytical testing industry.
  • the isolated compound used for an API would include one or more of the following compounds: alkaloids, flavonoids, terpenes, isoxazoles, phenolic compounds, or other medicinal bioactive compounds.
  • alkaloids flavonoids
  • terpenes isoxazoles
  • phenolic compounds or other medicinal bioactive compounds.
  • the described invention is specific to the Cannabis species, it will be appreciated to one skilled in the art that this methodology disclosed herein could be applied to other organisms for which a bio-active free matrix blank material would be useful to achieve accurate analytical testing and comparative studies. Examples of such organisms ta include, but are not limited to: Lion's Mane mushroom species such as Hericium erinaceus. Hericium coralloides, and Hericium americanum: Reishi mushroom species including Ganoderma hicidum.
  • Ganoderma tsugae, and Ganoderma applanalum Chaga mushroom specifically Inonotus obliquus,' Cordycep mushrooms such as Cordyceps militaris, Cordyceps gunni, and Cordyceps dipterigena; Amanita mushroom species such as Amanita citrina, Amanita muscaria, Amanita gemmata, Amanita.
  • the process applies to psychedelic cacti like Lophophora williamsii, Lophophora diffusa, Lophophora fricii, Trichocereus pachanoi, and Trichocereus peruvianus,' dimethyltryptamine containing species such as but not limited to Mimosa hostilis/tenuiflora and Acacia confusa, beta-carboline containing species such as but not limited to Banisteriopsis caapi, Passiflora incarnata, and Peganum harmala.
  • the application extends to other high-value natural products like Salvia divinorum (Salvia), Panax ginseng (Ginseng), Withania somnifera (Ashwagandha), Camellia sinensis (Tea plant), and coffee plants such as Coffea arabica and Coffea canephora.
  • the primary objective of embodiments disclosed herein is to create variants of these plants and fungi that retain their traditional uses, entourage biochemical profiles, nutritional value, and/or ornamental qualities without producing the psychoactive, toxin, or other specific bioactive compounds they are typically known for.
  • a further benefit of the matrix-blank organisms disclosed herein is that they can be used to create matrix-blank materials containing other bioactive molecules in quantities and purities, which could not be achieved naturally.
  • Such matrix-blank materials provide benefits arising from the presence of one or more selected entourage compounds.
  • cannabinoids which can be introduced to the cannabinoid free extracts, include one or more natural cannabinoids, synthetic cannabinoids, or degradation cannabinoids (modified natural cannabinoids). This produces a “designer” plant extract that can be used in analytical method validation and proficiency testing or for the production of customized cannabinoid-ratio materials that can not be achieved naturally.
  • this is achieved through solubilizing the cannabinoid in a known volume of liquid solvent, such as ethanol, methanol, or other solvents in which cannabinoids are soluble, that is then applied to the dry Cannabis based on the target dry weight percentage to infuse known amounts of a cannabinoid onto the dried matrix blank material.
  • a similar infusion process can be used to incorporate one or more compounds from the following classes: chemical contaminants, environmental contaminants, bioactive compounds derived from Cannabis, bioactive compounds not found in Cannabis, or non-bioactive compounds.
  • this disclosure enables the use of these methods in organisms other than Cannabis, such as in other medicinal plants or fungi.
  • psilocybin-free Psilocybe cubensis mushrooms can be used to create novel ratios of tryptamines that would not be found in nature, but may provide enhanced or improved medicinal properties.
  • CBGA being a precursor to THC A
  • Norbaeocystin and Baeocystin are precursors to Psilocybin, which prevents a psilocybin-containing mushroom from being baeocystin-free in nature.
  • a psilocybin-free fungal matrix-blank can be used to produce norbaeocystin and psilocybin containing biomass that is free of baeocystin, as well as create other non-natural combinations of tryptamines spiked into the natural background of metabolites found in the organism to allow for reproducible and targeted medicinal formulations of natural products to achieve spiked biomass or extracts derived thereof.
  • Other examples of this technology include producing mescaline-free cacti through disruption of the mescaline biosynthesis pathway, ganoderic acid free reishi mushrooms through disruption of the ganoderic biosynthetic pathway, cordycepin-free cordyceps through disruption of the cordycepin biosynthetic pathway, alkaloid-free ibogaine through disruption of alkaloid biosynthetic genes, and hericenone-free and/or erinacine-free lions mane material through the disruption of the triterpenoid biosynthetic pathway.
  • This disruption occurs through numerous selectively bred genotypes including, but not limited to, truncated genes in the biosynthetic pathway, loss-of-function genes in the biosynthetic pathway, upstream and downstream mutations causing differential regulation of the biosynthetic pathway, and mutations in regulatory factors associated with the expression of these biosynthetic genes. Additionally, these same mutations can be introduced through non-selective breeding methods as well, such as CRISPR-CAS, Cre-Lox, and other known gene modification techniques to introduce non-naturally occurring knockouts of these organisms to be used in the disclosed applications of the bioactive-free matrix blank material.
  • cannabinoid-free matrix blank material over extracted Cannabis or similar cannabinoid-free plant material lies in the fact that with over 500 unique bioactive compounds in Cannabis, any extraction is likely to remove other compounds that would prevent the matrix blank from being comparable to non-extracted material. This is important due to the ability to accurately detect and quantify all other biological and synthetic molecules from the cannabinoids. Furthermore, this provides an ideal matrix for proficiency testing and quality assurance testing that allows for the spiking of additives, contaminants or bioactive molecules that allow for the proficiency tester to accurately provide known material to testing laboratories to evaluate their ability to detect and quantify these compounds.
  • This spiking of the sample can be extended to live and/or dead organisms as well in the case of microbial testing proficiency testing, or even viroids or viruses to determine a lab’s ability to detect pathogens of Cannabis. This can also be done by spiking known amounts of synthetic and extracted DNA and/or RNA from that given organism, virus, or viroid.
  • Similar logic can be applied in many other species and genera of plants and fungi, to identify, selectively breed, and produce organisms devoid of medically or recreationally relevant compounds and their respective degradants.
  • Psilocybin-free Psilocybe species one would select or knockout the genes in the psilocybin biosynthesis pathway that would prevent the accumulation of tryptamines including norbaeocystin, baeocystin, psilocybin, psilocin, aeruginascin, and 4-hydroxy-trimethyltryptamine.
  • hericenone diterpene biosynthetic pathway is selected against to produce hericenone-free Hericium spp. Similar post-harvest processing and packaging steps are applied for these organisms to produce a research-grade or certified reference material grade bioactive- free matrix blank biomass material or extracts thereof that are used according to this disclosure.
  • the matrix-blank organism is spiked with pathogenic or contaminant organisms or metabolites indicative of those organisms to create a proficiency test to test a laboratory’s target species specificity of their assay.
  • two mixtures of the matrix-blank organism are prepared: one spiked with shiga-toxin producing E. Coli and one spiked with non-pathogenic E. Coli, and sent to a laboratory as an unknown sample to determine the accuracy and specificity of microbial testing specifically in regards to testing products containing the same organism as the matrix-blank organism.
  • the matrix blank organism are spiked with one or more species of pathogenic aspergillus species, which enables a laboratory to validate the specificity, accuracy and matrix compatibility of microbial testing methods in new organisms where the matrix of that organism has not been tested.
  • the backcrossed population provided a higher percentage of cannabinoid-free plants (-50% of offspring retaining zero cannabinoids); whereas the inbred siblings allowed for a greater introgression and recombination of genes related to yield and dioecy, while still producing 25% of the offspring as zero-cannabinoid producing plants.
  • the newly obtained plants (ZCSB1 & ZCBC1) increased yield by over 20% compared to the original ‘ZCP’ parental line, as well as showed multiple dioecious phenotypes that were selected for further breeding.
  • ‘ZCP’ additionally was bred to other lines for more agronomic qualities as well.
  • this plant was crossed with a high CBD lineage, CP21, that contained herbivory- induced plant volatiles, such as guaiol, a-farnesene and germacrene D. These compounds are known to be produced in response to pest or herbivore pressure to allow for less pesticide application during the cultivation cycle.
  • Fl offspring of that cross F2 populations were bred through sibling crosses as well as backcrosses to the ‘ZCP’ parent and the herbivory- induced volatile terpene containing plants were selected for future breeding.
  • ZCSB1 is the F2 seed lot derived from the sibling breeding of the ZCO1 offspring that originated from the ZCPxCK15 cross, and then ZCSB2 refers to the F2 seed lot derived from the sibling breeding from the ZCPxCP21 cross, which both F2 lots contained 25% of the individuals having a cannabinoid-free phenotype.
  • a similar strategy was used in the backcrossing of Fl offspring to the ‘ZCP’ parental line, which lead to the production of ZCB1 F2 seed lot derived from the outcross to CK15, as well as the seed lot ZCB2, which contained 50% of the individuals having a cannabinoid-free phenotype.
  • This reciprocal breeding strategy employed traditional inbreeding methods to produce 100% cannabinoid-free F3 lots of each of the sibling bred and back crossed lots individually allowing for the selection and isolation of traits in these inbred lines, as well as employing the “cousin cross” strategy disclosed here where an Fl -like plant with increased genetic diversity and heterozygosity was produced through crossing the ZCSB1 x ZCSB2.
  • the F3 crosses between the back crossed lots, crosses within a sibling bred lot, or the crosses between the sibling bred lots and the back crossed lots produced segregating inbred offspring that allowed for us to select for various subsets of the phenotypes traits of the parental line CK15 and CP21 in the cannabinoid-free offspring.
  • ‘ZCP’ lineage through selectively breeding for increased cannflavin production when compared to the parental lines.
  • ‘ZCSB1 & ZCBC1’ were crossed with a known high cannflavin A (CP21) producing plant that contained greater than 0.05% (weight/weight) cannflavin A in outdoor cultivation trials.
  • the hybrid Fl offspring was then subsequently inbred through a combination of sibling breeding and backcrossing to the ‘ZCP’ lineage to remove the cannabinoid biosynthetic capacity while retaining or improving the flavonoid production ability of CP21.
  • ZCF1 a plant, ZCF1 was produced that had the cannabinoid-free trait of the ‘ZCP’ lineage parental line, while also showing increased production of cannflavin A at nearly double the concentration of the CP21 cannabinoid-containing parental line.
  • ZCF1 was shown by HPLC analysis to produce greater than 0.1% cannflavin A by dry weight of the biomass material.
  • ‘ZPC’ This growth conditions were used for most widely applicable versions of ‘ZPC’ that can be used as a matrix blank for all regulatory testing employed by states with recreational or medical Cannabis programs currently as well as for spiked validation studies and services that are routinely employed during proficiency testing of Cannabis laboratories, where known concentrations of compounds or organisms were added to the ‘ZCP’ material that was then analyzed by a laboratory undergoing accuracy and precision assessments.
  • Bile-Tolerant Gram-Negative Bacteria Campylobacter spp, Candida albicans, Coliforms, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella spp, Shiga Toxin-Producing Escherichia coli (STEC), Staphylococcus aureus, Total Aerobic Bacteria, Total Enterob acteriaceae, Total Yeast and Mold, Yersinia spp, Hop Latent Viroid (HLVd), Lettuce Chlorosis Virus (LCV), Cannabis Cryptic Virus (CCV) Tobacco Mosaic Virus, Tocopheryl Acetate, a-Tocopherol, 5-Tocopherol, P-Tocopherol / y- Tocopherol, A8THC, A8THCV, A8-iso-THC, A9THC, A9THC Acetate, A10THC,
  • the plant can be grown indoors or outdoors without the need for a positive pressure environment. This allows for a laboratory to ensure that no additives, such as a pesticides, being added to the plant during cultivation are being detected as a cannabinoid and provides a cheaper alternative to cultivation of ‘ZCP’.
  • extracts derived from plants nurtured via technique (i) are conspicuously devoid of cannabinoids, pesticides, fungicides, mycotoxins, endotoxins, microbial contaminants, viruses, synthetic drugs, residual solvents, Per and Polyfluorinated Substances (PF AS), and heavy metals.
  • PF AS Per and Polyfluorinated Substances
  • the cannabinoid-free Cannabis allows for the creation of a cannabinoid-free extract that still contains these metabolites that were previously degraded or lost during the separation of cannabinoids and other metabolites found in the plant or extract thereof.
  • Example 3 In order to characterize the chemotypes of the parental and produced offspring plants and the resulting extracts, a chemical analysis of both the cannabinoid content, and selected other chemicals, was undertaken as set out in Example 3: Example 3
  • Impeding certain bioactive pathways does not necessarily impact bioactive synthesis, depending upon whether the pathway is impeded far upstream of the final bioactive, or is impeded closer to the final bioactive. In many cases, a disrupted pathway can result in reduced levels of precursors. On the other hand, a disruption in a downstream portion of the pathway hinders the formation of more direct precursors. Where the biosynthetic pathways are well known, analysis of precursor accumulation indicates which point(s) in the pathway are impeded.
  • the matrix-blank organism is spiked with pathogenic or contaminant organisms or metabolites indicative of those organisms to create a proficiency test to assess the specificity of a laboratory’s target species assay.
  • two mixtures of the matrix-blank organism are prepared: one spiked with shiga- toxin producing E. Coli and one spiked with non-pathogenic E. Coli, and are sent to a laboratory as an unknown sample to determine the accuracy and specificity of microbial testing protocols specifically in regards to testing products containing the same organism as the matrix-blank organism.
  • the matrix blank organism is spiked with one or more than one species of pathogenic Aspergillus species, which using this spiked matrixblank organism enables a laboratory to validate microbial methods in new organisms where the matrix has not been tested.
  • these same mutations are introduced through non-selective breeding methods as well, such as CRISPR-CAS, Cre- Lox, and other known gene modification techniques to introduce non-naturally occurring knockouts of these organisms to be used in the disclosed applications of the bioactive-free matrix blank material.
  • the methods disclosed herein are applicable to all producing a multiplicity of matrix-blank cacti, as has been shown in the exemplary bioactive-free matrix -blank organism, Cannabis. This is achieved through the creation and use of the specific selectively bred varieties and their progeny which are specialized for the various applications disclosed herein. Such selections are used in industries such as pharmaceuticals, nutraceuticals, and agriculture, as well as applications in analytical testing methodologies that require a matrix-blank material for accurate quantification of the bioactive molecules.
  • bioactive-free segregants resulting from backcrosses with a high bioactivecontent variety have biochemical profiles that appear to be typical as compared with the parent varieties, except as to the target bioactives which are selected for reduction and/or removal through such breeding.
  • Impeding certain bioactive pathways does not necessarily impact bioactive synthesis, depending upon whether the pathway is impeded far upstream of the final bioactive, or is impeded closer to the final bioactive. In many cases, a disrupted pathway can result in reduced levels of precursors. On the other hand, a disruption in a downstream portion of the pathway hinders the formation of more direct precursors. Where the biosynthetic pathways are well known, analysis of precursor accumulation indicates which point(s) in the pathway are impeded.
  • a further use of the cacti produced by this approach is that they can be used to create cacti -extracts matrix-blank materials containing other bioactive molecules than target bioactives in quantities and purities, which could not be achieved naturally.
  • Such cacti-extracts matrix-blank materials provide the benefits arising from the presence of one or more selected other compounds. This produces a “designer” cactus extract that can be used in analytical method validation and proficiency testing or for the production of customized bioactive-ratio materials that cannot be achieved naturally.
  • the zero- mescaline cacti would be derived from an interspecific hybrid line of one or more than one of the following species: San Pedro (Echinopsis pachanoi), Peruvian Torch (Echinopsis peruviana), Venezuelan Torch (Echinopsis lageniformis), Peyote (Lophophora williamsii), Lophophora diffusa, Echinopsis scopulicola, Echinopsis santaensis, Echinopsis macrogona, Trichocereus bridgesii, Trichocereus terscheckii, Trichocereus cuzcoensis, and/or Trichocereus validus.
  • the Zero-mescaline Cactus biomass or extract derived thereof is spiked with >0.1% by dry weight of a target bioactive while remaining absent of other target bioactives. Due to the nature of bioactive biosynthesis, creating cacti without certain bioactives but having other bioactives is normally impossible if a given bioactive is a precursor to the terminal bioactives. Thus, through these methods of using the fungal materials of this invention, it is possible to create non-naturally occurring bioactive profiles through infusing naturally derived, synthetic, or modified bioactives into the fungal biomass. Extracts derived therefrom are possible using the matrix-blank organisms and materials and methods disclosed herein.
  • this is achieved through solubilizing the bioactives in a known volume of liquid solvent, such as ethanol, methanol, or other solvents in which the target bioactives are soluble.
  • a similar infusion process can be used to incorporate one or more compounds from the following classes: chemical contaminants, environmental contaminants, bioactive compounds derived from the cactus, bioactive compounds not found in the cactus, or non-bioactive compounds.
  • the matrix-blank organism is spiked with pathogenic or contaminant organisms or metabolites indicative of those organisms to create a proficiency test to assess the specificity of a laboratory’s target species assay.
  • two mixtures of the matrix-blank organism are prepared: one spiked with shiga- toxin producing E. Coli and one spiked with non-pathogenic E. Coli, and are sent to a laboratory as an unknown sample to determine the accuracy and specificity of microbial testing specifically in regards to testing products containing the same organism as the matrix-blank organism.
  • the matrix blank organism is spiked with one or more than one species of pathogenic Aspergillus species, which using this spiked matrix-blank organism enables a laboratory to validate microbial methods in new organisms where the matrix has not been tested.
  • the technology disclosed herein is used to make zero-bioactive organisms including ganoderic acid free Ganoderma spp mushrooms through disruption of the ganoderic biosynthetic pathway, cordycepin-free cordyceps spp through disruption of the cordycepin biosynthetic pathway, alkaloid-free ibogaine through disruption of alkaloid biosynthetic genes, isoxazole-free amanita spp through the disruption of the ibotenic acid biosynthetic pathway, and hericenone-free and/or erinacine-free lions mane material through the disruption of the triterpenoid biosynthetic pathway. Similar observations and presumptions are applied, and similar conclusions are reached, as those discussed in Examples 5 and 6.
  • Cannabis and Cannabis extracts greater than the sum of their parts?. J Cannabis Therapeutics. 2001;1 : 103-32.
  • Taura et al. First direct evidence for the mechanism of .DELTA.1-tetrahydrocannabinolic acid biosynthesis. J Am Chem Soc. Sep. 1995;117(38):9766-7.
  • Taura et al. Purification and characterization of cannabidiolic-acid synthase from Cannabis spp L.. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid. J Biol Chem. Jul. 19, 1996;271(29): 17411-6. Virovets, Interview. J Int Hemp Assoc. 1998;5:32-4.
  • Cannabis as a medicine evidence for synergy. In Medicinal uses of Cannabis, 26 th LOF Symposium, Leiden 2002.

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Abstract

La présente invention concerne des organismes de blanc de matrice développés de manière sélective pour : a) ne pas exprimer un composé ou un groupe de composés bioactifs ; ou b) exprimer, au moins de manière substantiellement qualitative, la plupart des autres composés non bioactifs présents dans un organisme de comparaison analytiquement actif, les organismes servant à générer un matériau de référence de blanc de matrice avec un profil chimique qui ressemble à celui de l'organisme de comparaison moins le composé ou le groupe de composés actifs, ainsi que des procédés de fabrication des organismes et leurs utilisations. La présente invention concerne également les matériaux de référence de blanc de matrice, ainsi que leurs procédés de fabrication et leurs utilisations. La présente invention concerne des organismes de blanc de matrice développés de manière sélective pour : a) ne pas exprimer un composé ou un groupe de composés bioactifs ; mais b) exprimer, au moins de manière substantiellement qualitative, la plupart des autres composés non bioactifs présents dans un organisme de comparaison analytiquement actif, les organismes servant à générer un matériau de référence de blanc de matrice dont le profil chimique ressemble à celui de l'organisme de comparaison moins le composé ou le groupe de composés actifs, ainsi que des procédés de fabrication des organismes et leurs utilisations. L'invention concerne également les matériaux de référence de blanc de matrice, leurs procédés de fabrication et leurs utilisations.
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