[go: up one dir, main page]

WO2022064527A1 - Bio-formulation pour la production de composés organiques volatils pour améliorer la durée de conservation de produits de post-récolte - Google Patents

Bio-formulation pour la production de composés organiques volatils pour améliorer la durée de conservation de produits de post-récolte Download PDF

Info

Publication number
WO2022064527A1
WO2022064527A1 PCT/IN2021/050941 IN2021050941W WO2022064527A1 WO 2022064527 A1 WO2022064527 A1 WO 2022064527A1 IN 2021050941 W IN2021050941 W IN 2021050941W WO 2022064527 A1 WO2022064527 A1 WO 2022064527A1
Authority
WO
WIPO (PCT)
Prior art keywords
bio
formulation
substrate
muscodor
kashayum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2021/050941
Other languages
English (en)
Inventor
Sanjai Saxena
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agpharm Bioinnovations LLP
THAPAR INSTITUTE OF ENGINEERING AND TECHNOLOGY
Original Assignee
Agpharm Bioinnovations LLP
THAPAR INSTITUTE OF ENGINEERING AND TECHNOLOGY
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agpharm Bioinnovations LLP, THAPAR INSTITUTE OF ENGINEERING AND TECHNOLOGY filed Critical Agpharm Bioinnovations LLP
Publication of WO2022064527A1 publication Critical patent/WO2022064527A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • the present invention broadly lies in the field of biotechnology and microbiology. Particularly, the present invention relates to a bio-formulation comprising the biobased substrate and a novel fungus which produces a unique spectrum of volatile compounds possessing a very potential broad-spectrum activity. Further, the present invention also provides method of producing such unique spectrum of volatile compounds, the bio-substrate and the bio-formulation and uses thereof.
  • PHL post-harvest losses
  • Majority of the techniques involved in enhancing the shelf life of F&V primarily involve two facets (a) the action of microbes as contaminants (pathogens and spoilage) during storage which leads to rotting and deterioration making it un-consumable by living beings, or (b) production of toxic secondary metabolites, prominently mycotoxins which are harmful to both humans and animals being difficult to remove/detoxify.
  • the former problem happens due to direct microbial contamination of the crop at the harvest or at storage and generally remains undetected until the crop is taken in the retail chain or food processing.
  • There are several techniques available providing modified atmosphere with different gases for forced flow in the F&V storage boxes along with humidity control to arrest the microbial growth and deterioration.
  • these technologies are very expensive and out of the reach of small holding farmers.
  • Aflatoxins are more toxic as compared to Fumonisins and are found to be teratogenic, carcinogenic, mutagenic and immunosuppressive. These have residual effects similar to the other chemical pesticides and synthetic preservatives, apart from their lethal attributes.
  • Food coatings is yet another method of keeping F&V fresh for longer duration of protection against chemicals, microbial contamination and oxidative decay.
  • researchers have developed edible coatings for preserving the quality and freshness of fruits as well as prevent them from microbial spoilage or degradation (Indian Patent 702/Del/1999).
  • Phosphatides like lecithin have also been developed into film coatings to enhance the shelf life of F&V which also helps in preventing them from drying out.
  • coatings materials comprise of carob beans, pullulan, carrageenan, chitosan, alginates, pectin’s, cellulose and starches (Carbohydrates), collagen and keratin (proteins) and bees wax, polycosanols, carnauba wax (Solid fats).
  • synthetic polymers have also been used for the purpose of using as a coating such as polyethylene, polypropylene, polyvinyl acetate (PVAc), polyvinyl alcohol (PVA) or polyacrylates.
  • Interventions like a composition comprising of a hydrocolloid, an edible wax, a fatty acid and an edible alkaline component have been developed for coating plant matter and thereby reduce the plant's postharvest water losses, weight and quality reduction.
  • these interventions make the end product quite expensive.
  • Citral 7-dimethyl-2, 6-octadienal
  • USFDA a safe additive
  • US 7465469B2 an unsaturated terpene from citrus fruits which is recognized as a safe additive by USFDA and exhibits a broad range of antimicrobial and anti-fungal activity
  • Essential oils, plant extracts and ethanol have been exploited due to their antimicrobial action and volatile nature as they in headspace of food packs increase microbial stability while mildly affecting the sensory attributes (Kapetanakou & Skandanis, Current Opinion in Food Science, 2016, 12: 1-12).
  • VOCs volatile organic compounds
  • Muscodor albus CZ620 Muscodor crispans
  • Muscodor vitigenus have been exploited till date which have been exclusively isolated from Cinnamomum zeylanicum (from Honduras), Ananas ananassoides (Bolivian Amazon Basin) and Paulliana paullinioides (Peruvian amazon forest).
  • Composition of volatile organic compounds (VOC’s) from these Muscodor isolates possess biological activity and can kill or pathogenic microbes i.e. bacteria and fungi apart from some insects.
  • VOC VOC
  • the major limitation of the existing technologies is the modified atmosphere i.e. an environment in which pathogenic and spoilage microorganisms cannot survive, thereby drastically reducing the chances of decay and deterioration due to accumulation of volatile inside fruit & vegetable packing.
  • the present inventors have provided such bio-formulation which enhances the shelf life of fruits and vegetables and also delays the ripening process.
  • An objective of the present invention is to provide a novel bio-formulation.
  • Yet another objective is to provide a novel bio-substrate.
  • Another objective of the invention is to provide the bio-formulation comprising the novel bio- substrate and a novel fungus which produces a unique spectrum of volatile compounds possessing a very potential broad-spectrum antimicrobial activity.
  • Still another objective of the present invention is to provide a method of producing the bio- substrate and the bio- formulation.
  • Yet another objective of the present invention is to provide a novel fungus.
  • the present invention relates to a novel bio-formulation comprising the bio-substrate and a novel fungus which produces a unique spectrum of volatile compounds possessing a very potential broad-spectrum activity.
  • the novel fungus is Muscodor kashayum #16AMLWLS.
  • the bio-formulation of the invention exhibits anti-microbial activity and inhibits bacteria, fungi, yeasts thus, exhibiting a broad-spectrum antimicrobial activity.
  • the bio- formulation of the invention is “Bactericidal”, “Fungicidal” and “Anti-candida” in nature.
  • the bio-formulation of the invention is a synergistic composition which is capable of eradicating or drastically reducing the count of spoilage and pathogenic microorganisms inhabiting or infecting F&V, grains, pulses, dairy products (such as eggs), unconsumed cooked food, raw meat, fish etc.
  • the invention also relates to a bio-substrate that aids in production of the unique spectrum of volatile organic compounds possessing a very potential broad-spectrum activity.
  • the novel fungal strain of the invention has been deposited at IMTECH, Chandigarh. The deposition number of the strain is MTCC-25403
  • Figure 1 Phylogenetic analysis of Muscodor kashayum. Phvlogeneticallv. M. kashayum clearly confirmed its basal position in clade A. with significantly high bootstrap value distinct from M. suthepensis which was much lower, indicated genetic and evolutionary differences.
  • Figure 2a and 2b Scanning electron micrographs of Muscodor kashayum #16AMLWLS grown on PDA for 10 days exhibiting the mycelial arrangement and morphological structures.
  • FIG. 3 In vitro assay for antimicrobial activity of the VOC’s against spoilage and pathogenic microorganisms Candida albicans, Cercospora beticola, Fusarium species, Staphylococcus aureus.
  • modified atmosphere means a closed environment where the volatiles accumulates inside the closed container and create an environment in which pathogenic and spoilage microorganisms cannot survive, thereby drastically reducing the chances of decay and deterioration.
  • the present invention relates to a bio-formulation comprising of a novel bio-substrate and a novel fungus which produces a unique spectrum of VOC’s possessing a very potential broad- spectrum activity.
  • the novel fungi are Muscodor kashayum #16AMLWLS.
  • the bio- formulation of the invention exhibits anti-microbial activity and inhibits bacteria, fungi, yeasts thus, exhibiting a broad-spectrum antimicrobial activity.
  • the bio-formulation of the invention is “Bactericidal”, “Fungicidal” and “Anti-candida” in nature.
  • the bio-formulation of the invention is a synergistic composition which is capable of eradicating or drastically reducing the count of spoilage and pathogenic microorganisms inhabiting or infecting fruits and vegetables, grains, pulses, dairy products (such as eggs), unconsumed cooked food, raw meat, fish etc.
  • Muscodor kashayum #16AMLWLS is a dueteromycetous fungus (Mycelia sterilia) and exists as an endophytic species which belongs to the group Xylaria. It is genetically distinct from other reported Muscodor species from different parts across the globe.
  • Muscodor fungi Muscodor kashayum sp. nov is isolated by using the volatiles of previously discovered Muscodor albus cz620 (received from Gary Strobel) as a stress tool since, only Muscodor species can survive or resist the presence of volatiles of another Muscodor species while other fungi is eradicated or drastically inhibited by the volatile organic compound’s gaseous mixture.
  • the Muscodor kashayum #16AMLWLS. has been deposited at the International Depository Authority (IDA), India at Microbial Type Culture Collection (MTCC) & Gene Bank at CSIR- Institute of Microbial Technology (IMTECH), Chandigarh, India under the accession number MTCC 25403.
  • the present invention establishes the fact that the signature VOC’s produced by Muscodor kashayum #16AMLWLS inhibits bacteria, fungi, yeasts and hence, exhibits a broad-spectrum antimicrobial activity (Table 1).
  • the volatiles completely destroy the growth of Aspergillus spp., Penicillium spp. and Fusarium spp. which are mycotoxigenic.
  • VOC Muscodor kashayum
  • Bactericidal Muscodor kashayum
  • Anti-candida the VOC’s produced by M. kashayum are unique, and have not been previously reported by any other Muscodor species (Table 2) or not been exploited so far for sanitization and extending the shelf life of food materials (raw, fresh and processed) in India or globe.
  • the present invention provides a unique bio-substrate for the production of volatile organic compounds, wherein said bio-substrate comprises carrier selected from wood powder or turnings, rice husk, Isabgol husk, or agricultural by-products or agricultural wastes, ground pulverized grains, potato dextrose broth (PDB) and stabilizing agents, and wherein the ratio of wood tumings/wood powder: rice husk : Isabgol husk: ground pulverized grains is 0.75:1:0.75:05.
  • carrier selected from wood powder or turnings, rice husk, Isabgol husk, or agricultural by-products or agricultural wastes, ground pulverized grains, potato dextrose broth (PDB) and stabilizing agents, and wherein the ratio of wood tumings/wood powder: rice husk : Isabgol husk: ground pulverized grains is 0.75:1:0.75:05.
  • the potato dextrose broth (PDB) is in a ratio of 5% (v/w).
  • the potato dextrose broth is freshly prepared.
  • said agricultural waste/ byproduct is hulls of seeds, pulverized rice /wheat husk and grains.
  • said bio-substrate comprises 1 Rice husk: 0.75 Saw dust: 0.5 seed hulls: 0.5 pulverized grain.
  • said grain is wheat, rice, rye or barley.
  • said stabilizing agent is a carbohydrate.
  • said carbohydrate is selected from the group consisting of dextrose, sucrose, trehalose or raffinose.
  • the stabilizing agent is selected from 1% trehalose, 0.5 polyethylene glycol (6000) and 0.5% Lactose/sucrose.
  • the bio-substrate comprises wood powder or turnings, Rice husk, Isabgol Husk and Rye/wheat grain in ratio of 0.75:1.00: 0.75:0.5 w/w, freshly prepared potato dextrose broth (PDB) comprising of 5% (v/w) and stabilizing agents.
  • PDB potato dextrose broth
  • the present invention provides a bio-formulation comprising the bio-substrate as claimed in any of the claims 1-8 and a culture of Muscodor kashayam (MTCC-25403).
  • the culture of Muscodor kashayam (MTCC-25403) produces a unique spectrum of Volatile Organic Compounds (VOCs).
  • said bio-formulation is in the form of a wettable powder (WP), an emulsifiable concentrates, gel, pellets or soluble liquids.
  • WP wettable powder
  • emulsifiable concentrates gel, pellets or soluble liquids.
  • said formulation is in the form of a filter paper, pad or sachet.
  • said formulation comprises a mixture of volatile compounds (VOCs) comprising: i. 3-cyclohexen-1-ol, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- (Synonym: ⁇ -Bisabolol); ii. 2,6-Bis (1,-dimethylethyl)-4-(1 -oxypropyl) phenol; iii. 1,6- Dioxacyclododecane-7, 12-dione; iv. 2,3-dihydro-1,1-dimethyl-6-tert-butyl-1H-indene-4-acetic acid; v. 2,4-Di-tert-butylthiophenol; vi. 4-Octadecylmorpholine and, vii. 2-(4-morpholinyl) ethanamine.
  • VOCs volatile compounds
  • said formulation is effective in enhancing the shelf life of fruits, vegetables and grains such as Strawberry’s, Apples, Grapes, Mandarins, Oranges, Guavas, Mangoes, Lychees, Tomatoes, Onions, Okra, pulses, chickpea, wheat and rice.
  • fruits, vegetables and grains such as Strawberry’s, Apples, Grapes, Mandarins, Oranges, Guavas, Mangoes, Lychees, Tomatoes, Onions, Okra, pulses, chickpea, wheat and rice.
  • the present invention provides a method for preparing a bioformulation comprising the steps of:
  • the present invention provides a method of inhibiting the growth of a microorganisms in post-harvest products, said method comprising the delivery of the composition of naturally produced VOC’s through the bioformulation as claimed in claim 9.
  • the present invention provides a method of protecting and enhancing the shelf life of fruits, vegetables, seeds and grains from infestation by a microorganism viz. fungus, a bacterium, a yeast or an actinomycetes, wherein the method comprises delivery of the composition of naturally produced VOC’s through the bioformulation as claimed in claim 9, causing a delayed process of ripening of fruits and vegetables apart from antimicrobial action thereby extending their shelf life.
  • the present invention also provides a novel strain of Muscodor kashayam with the deposition number MTCC 25403
  • Example 1 Isolation of the fungus
  • Aegle marmelos also known as Bael
  • a medicinal plant in Ayurvedic literature was collected from Wayanad Wildlife Sanctuary, Muthanga, Huawei, during July 2019. The leaves were stored in sterile packets and stored at 4 ⁇ 2°C till further use. Stress VOC based assay was used for isolation of Muscodor kashayum sp.nov., using volatiles of Muscodor albus CZ620 Woropong, Strobel and Hess, as an isolation tool as reported by Ezra et al. (2004).
  • Adaxial and abaxial sides of leaves of Aegle marmelos were disinfected, made sterile using 75% ethanol for 30 s, 2% sodium hypochlorite for 3 min and 95% ethanol under a laminar flow hood. These were then sectioned into segments of 5 mm x 5mm and then placed in three sections of the four sectioned commercially available petri dishes containing pre-sterilized water agar (WA). In the fourth quadrant a 5 mm mycelial plug of actively growing M. albus CZ620 was placed on a pre-sterilized Fresh Potato Dextrose Agar (FPDA).
  • FPDA Fresh Potato Dextrose Agar
  • the Muscodor albus CZ 620 was inoculated into the petri-dish 3 days at 23 ⁇ 2 °C for the production of VOCs (volatile organic compounds) prior to the inoculation of the sterilized leaf segments.
  • VOCs volatile organic compounds
  • the fungi emerging out of the host tissue was aseptically sub-cultured onto a fresh PDA plate so as to obtain pure isolate(s).
  • the only organisms developing from the plant material were the ones which were resistant to M. albus CZ620, which are possibly other volatile antibiotic producers or possibly related to the genus Muscodor in the Xylariaceous group (Strobel et al., 2001). This isolated Muscodor strain was initially designated as #16AMLWLS.
  • the morphological identification of the fungi generally involves induction of fruiting bodies or spore production on basis of which a presumptive identification is generally made.
  • the fungus #16 AMLWLS was inoculated on a host of different solid media such as MEA, CMA, PDA, CDA, WA, BLA. There was no spore production on any of the media.
  • the fungus was stored using paper disc cultures and glycerol stocks.
  • a filter paper discs of Whatman no.42 were sterilized and place on sterile PDA plate.
  • An agar plug of Muscodor kashayum was placed in the middle of the disc and incubated at 24 ⁇ 2 °C for 18 days.
  • the paper disc was removed from the plate and kept in air under sterile conditions for approx. 48 hours until it became completely dry. It was then cut into small pieces using sterile scalpel under the laminar flow.
  • These paper cultures were then stored at 24 ⁇ 2 °C, 4 °C, 0 °C, and -20°C.
  • the agar plugs of the actively growing fungus were also stored in 15% glycerol and stored at -20 °C till further use.
  • the viability of the paper culture as well as glycerol stocks were placed on PDA and incubated for 3-5 days for examining the fungal growth.
  • the purified DNA was quantified using a nano- drop spectrophotometer to assess the purity.
  • Muscodor specific primers developed by Ezra et al. (2009) viz. M. albus F (5' GGGAGGCTACCCTATAGGGGATAC3') and M. albus R (5'CAGGGGCCGGAACCACTACAGAGG3') were used for amplifying the ITS-5.8S rDNA region of #16AMLWLS.
  • the PCR mixture comprised 25 ng of extracted genomic DNA, 25 mM MgCh, 2.5 mM dNTP, 10 pmol / ⁇ l of each primer, 1.5 U of Taq DNA polymerase in 10X Taq buffer while the PCR amplification conditions used have been previously described by Ezra et al. (2009).
  • the ITS rDNA PCR amplicon of approximately 500-600 bp was resolved onto 1.5% agarose gel and further purified by Wizard® SV Gel and PCR clean-up system kit (Promega). The purified ITS rDNA amplicons were then sent for sequencing to Xcleris Genomics, Xcleris labs, Gujarat, India.
  • Sequencher ver.5 was used for assembling the obtained sequences and subsequently submitted to GenBank with accession no. KC481680.
  • the sequence so obtained was compared to sequences of other type species of Muscodor reported across the globe. Briefly the sequences were aligned with the obtained ITS sequence of #16AMLWLS using Clustal W option in MEGA 5.0 (Tamura et al. 2011).
  • the phylogenetic map was constructed by using all 12 reported species of Muscodor. Xylaria arbuscular and Peziza badia were taken as outgroup in the phylogenetic analysis.
  • the Clustal W aligned sequences were made uniform by manually correcting the aligned sequences.
  • the alignment was exported to the MEGA format.
  • the evolutionary distances were inferred by using maximum likelihood method based upon the Kimura-2-parameter model.
  • Muscodor kashayum ITS sequence in BLAST Analysis exhibited 99% sequence similarity with Muscodor sp. strain J8, Muscodor sp. strain Y-L-54, Muscodor oryzae JCM18231, Muscodor musae CMU MU3, Xylariaceae species ARIZ FL 1963, Muscodor crispans.
  • its exact phylogenetic position could be inferred only after phylogenetic analysis and mapping of its taxonomic identity with already know type/ novel species reported so far.
  • the phylogram (Fig.l) clearly indicates Muscodor kashayum (#16 AMLWLS) is a novel type species.
  • Agar plugs of 10 days old culture of #16AMLWLS was kept in 2.5 % glutaraldehyde in 0.1M phosphate buffer (pH 7.2) overnight at 4°C. Next day, they were washed twice with 0.1 M phosphate buffer at a 10 min interval. Thereafter, the samples were slowly dehydrated using acetone-graded series (10 min each at 30%, 40%, 50%, 60%, 70%, 80%, 95% and 100%). The samples were then brought to critical point drying and coated with gold palladium using a sputter coater.
  • Muscodor kashayum #16 AMLWLS is different from M. yucantanesis and M. equiseti as it does not have swollen hyphae. Further, it is different from M. cinnamomi, M. crispans and M. sutura by the absence of cauliflower-like or non-descript structures. Muscodor kashayum does not have wavy margins or hairy mycelium which makes it distinct from Muscodor musae. Further it does not produce any pigment as produced by M. roseus, M. suthepensis, M. oryzae and M. sutura. The fungus in nature is associated with Aegle marmelos and an Ascomycete with sterile mycelium.
  • Fungal colonies are milky white and form a floccose mycelium pattern on PDA when grown in a 12-h photoperiod for 10 days at 26 ⁇ 2°C.
  • Mycelium on PDA produced a growth rate of 4.5-4.7 ⁇ 1.41 mm day-1 having a pungent smell. Spores and fruiting bodies did not develop under any tested conditions.
  • the fungal culture produces VOCs with a strong pungent odor only in PDA, MEA and CDA medium. Volatile production was comparably higher in PDA than to MEA and CDA. Spores and fruiting bodies did not develop under any of the tested conditions.
  • Muscodor yucatanensis has a ropy structure with swollen hyphae whereas Muscodor albus only exhibits a ropy mycelium without any fruiting bodies, spores or sterile structures.
  • Muscodor satura forms knitting pattern of mycelial network over the PDA plate making it remarkably dissimilar from M. kashayum #16 AMLWLS.
  • Muscodor kashayum sp. nov. #16AMLWLS was grown on different substrates such as PDA, Rye grains, Wheat grains, combination of novel bio-substrates like rice husk, psyllium husk, wood powder, wood turnings etc. to produce volatile organic compounds. Further the VOC’s so produced were tested for their antimicrobial profile. For instance, in case of PDA the medium was poured in four sections of a sterile commercial petri plate and allowed to solidify under aseptic conditions. On solidification, one quadrant was inoculated with a mycelial plug of #16 AMLWLS and incubated for 3 days at 26 ⁇ 2 °C.
  • PDA the medium was poured in four sections of a sterile commercial petri plate and allowed to solidify under aseptic conditions. On solidification, one quadrant was inoculated with a mycelial plug of #16 AMLWLS and incubated for 3 days at 26 ⁇ 2 °C.
  • Bioassay method adopted for evaluating the volatiles produced by Muscodor kashayum revealed a potent broad-spectrum antimicrobial activity against a test panel of microorganisms comprising of both gram+ve and gram-ve bacteria, fungi and yeasts. A total of 29 microorganisms were tested of which 15 were fungi. 04 were yeasts and 10 were bacteria. Only 66.6% fungal isolates in the test panel exhibited 100 % reduction in the growth i.e. no growth was observed as they were completely inhibited the VOC’s produced by Muscodor kashayum #16 AMLWLS. 20% of the fungal isolates exhibited a 50% reduction on colony size due to VOC’s of M. kashayum.
  • yeasts 100% inhibition was obtained in three isolates while 20% inhibition in colony diameter was observed in the case of Saccharomyces cerevisiae which is generally recognized as GRAS.
  • bacterial isolates tested 80% exhibited 100% kill/inhibition in the presence of volatile antibiotics of Muscodor kashayum.
  • Bacillus subtilis only exhibited 20% reduction in the growth while Staphylococcus epidermidis exhibited 50% reduction in colony size/growth when compared to the control.
  • Example 7 Analysis of volatiles produced by Muscodor kashayum sp. nov. #16AMLWLS
  • the method to analyze the air space above the mycelium of Muscodor kashayum was trapping via using solid phase microextraction as well as by trapping them on amorphous carbon columns.
  • the solid phase microextraction method comprised of using a “Solid Phase Micro Extraction” syringe which was used to trap the fungal volatiles.
  • the syringe was having a stable flex fiber made up of 50/30 divinylbenzene/carboxen on polydimethylsiloxane (Supelco, Sigma Aldrich, St. Louis, MO, USA).
  • the syringe was placed through a small hole drilled in the side of the Petri plate and exposed to the vapor phase for approx. 45 min. Subsequently the syringe was then inserted for 30 s exposing the flex fiber in the Shimadzu QP 2010 (Columbia, MD, USA) plus Gas chromatograph with TD-20 (Shimadzu, Columbia, Maryland, USA) thermal desorption system.
  • the GC column comprised of (diphenyl (95%) and dimethyl polysiloxane (5%) as the stationary phase. The length of the column was 30 m with 0.25mm internal diameter and 0.25 mm film diameter.
  • the column was programmed at 100°C for 2 min and the temperature was then raised to 250°C for 2 min and then finally to 300°C for 13 min.
  • the carrier gas was helium and the initial column head pressure as 94.4 KPa.
  • Data acquisition and processing was done on GC-MS (Shimadzu, Columbia, Maryland, USA) solution software. Comparable analysis was conducted on un-inoculated substrate as PDA or complex bio-substrate in the petri dish to explore the background volatiles, which were later on subtracted from the analysis of fungus containing plate.
  • the compounds were identified using NIST (National Institute of Standard and Technology) database with a high-quality scoring of over 75% similarity. Further they were compared with all the reported species of Muscodor till date (Ezra et al. 2004; Kudalkar et al. 2012).
  • Muscodor kashayum #16 AMLWLS produced a mixture of 20-26 volatile organic compounds (VOC’s) which were identified using the GC-MS spectral analysis with support of the NIST database.
  • VOC volatile organic compounds
  • the characteristic volatile signatures produced by M. kashayum comprised of ⁇ - Bisabolol (3-Cyclohexen-1-ol, 1-(1, 5-dimethyl-4-hexanyl)-4-methyl). The other prominent volatiles produced by M.
  • kashayum comprised of 2, 6-Bis(1,1-dimethylethyl)-4-(1 -oxopropyl) phenol; 1 , 6, -Dioxacyclododecane-7, 12-dione and 2,3,-dihydro-1,1-dimethyl-6-tert-butyl-1 H indene-4-acetic acid (Table 2)
  • Muscodor kashayum is entirely different from other Muscodor species.
  • the signature compounds produced by other species of Muscodor comprise of 2-methylpropanoic acid; Azuelene, Naphthalene and its derivatives, Thujopsene etc.
  • the VOC’s produced by Muscodor kashayum are unique but are not reported previously by other reported species of Muscodor till date.
  • Table 2 Major volatiles produced by Muscodor kashayum after 10 days of incubation using solid phase micro-extraction (SPME) fibre for GC-MS analysis.
  • SPME solid phase micro-extraction
  • the volatile exhibiting the broad-spectrum antimicrobial activity are induced by the novel biosubstrate and bio-formulation which are released naturally. So, the biologically designed biosubstrate is the driver for production of these compounds. When this organism is grown on a regular culture plate then the spectrum changes, which affects the antimicrobial activity of the volatiles so produced.
  • the inventors did a very preliminary study on some compounds which were available with the inventors under in vitro conditions, to ascertain their activity. The inventors found that though the VOCs are active but not as potential as the naturally produced admixture.
  • the inventors entrapped the VOC’s through a solid phase microextraction needle (SPME) and then went for a GC-MS/MS analysis to identify the compounds based on NIST Library.
  • SPME solid phase microextraction needle
  • Example 8 Bio-formulation of Muscodor kashayum sp. nov. and its activity as a post-harvest preservative/sanitizer to enhance the shelf life
  • Muscodor kashayum is grown in bulk in a 5-liter flask comprising of 3 liters of fresh potato dextrose broth (pH 3-7).
  • the inoculated flask is then incubated at between 23-30 °C (optimal 26 ⁇ 2 °C), 120 rpm (for agitation as well as aeration), 12 hours photoperiod for a period of 05-14 days.
  • the highest biomass is preferably achieved on 10 th day
  • the sterile composite substrates were generally composed of combinations of different agricultural materials (waste and non-wastes in 1 : 1 ratio) such as rye grain: rice husk, rice husk: wood powder; rye grain: wood powder; wheat grain: rye grain; wheat grain: rice husk; wheat grain: wood powder).
  • the formulation is further incubated between 24 to 28 °C for a period of 6-12 days, preferably for 8 days.
  • Example 9 Activity of M. kashayum formulation on Strawberries
  • the concentration was adjusted to 1x 10 4 spores/ml using a hemocytometer (Mercier and Jimenez, 2004). Subsequently fruits were infected with Botrytis cineria by creating a lesion in the equator of Strawberries with a sterile nail of 4mm tip. Then 10 ⁇ l of spore suspension of B. cineria was dropped either 24 hrs before (pre-inoculation) or immediately before the experiment. Then these were placed in an 8 L plastic boxes with wounded side up. 8-10 strawberries were kept in a row inside the box i.e. there was two rows per box. (i.e. 16-20 strawberries/ box). There were three such boxes per treatment.
  • spiked i.e. inoculated with grey mold were also tested for their perishability in presence and absence of VOC’s of M. kashayum.
  • the infection developed rapidly by the 7th day and complete deterioration occurred by 14th day and decay happened by 21st day. While in the presence of the VOC producing formulation of M. kashayum no infection, death or decay was observed till 21st day (Table 3).
  • the present invention provides a non-residual technology which does not have any residual effects and standardized as per existing food safety standards.
  • the present invention discloses a method of using naturally produced volatile organic compounds which is least energy intensive, cost effective and does not require any infrastructural setup to sanitize post-harvest products.
  • the present invention also provides a method that does not require special skill set and training for end user application as compared to other physical and chemical technologies identified in the prior art.
  • bio-substrate of the invention as well as the bio-formulation is eco-friendly and easy to use.
  • bio-substrate of the invention as well as the bio-formulation are cost effective and does not require any infrastructural setup to sanitize post-harvest products.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Pest Control & Pesticides (AREA)
  • Environmental Sciences (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne une nouvelle bio-formulation comprenant le bio-substrat et un nouveau champignon qui produit un spectre unique de composés volatils possédant une activité à large spectre très potentielle. Les nouveaux champignons sont de type Muscodor kashayum #16AMLWLS. La bio-formulation de l'invention présente une activité antimicrobienne et inhibe les bactéries, les champignons et les levures, présentant ainsi une activité antimicrobienne à large spectre. En résumé, la bio-formulation de l'invention est de nature "bactéricide", "fongicide" et "anti-candida". La bio-formulation de l'invention est une composition synergique qui est capable d'éradiquer ou de réduire radicalement le nombre de micro-organismes d'altération et pathogènes habitant ou infectant les fruits et légumes, les céréales, les légumineuses, les produits laitiers (tels que les œufs), les aliments cuits non consommés, la viande crue, le poisson, etc., empêchant ainsi leur décomposition et prolongeant leur durée de conservation. L'invention concerne également un bio-substrat contribuant à la production du spectre unique de composés organiques volatils possédant une très forte activité potentielle à large spectre. La nouvelle souche fongique de l'invention a été déposée à l'IMTECH, Chandigarh. Le numéro de dépôt de la souche est MTCC-25403.
PCT/IN2021/050941 2020-09-25 2021-09-27 Bio-formulation pour la production de composés organiques volatils pour améliorer la durée de conservation de produits de post-récolte Ceased WO2022064527A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202011041628 2020-09-25
IN202011041628 2020-09-25

Publications (1)

Publication Number Publication Date
WO2022064527A1 true WO2022064527A1 (fr) 2022-03-31

Family

ID=80845593

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2021/050941 Ceased WO2022064527A1 (fr) 2020-09-25 2021-09-27 Bio-formulation pour la production de composés organiques volatils pour améliorer la durée de conservation de produits de post-récolte

Country Status (1)

Country Link
WO (1) WO2022064527A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110156528A (zh) * 2018-03-21 2019-08-23 张路 一种利用农牧业废弃物制成生物有机碳基肥及其方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110156528A (zh) * 2018-03-21 2019-08-23 张路 一种利用农牧业废弃物制成生物有机碳基肥及其方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MESHRAM VINEET, KAPOOR NEHA, SAXENA SANJAI: "Muscodor kashayum sp. nov. – a new volatile anti-microbial producing endophytic fungus", MYCOLOGY, vol. 4, no. 4, 1 December 2013 (2013-12-01), pages 196 - 204, XP055922393, ISSN: 2150-1203, DOI: 10.1080/21501203.2013.877990 *
STROBEL, GARY: "Muscodor albus and its biological promise", JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY, vol. 33, no. 7, 2006, pages 514, XP019388171, DOI: 10.1007/s10295-006-0090-7 *

Similar Documents

Publication Publication Date Title
Dukare et al. Exploitation of microbial antagonists for the control of postharvest diseases of fruits: a review
Ramos-García et al. Use of chitosan-based edible coatings in combination with other natural compounds, to control Rhizopus stolonifer and Escherichia coli DH5α in fresh tomatoes
Dukare et al. Non-fungicides-based promising technologies for managing post-production penicillium induced spoilage in horticultural commodities: a comprehensive review
Zhang et al. Control of ochratoxin A-producing fungi in grape berry by microbial antagonists: A review
Ekpenyong et al. Use of Cymbopogon citratus essential oil in food preservation: Recent advances and future perspectives
Nicosia et al. Control of postharvest fungal rots on citrus fruit and sweet cherries using a pomegranate peel extract
Tzortzakis Impact of cinnamon oil-enrichment on microbial spoilage of fresh produce
Enyiukwu et al. Efficacy of plant-derived pesticides in the control of myco-induced postharvest rots of tubers and agricultural products: A review
Zhang et al. Antifungal activity of trans‐2‐hexenal against Penicillium cyclopium by a membrane damage mechanism
CN103621621B (zh) 一种复配生物防腐保鲜剂及其使用方法
JP2004528030A (ja) 農産物の損傷を制御するのに有用な新規の拮抗酵母、その使用方法及びそれを含む組成物
JP2004528030A5 (fr)
Qadri et al. Conventional and modern technologies for the management of post-harvest diseases
Lamenew et al. Biocontrol potential of Trichoderma and yeast against post harvest fruit fungal diseases: A review
Suasa-ard et al. A novel biological control agent against postharvest mango disease caused by Lasiodioplodia theobromae
Raynaldo et al. Biological control and other alternatives to chemical fungicides in controlling postharvest disease of fruits caused by Alternaria alternata and Botrytis cinerea
Sharma et al. Bio-management of postharvest diseases and Mycotoxigenic Fungi
Šaric et al. Potential of lactic acid bacteria in biocontrol of Aspergillus niger, Penicillium chrysogenum and Fusarium graminearum in culture media and natural substrate.
Al-Ansary et al. Evaluation of antifungal activity of some natural essential oils against fungal pathogens associated with maize grains.
WO2022064527A1 (fr) Bio-formulation pour la production de composés organiques volatils pour améliorer la durée de conservation de produits de post-récolte
Hamza et al. New Trends for Biological and Non-biological Control of Tomato Root Rot, Caused by Fusarium solani, under Greenhouse Conditions.
Kena Antifungal activities of Monsonia burkeana and Euphorbia ingens extracts against Penicillium digitatum, the causal agent of citrus green mould
Sriram et al. Volatile Organic Compounds Produced by Microbes in the
Mrvová et al. In vitro antifungal effect of twelve essential oils on Penicillium expansum growth
Rani et al. Management of postharvest blue mould of apple caused by Penicillium fuscoglaucum using a gel formulation containing Monarda citriodora essential oil and linalool

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21871861

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21871861

Country of ref document: EP

Kind code of ref document: A1