Title of Invention: A Microbial Sanitiser with Broad Applications

Technical Field

THIS INVENTION relates to sanitisers, food preservatives, probiotics, insecticides and composting inoculants. More particularly, this invention relates to the discovery of an isolate of Lactococcus lactis bacteria that was further enhanced through serial sub-culturing under specified laboratory conditions, creating a derivative mutant isolate that presents improved properties and molecular components thereof, for use as a sanitiser, food preservative, probiotic, insecticide and/or a composting inoculant.

Background Art

Background to Problem 1

Many foods, particularly fruits, vegetables, meat, seafood, oils and products produced from these may have a limited“shelf-life” as a result of microbial contamination, ultimately leading to oxidation, deterioration and/or spoilage. This can make these foods unsafe for human consumption, or at least diminish the attractiveness of these foods to potential consumers. It is well acknowledged that foods such as fruits, vegetables, meats and seafood having a fresh and appealing appearance are far more likely to be chosen for purchase by consumers. Also, outbreaks due to microbial contamination of foods, such as Listeria contamination of fruits and Salmonella contamination of eggs among others, can have fatal health consequences while also devastating the livelihoods of primary producers.

Accordingly, foods may be subjected to treatment with chemicals, sanitisers, antioxidants or preservative agents that promote decontamination and ultimately enhance appearance and/or improve shelf-life, eitherwhen used alone or in combination with each other. However, consumers have become concerned about the potentially adverse health and environmental effects, or lack of consistent function and/or direct health benefits of chemicals and other agents used in food decontamination and/or shelf-life extension.

Background to Problem 2

Furthermore, challenges posed by fruit flies and other invasive insects threaten Australia’s horticultural capacity and its ability to trade in domestic and international horticultural markets. National fruit fly research groups have developed sterile insects, invertebrate-based biological control, and chemicals/attractants for use in traps and sprays. International research & development is also testing molecular methods to modify insects' biology (i.e. CRISPR technology), or developing traps that contain movement sensors. Despite all efforts, insects are remarkable at rapid adaption, evasion and evolution of survival mechanisms that enable their reproduction. In addition, some of the research that has been conducted is impracticable in the field, as implementation costs surpass their affordability. Similarly, Additionally, challenges posed by invasive insects cost our society approximately US$70 billion globally, due to 1) infrastructure damage, 2) crop destruction and 3) public health issues. Pest control promoted by chemicals and/or predators can lead to ecological disasters due to environmental selection. Finally, to our knowledge no organic-certified, safe and low- cost methods exist that support simultaneously both organic and conventional agriculture, while preventing infections and agricultural infestation caused by various insects.

Background to Problem 3

In addition to all this, organic waste management is another significant global issue, as landfills are full or decomposing waste constantly generating methane due to microbial activity. This greenhouse gas when released into the atmosphere is more potent than carbon dioxide. However, when organic waste is treated to make compost, fertilisers and/or soil conditioning products, it can be transformed in a nutrient-rich resource that can be used for plant growth and ultimately to increase global food production.

Summary of Invention

General summary of the enhanced microbe, and of how the invention addresses Problem 1

The invention provides an isolate of Lactococcus lactis bacterium that has particularly useful properties as an ingredient in the elaboration of a food sanitiser that ultimately extends food shelf-life. The Lactococcus lactis bacterium was originally isolated from a mixture of fox milk and mammary tissues, and subjected to a selection process that produced a Lactococcus lactis bacterium different from the native isolate, without using any gene-specific genetic manipulation, and which led to the new derivative isolate having enhanced antimicrobial and/or antioxidant and/or biofilm formation properties that facilitate food sanitation and preservation. The Lactococcus lactis bacterium may also confer health-promoting probiotic effects on foods treated with the bacterium. The Lactococcus lactis bacterium may also confer health-promoting effects in skincare, mucosal tissue repair, skin regeneration, wound healing, probiotic effect, and other applications comprising the bacterium.

A first aspect of the invention therefore provides a native and parental isolate Lactococcus lactis 2016QLDWLFx bacterium (also known as 16QF), or one or more molecular components thereof.

A second aspect of the invention provides a method of producing an enhanced Lactococcus lactis bacterium, said method including the steps of obtaining one or more Lactococcus lactis bacteria from a mixture of fox milk and mammary tissues, sub-culturing in an manner that uses natural selection to induce adaptation to environmental cues that lead to enhancement of the isolate’s phenotypic traits, and selection of a new Lactococcus lactis bacterium that presents enhancement of one or more desired properties, and which is different from the native and parental isolate and is therefore designated 16QF-e.

A third aspect of the invention provides an enhanced Lactococcus lactis bacterium, or one or more molecular components thereof, produced by the method of the second aspect.

A fourth aspect of the invention provides a composition comprising an enhanced Lactococcus lactis bacterium according to the second or third aspects, or one or more molecular components thereof. A fifth aspect of the invention provides a method of at least partly sanitising and preserving foods, said method including the step of contacting the food with the enhanced Lactococcus lactis bacterium of the second aspect or the third aspect, or one or more molecular components thereof, or with the composition of the fourth aspect to thereby at least partly sanitise and preserve the food.

A sixth aspect of the invention provides a method of stimulating, enhancing and/or improving one or more probiotic properties of a food, including the step of contacting the food with the enhanced Lactococcus lactis bacterium of the second aspect or the third aspect or one or more molecular components thereof, or with the composition of the fourth aspect, to thereby at least partly preserve the food and stimulate, enhance and/or improve one or more probiotic properties of the food.

A seventh aspect of the invention provides a method of treating skin, hair, intestines and/or any other mucosal tissues including the step of administering a composition comprising the enhanced Lactococcus lactis bacterium of the second aspect or the third aspect or one or more molecular components thereof, or with the composition of the fourth aspect, to the skin, hair, intestines and/or any other mucosal tissue.

In an embodiment of the aforementioned aspects, the enhanced Lactococcus lactis bacterium is Lactococcus lactis 16QF-e, which may be characterised by the partial genome sequence and by the protein-coding nucleotide sequences set forth in Table 1 , which constitute the unique portion of the genome of Lactococcus lactis 16QF-e.

A further aspect of the invention provides an isolated nucleic acid set forth in Table 1 , or a variant or fragment thereof.

Another further aspect of the invention provides an isolated protein encoded by an isolated nucleic acid set forth in T able 1 , or a variant or fragment thereof.

Related aspects of the invention provide a genetic construct comprising one or more isolated nucleic acids of the aforementioned aspect, and/or a host cell comprising the genetic construct.

Throughout this specification, unless otherwise indicated, “comprise”, “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

It will also be appreciated that the indefinite articles“a” and“an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example,“a” bacterium includes one bacterium, one or more bacteria or a plurality of bacteria.

Summary of how the invention addresses Problem 2

A further aspect relates to the use of isolate 16QF-e of Lactococcus lactis bacterium of the second aspect or the third aspect, or one or more molecular components thereof, or with the composition of the fourth aspect as an ingredient in the elaboration of insecticides, pesticides, repellents and/or larvicides that at least reduce and ultimately prevent the infestation, consumption, destruction, damage, degradation and/or colonisation of fruits, vegetables, seeds, seedlings, plants (or parts of all these) or any surfaces by eggs, larvae, juvenile stages and/or adults of fruit flies, moths, aphids, weevils, earwigs and any other insects.

Another aspect relates to the use of isolate 16QF-e of Lactococcus lactis bacterium of the second aspect or the third aspect, or one or more molecular components thereof, or with the composition of the fourth aspect as an ingredient in the elaboration of antioxidants for preharvest application, which slow and/or decelerate the maturation of fruits and vegetables and/or reduce their ripening pace, including when also used as an ingredient in the elaboration of insecticides, pesticides, repellents and/or larvicides.

A further aspect of the invention provides a method of at least partly protecting fruits, vegetables, seeds, seedlings, plants (or parts of all these) or any surfaces against all life stages of any insects, and/or at least slowing and/or decelerating their ripening pace, said method including the step of contacting fruits, vegetables, seeds, seedlings, plants (or parts of all these) or any surfaces with the enhanced Lactococcus lactis bacterium of the second aspect or the third aspect, or one or more molecular components thereof, or with the composition of the fourth aspect.

Summary of how the invention addresses Problem 3

A further aspect relates to the use of isolate 16QF-e of Lactococcus lactis bacterium of the second aspect or the third aspect, or one or more molecular components thereof, or with the composition of the fourth aspect as an ingredient in the elaboration of inoculants and/or products for digestion, composting, degradation, deterioration, reduction, recycling, conversion, transformation, break down and/or pasteurisation of foods, food waste, food scraps, paper, paper waste, cellulose, cellulose waste, gardening waste, agricultural waste, livestock-derived waste, and/or any other waste characterised as organic or green, including when these come into contact with chemicals and/or synthetic compounds, ultimately leading to and/or contributing towards an outcome, by-product and/or end-product of the referred processes, while promoting, contributing to and/or assisting with sanitation.

Brief Description of Drawings

Fig. 1

[fig. 1] provides an overview of isolation of Lactococcus lactis bacterium 16QF, and selection of enhanced isolate 16QF-e.

Fig. 2

[fig. 2] compares Lactococcus lactis bacterium 16QF-e full genome sequence to its closest Lactococcus lactis relative, whose full genome sequence was retrieved from a public non- redundant nucleotide database. Predicted genes and coding sequences, including those present within the two large unique genomic insertions (arrows) were further aligned to a public database of global nucleotide sequences (NT), and the unique coding sequences present within the two large unique genomic insertions were used to elaborate Table 1. In silico translated amino acid sequences (not shown) were also aligned to a public non- redundant protein database available at GenBank using BlastP (Camacho et al., 2009), and in both cases results were filtered using an e-threshold of 1e-5.

Fig. 3

[fig. 3] is a growth kinetics in different manufacturing media. The media that may or may not be used for manufacturing of Lactococcus lactis 16QF-e include CBA (Whey protein concentrate ranging from 3 to 5%), F2 (powder milk ranging from 3 to 5%), F3 (powder milk ranging from 3 to 5% supplemented with sucrose ranging from 0.25 to 5%) and F4 (powder milk ranging from 3 to 5% supplemented with a mix of beef heart, sodium chloride and tryptose ranging from 0.05 to 0.5%).

Fig. 4

[fig. 4] presents the increased shelf life of strawberries, raspberries and Flabanero Chilli treated with Lactococcus lactis 16QF-e.

Fig. 5

[fig. 5] shows protection of canola oil against rancidity and oxidation by treatment with Lactococcus lactis 16QF-e. A Rancimat test was used to determine the prevention of rancidity of canola oil by isolates 1 and 2. Of note, isolate 1 corresponds to the initial Lactococcus lactis bacterium isolate that was directly recovered from a mixture of fox milk and mammary tissues sub-cultured in culture media only (native and parental), and isolate 2 corresponds to Lactococcus lactis 16QF-e that was obtained after its serial sub-culturing within porous membrane bags, in growth media supplemented with a multidrug resistant isolate of Staphylococcus aureus, aimed to create specific selection pressure that led to adaptation, enhancement of biological traits, and prompt response of isolate 16QF-e to the presence of Gram-positive pathogens (and probably also Gram-negative pathogenic microbes) (see FIG. 1 Step 3).

Fig. 6

[fig. 6] summarizes the mechanisms and properties underlying sanitation of fruits, foods in general and surfaces by Lactococcus lactis 16QF-e (6A-B). Antimicrobial action such as the one observed against Staphylococcus aureus resistant to oxacillin and methicillin (black arrow, 6C) led to sanitation of raw chicken minced meat and significant plate count reduction by day 3 post-best by date (6D). The same was observed among premium minced beef that was treated with product containing 16QF-e, which presented 15% less microbial contamination and extended shelf life in relation to untreated control in an independent laboratory analysis (6E-F).

Fig. 7

[fig. 7] demonstrates killing of QLD fruit fly adults (7A), and delay in pupation rates promoted by Lactococcus lactis 16QF-e during laboratory tests (7B). Results were separated per gender whenever possible, and formed the basis for independently conducted qualitative and quantitative field trials that confirmed the success of 16QF-e as a fruit fly pesticide ingredient (7C). White arrows point at infestation occurring in an untreated peach.

Fig. 8 [fig. 8] uses Lactococcus lactis 16QF-e as an inoculant for composting of food waste within a composter machine at 50°C, and confirms that Lactococcus lactis 16QF-e kills pathogens, persists into the chamber (responding for improved composting outcome), and assists the achievement of complete sanitation during the process.

Description of Embodiments

The present disclosure relates to a newly discovered and unique isolate of Lactococcus lactis originally obtained from fox milk and mammary tissues, which was initially found to sanitise foods such as fruits, vegetables, leaves, herbs, roots, cut produce, meats, seafood, oils, fats, dips, sauces and pre-harvest produce types ultimately protecting against pests and diseases, and extending and enhancing their preservation and/or shelf life. Preferably, the Lactococcus lactis isolate is used in a liquid composition for topical, penetrable and/or injectable application to foods. It is proposed that the antimicrobial and/or antioxidant properties of the Lactococcus lactis isolate at least partly contribute to its ability to sanitise foods, and as a result extend and enhance the preservation and/or shelf life of such foods. The Lactococcus lactis isolate appears to be about 1.85% different from all other L lactis isolate nucleotide sequences deposited on GenBank, at the DNA level. These genetic differences were found to be particularly present in two unique genomic islands which contain genes that appear to be absent from other L. lactis isolates, and to a minor extent were found as point mutations scattered across the L lactis 16QF-e genome based on genome alignment analyses. The Lactococcus lactis bacterium disclosed herein may also confer health-promoting probiotic effects to individuals ingesting foods treated with the bacterium, as Lactococcus lactis isolates are notorious for lactose reduction in foods.

The present disclosure also relates to the use of isolate 16QF-e of L. lactis bacterium as an ingredient in the elaboration of larvicides, pesticides and/or insecticides that were found to at least reduce infestation, consumption, destruction, damage, degradation and/or colonisation, and at least partly protect fruits, vegetables, seeds, seedlings, plants (or parts of all these) or any surfaces against eggs, larvae, juvenile stages and/or adult fruit flies, moths, aphids, weevils, earwigs and any other insects. Preferably, the Lactococcus lactis isolate is used in a liquid composition for topical, penetrable and/or injectable application to the produce as a product to be applied pre-harvest. It is proposed that the antioxidant properties of the Lactococcus lactis isolate at least partly contribute to its insecticide, larvicide and/or pesticide ability, and as a result at least disorientate, repel, inhibit and/or kill invertebrates.

Furthermore, the present invention also relates to the use of L lactis isolate 16QF-e as an ingredient in the elaboration of inoculants and/or products that were found to digest, compost, degrade, deteriorate, reduce, recycle, convert, transform, break down, pasteurise and/or sanitise foods, food waste, food scraps, paper, paper waste, cellulose, cellulose waste, gardening waste, agricultural waste, livestock-derived waste and/or any other waste characterised as organic or green in a compost, soil conditioner, fine powder and/or a fertiliser with high nutritional value, including when this comes into contact with chemicals and/or synthetic compounds, and when 16QF-e is used as an inoculant, product and/or additive in a composting machine at 50-55°C, or in any other composting systems such as rows, piles, holes and others. Preferably, the Lactococcus lactis 16QF-e isolate is used in a liquid composition for superficial application onto waste material that was deposited into chambers, tanks, reactors, composters, units, digestors, piles and/or rows. It is proposed that the enzymatic activity and/or the biofilm formation properties of the Lactococcus lactis 16QF- e isolate at least partly contribute to its composting and/or waste degradation ability.

For the purposes of this invention, by“isolated” is meant material (e.g. a bacterium or a molecular component thereof) that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated molecules may be in native, chemical synthetic or recombinant form. By “enriched” or “purified” is meant isolated material having a higher incidence, representation or frequency in a particular state, or free of, or separated from most unspecific molecules not desired for particular applications (e.g. an enriched or purified state) compared to a previous state prior to enrichment or purification.

The isolated L. lactis bacterium was produced by isolating L lactis bacteria from the milk of a fox ( Vulpes vulpes), which was followed by its sub-culturing under specified conditions using a method that simulates realistic environmental selection pressure, and which led to its adaptation and ultimate selection of L. lactis bacteria having one or more enhanced properties for food sanitation and preservation, produce and surface protection against diseases and insects, and waste composting and transformation into a pathogen-free fertiliser. A more detailed description of the isolation and selection of the L lactis bacterium is provided in the Examples and is summarised schematically in FIG. 1. A particular embodiment of the isolated Lactococcus lactis bacterium produced according to this method is designated “Lactococcus lactis 16QF-e”. Although not wishing to be bound by any particular theory, it is proposed that the isolated L lactis bacterium has enhanced antimicrobial, antioxidant, biofilm formation and/or enzymatic properties that are particularly advantageous for all aforementioned applications. The isolated L. lactis bacterium also has health-promoting probiotic properties.

Accordingly, the isolated L. lactis bacterium disclosed herein is particularly useful for the sanitation and preservation of food, food items, food products and/or surfaces. As used herein, terms such as“food”,“food items” and“food products” relate to any items of nutritional value or content that may be eaten by mammals such as humans. Typically, in this context the food is perishable, susceptible to spoilage, microbial contamination, oxidation, deterioration in quality and/or of limited shelf-life. Non-limiting examples of foods are fruits, vegetables, cut produce, vines, leaves, herbs, roots, meats, seafood, oils, fats, dips, sauces and/or edible or otherwise consumable products containing, processed, derived or made from one or more of the aforementioned foods and/or combinations thereof, including beverages such as wines and fermented drinks.

In this context“sanitation” refers to at least partly reducing, preventing or inhibiting microbial presence and/or food or surface contamination and/or their microbial colonisation, while “preservation” and“preservative” refer to at least partly reducing, preventing or inhibiting the spoilage, deterioration in quality, microbial contamination, oxidation and/or the limited shelf- life of perishable food, food items and/or food products.

For the purposes of food and surface sanitation and food preservation, the L lactis 16QF-e bacterium disclosed herein may be provided“intact” or otherwise as a viable, living bacterium or as an extract, fraction, preparation or isolate comprising one or more molecular components of the L. lactis bacterium. These molecular components may include proteins, nucleic acids, lipids, carbohydrates, lipoproteins, proteoglycans, glycolipids, metabolites or other metabolic by-products and/or other molecules produced by, or components of, the L lactis bacterium disclosed herein. By way of example, metabolites may include antimicrobial metabolites such as phenylacetic acid and its derivatives, hydroxyphenylacetic acid and its derivatives, hydroxyphenylacetic acid and its derivatives, hydroxypropanaldehyde and its derivatives, propandiol and its derivatives, hydrogen peroxide, ethanol, acetic acid, carbon dioxide, carbonic acid, propanoic acid, butyric acid, cyclic dipeptides, cyclic compounds, aromatic compounds, hydroxydecanoic acid and its derivatives, hydroxydodecanoic acid and its derivatives, and hydroxytetradecanoic acid and its derivatives.

Other metabolites or molecular components may include at least one bacteriocin selected from a lantibiotic, a lacticin, a salivaricin, streptin, a Streptococcin, a mutacin, a bovicin, a macedocin, a plantaricin, a lactocin, a cytolysin, a enterocin, a divercin a bavaricin, a coagulin, a pediocin, a mundticin, a piscicocin, a sakacin, a leucocin, a avicin, a ubericin, a mesentericin, a curvacin, a carnobacteriocin, a penocin and/or a hiracin.

As will be described in more detail hereinafter, the L. lactis 16QF-e bacterium disclosed herein comprises a unique genome that may provide molecular components (e.g., encoded proteins, metabolites) that are unique to the L. lactis bacterium disclosed herein. These molecular components may provide enhanced food sanitation and/or preservation properties whether present in intact 16QF-e bacteria that was cultured and/or added to foods either alone or in combination with other ingredients and/or microorganisms, or when provided as an extract, isolate or other fraction obtained from intact 16QF-e bacteria that was cultured and/or added to foods either alone or in combination with other ingredients and/or microbial extracts. In other embodiments, the molecular components may be provided in a synthetic form, such as produced by recombinant DNA technology or in chemical synthetic form.

More particularly, Lactococcus lactis 16QF-e has been subjected to genetic analysis to determine the genetic“fingerprint” of this L lactis isolate by comparing its genomic DNA sequence with that of other prior art L lactis isolates. The unique genome sequence is shown in Table 1. Distinct genetic regions were located in a single contig, and they appear to contain 232,157 bp of novel sequence (169,298 bp and 62,859 bp, respectively; FIG. 2), and a total of 238 coding sequences were found across both insertions (Table 1). Mobile element proteins were also found across the insertions, suggesting a transposon-related origin. Additionally, several smaller differences such as point mutations can be found across the genome, although the most significant appear to be the two aforementioned insertions.

Accordingly, a further aspect of the invention provides an isolated nucleic acid comprising a nucleotide sequence set forth in Table 1 , or a fragment or variant thereof, or an isolated protein encoded by a nucleotide sequence set forth in Table 1 , or a variant or fragment thereof. In a particular embodiment, the isolated nucleic acid comprises a nucleotide sequence contained within the distinct genetic regions referred to above (FIG. 2; Table 1).

By“protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.

The term“protein” includes and encompasses“peptide”, which is typically used to describe a protein having no more than fifty (50) amino acids and“polypeptide”, which is typically used to describe a protein having more than fifty (50) amino acids.

The term“nucleic acid” as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto. Reference to a nucleotide sequence also includes a complementary nucleotide sequence and both possible orientations (i.e. 5‘ to 3’ and 3’ to 5’).

A“gene” is a structural unit or region of a genome that comprises a nucleotide sequence encoding an amino acid sequence of a protein. Typically, a bacterial gene comprises a single coding sequence that encodes a protein and non-coding genetic elements such as regulatory regions including a promoter, operator and/or enhancer.

By“variant” in the context of an isolated protein is meant a protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 90% or 95% identical to an amino acid sequence of an isolated protein encoded by a nucleotide sequence set forth in Table 1.

By“variant” in the context of an isolated nucleic acid is meant an isolated nucleic acid having a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to a nucleotide sequence set forth in Table 1.

The term“fragment” refers to a portion, domain, sub-sequence or region of an isolated protein or isolated nucleic acid that constitutes no more than 99%, 95%, 90%, 85%, 80%,, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the entire amino acid sequence of the isolated protein or the entire nucleotide sequence of the isolated nucleic acid.

Non-limiting examples of variants include natural variants such as orthologues, homologues and/or allelic variants and/or artificial variants such as produced by in vitro mutagenesis, although without limitation thereto.

A still further aspect of the invention provides a genetic construct comprising an isolated nucleic acid comprising a nucleotide sequence set forth in set forth in Table 1.

Suitably, the genetic construct is an“expression construct” wherein the isolated nucleic acid is operably linked or operably connected to one or more additional nucleotide sequences that control, facilitate or regulate expression of the isolated nucleic acid. By“operably linked” is meant that said additional nucleotide sequence(s) is/are positioned relative to the isolated nucleic acid preferably to initiate, regulate or otherwise control transcription of the isolated nucleic acid. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, origins of replication, polyadenylation sequences, transcriptional start and termination sequences, donor/acceptor splice sites, Kozak and translational start and termination sequences, and/or enhancer or activator sequences. The choice of said one or more regulatory nucleotide sequences may be at least partly dependent on the host cell type used for expression, particularly according to the origin of the host cell Broadly, the genetic construct may be in the form of, or comprise genetic components of, a plasmid, a transposon, a bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. The genetic construct may be either a self-replicating extra-chromosomal construct such as a plasmid, or more preferably a construct that integrates into a host cell genome.

Another further aspect of the invention provides a host cell comprising the aforementioned genetic construct. The host cell may be a bacterial host cell, mammalian host cell, a yeast host cell, a plant host cell and/or an insect host cell, although without limitation thereto. Host cells comprising the aforementioned genetic construct may be produced by any gene transfer method known in the art such as electroporation, heat shock, liposome-mediated gene transfer, although without limitation thereto.

The isolated L. lactis bacterium of the invention may be produced under culture conditions where the pH is between about 5.5 and about 8.5 or preferably about pH 6-8, whereby the preferred nutrient medium could be any among CBA (Whey protein concentrate ranging from 3 to 5%), F2 (powdered milk ranging from 3 to 5%), F3 (powdered milk ranging from 3 to 5% supplemented with sucrose ranging from 0.25 to 5%) and F4 (powdered milk ranging from 3 to 5% supplemented with a mix of beef heart, sodium chloride and tryptose ranging from 0.05 to 0.5%). A typical temperature is about 30°C. Preferably, the dissolved oxygen level is between about 15 and about 90 mM. Preferably, the concentration of the resulting bacterial culture is between about 1x10 ⁷ cfu/gram of said bacterial culture, and about 1x10 ¹⁰ cfu/gram of said bacterial culture. L lactis bacteria produced under these conditions may have a shelf- life of between about 6 months and 12 months when stored with moisture content of about 80%, and/or at least about 12 months when stored with a moisture content below about 20%.

By way of example, the L. lactis 16QF-e bacterium disclosed herein may be provided as a suspension in liquid form, or in a frozen, dried or dehydrated form (such as freeze dried, spray dried, vacuum dried, lyophilized or powdered) form.

In some embodiments, the L. lactis bacterium disclosed herein may be provided as a concentrate for subsequent dilution before delivery and/or use. A concentrate may be produced by sedimentation, centrifugation, aspiration, decantation, drying, freeze drying, spray drying, vacuum drying and/or evaporation, although without limitation. By way of example only, the concentrate may comprise L lactis bacteria at a concentration whereby 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10-fold-dilutions produce a“working” concentration suitable for food preservation. A preferred“working” concentration of L lactis bacterium disclosed herein is from 1x10 ⁴ cfu/gram to about 1x10 ⁸ cfu/gram of composition.

Typically, the isolated L lactis bacterium is utilised as a liquid composition. Suitably, the composition may be suitable for topical administration to surfaces, food, food item or food products. In some particular embodiments, the composition may be capable of penetrating an outer surface or layer of the food to thereby infiltrate or permeate the food. In other embodiments, the composition may be injectable into a surface, food, food item or food product.

The composition may comprise a carrier or diluent which facilitates delivery of the L. lactis bacterium disclosed herein, or one or more molecular components thereof. Non-limiting examples of carriers or diluents include one or a combination of water, solvents, buffers, salt solutions, water, acids such as acetic acid, flavonoids, bacterial culture media or components thereof, gelling and/or polymerizing agents, although without limitation thereto.

In some embodiments, gelling or polymerizing agents may include at least one member selected from a group comprising, but not limited to, gelatine, collagen and/or agar. Typically, the polymerizing or gelling agent may be 10%, 9%, 8%, 7%, 6%, 5% or less by weight of said composition.

The composition may further comprise at least one of a flavour enhancer, palatant, stabilizing agent, emulsifying agent, food coating stabilizer, fragrance, binder, colour, chemical preservative, colouring agent, chemical antimicrobial, aromatic molecule and/or antioxidant. However, it is preferred that the composition does not include exogenous or additional bactericides and/or antibiotics and/or bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties.

The composition may be delivered or applied to food, food items, food surfaces, general surfaces or food products by spraying, brushing, coating, immersing, dipping, soaking, dusting, injection, impregnation, smearing or any other delivery process that facilitates delivery of the enhanced L. lactis bacterium disclosed herein, or one or more molecular components thereof, in a manner which achieves at least partial sanitation, preservation, protection and/or coverage of a surface, food, food items and/or food products. Application rates of products containing the enhanced L. lactis bacterium for use in sanitation, preservation, protection and/or antioxidation may be in the range 1-100L per 150 kg food, food item or food product, preferably in the range 2-50L per 150 kg, more preferably in the range 5-20L per 150 kg or advantageously about 10L per 150 kg. Application rates of products containing the L. lactis bacterium for use in composting may be in the range 1 L-10L per 1 ,000 kg food waste or waste product.

In an embodiment, the food is a plant, plant part, derived from a plant, animal, animal part, derived from an animal and/or derived from cells grown in a laboratory. Suitably, the food is, comprises or is obtained from a leaf, fruit, vegetable, flower, tuber, root, seed, stem of a plant, or in relation to animal material food is, comprises or is obtained from muscle, tissue, organ, cells.

In some embodiments the plant may be a“crop species”. As used herein, the term“crop species” includes, but is not limited to, Abelmoschus esculentus (okra), Allium sativum (garlic), Allium spp. (onions), Anacardium spp. (cashew), Ananas comosus (pineapple), Anethum graveolens (dill), Arachis hypogaea (peanut), Azadirachta indica (neem), berries such as strawberries, raspberries and blueberries although without limitation thereto, Beta vulgaris (sugar beet), bok choy, Brassica napus (rapeseed), Brassica oleracea (cabbage, broccoli), Brassica pekensis (Wong bok or Chinese cabbage), Camellia sinensis (tea), Capsicum spp., (capsicums and chillies), Carthamus tinctorius (safflower), Carum carvi (caraway), Cassinia spp., Cicer arietinum (chickpea), Daucus carota (carrot), fruits such as apples, pears, citrus (e.g. oranges, lemons etc), Carica spp. (papaya), Diospyros spp. (persimmon), bananas, pineapples and stone fruits (e.g apricots, peaches etc), Glycine max (soybean), Glycine max (vegetable soybean), Hordeum vulgare (barley), Humulus lupulus (hops), Lens spp. (lentil), lettuce, Mentha X piperita (peppermint), Ocimum basilicum (basil), O/ea europaea (olive), Olearia sp., Origanum spp. (marjoram, oregano), Oryza spp. (rice), Phaseolus lunatus (lima bean), Phaseolus spp., Phaseolus vulgaris (culinary bean), Phaseolus vulgaris (navy bean), Phaseolus vulgaris (red kidney bean), Pisum sativum (field pea), Plantago ovata (psyllium), Rosmarinus officinalis (rosemary), Rungia klossii (rungia), Saccharum officinarum (sugar cane), Secale cereale (rye), Sesamum indicum (sesame), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Sorghum almum (sorghum), Triticum spp., (wheat) Vicia faba (faba bean), Vigna radiata (mung bean), Vigna spp., Vigna unguiculata (cowpea), Vitis spp. (grapes), Zea mays (maize), Zea mays (sweet corn), Zingiber officinale (ginger) and Zizania spp. (wild rice).

It will be appreciated that in the context of harvestable plant-derived foods such as fruit and vegetables, the composition may be delivered or applied before or after harvesting the fruit and/or vegetables. In embodiments where the composition is delivered or applied before harvesting, it may be delivered or applied at the rate of about 10L per 10,000 kg, whereby the bacterium concentration may vary between 1x10 ⁴ cfu/gram and about 1x10 ¹⁰ cfu/gram of composition. This may be repeated one, two or more times at intervals of at least one, two or more weeks prior to harvesting the fruits or vegetables.

In embodiments where the composition is delivered or applied to plants, plant material and/or said produce after harvesting, it may be delivered or applied at the rates varying between about 10L per 150 kg and about 10L per 50 Kg, whereby the functional bacterium concentration may vary between 1x10 ⁴ cfu/gram and about 1x10 ⁸ cfu/gram of composition. This is preferably a single treatment that may or may not be repeated depending on the level of pathogen contamination surrounding the treated food or within its environment.

Another aspect of the invention provides a method of enhancing or improving one or more probiotic properties of food, including the step of contacting the food with the enhanced Lactococcus lactis bacterium of the second aspect or the third aspect or one or more molecular components thereof, or with the composition of the fourth aspect, to thereby at least partly sanitise and preserve the food and enhance or improve one or more probiotic properties of the food.

As hereinbefore described, the enhanced Lactococcus lactis bacterium may also confer health-promoting probiotic effects on foods treated with the bacterium. Thus, ingestion of treated foods by a human or an animal may at least partly protect the gut, improve the biology of microvilli, regulate or reduce the concentration of blood glucose, regulate or reduce the concentration of lactose in a food or in the body, regulate or reduce the amount of body fat, resolve or alleviate skin and other allergies and/or positively affect immunomodulatory gene expression.

In one embodiment, the Lactococcus lactis bacterium of the second aspect or the third aspect or one or more molecular components thereof, may be in the form of a cellular extract. The cellular extract may be produced or obtained through any cell disruption or fractionation method including, but not limited to, sonication, mechanical agitation, fragmentation and/or heating of the enhanced Lactococcus lactis bacterium to thereby produce the cellular extract.

It will also be appreciated that according to the abovementioned aspects and embodiments, methods and compositions may include one or more other bacteria or fungi, or molecular components or extracts thereof, in addition to Lactococcus lactis 16QF-e. These may include other probiotic bacteria of genera such as Lactococcus, Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, Bacillus and Proprionibacterium, or fungi of the genera Saccharomyces, Pichia, Pisolithus although without limitation thereto.

A seventh aspect of the invention provides a method of treating skin or mucosal tissues including the step of administering a composition comprising isolated Lactococcus lactis 16QF-e bacteria to the skin or mucosal tissues.

Thus Lactococcus lactis 16QF-e may be formulated as an administrate composition such as a cosmetic, ointment, skincare cream, medication, spray, tablet, pill, powder and/or tonic to improve skin and mucosal health and/or treat or prevent one or more conditions. Such compositions may include carriers or diluents such as glycerine, pH buffers, sugar acids, emulsifiers, emollients, lubricants, colouring agents, glidants, fats and oils, hyaluronic acid, starch and other thickeners, vitamins and/or minerals, although without limitation thereto.

So that the invention may be readily and understood and put into practical effect, reference is made to the following non-limiting examples. Description of Examples

Example 1

Isolation and characterisation of Lactococcus lactis 16QF-e

An overview of the isolation of Lactococcus lactis 16QF-e is shown in FIG. 1. In step 1 , milk and mammary tissue samples were obtained from a fox ( V. vulpes the selection of bacteria from fox samples is part of an on-going program testing for bacteria of potential value in wild animals) and mixed with HI or LB or another agar preparation containing Gram negative and/or Gram positive bacterial extracts (E. coli and Bacillus subtilis, respectively) in a gasket device (FIG. 1 Step 2). These devices were wrapped in porous cellulose or silicone dialysis membranes or tubing of MW3000-10000 and incubated within a milk mixture in a refrigerator to allow microbial growth in our synthetic agar of choice. Four to six weeks later, agar plugs were colonised by fox milk microbes. In step 3, one particular colony was sub-cultured in laboratory medium (LB or HI or BHI or Starch) within membrane bags in growth media supplemented with a multidrug resistant isolate of Staphylococcus aureus, aiming to create specific selection pressure that leads to adaptation and prompt response of 16QF-e to the presence of Gram-positive pathogens (and potentially Gram-negative pathogenic microbes) (see FIG. 1 Step 3). Lactococcus lactis 16QF-e subsequently showed enhanced food sanitation and preservation qualities when sprayed onto tomatoes. DNA extracted from this colony was sequenced, and contigs were assembled.

Assembly: Lactococcus

Total length: 2,545,433

Number of sequences: 100

Average scaffold length: 25,454.30

N50: 616,324

Largest scaffold: 1 ,020,771

The contigs classified as Lactococcus lactis, in our de novo assembly, were aligned to the L lactis reference genome NCBI: NC_002662.1 using the module nucmer for the mummer package v4.0 (Kurtz et al., 2004). The alignment was further filtered and converted into graphical format, revealing two large insertions in isolate 16QF-e which were absent in the reference genome. Both regions were located in a single contig, and they appear to contain 232,157 bp of novel sequence (169,298 bp and 62,859 bp, respectively), and a total of 238 coding sequences across both insertions. Mobile element proteins were also found across the insertions, suggesting a transposon-related origin. Additionally, several smaller differences (point mutations, mismatches, etc) can be found across the genome, although the most significant appear to be the two aforementioned insertions. The whole genome alignment data and the unique genome annotations and sequences are shown in FIG. 2 and Table 1.

Predicted genes and coding sequences, including those present within the two large unique genomic insertions were aligned to a public database of global nucleotide sequences (NT), and the unique coding sequences present within the two large unique genomic insertions were used to elaborate Table 1. In silico translated amino acid sequences (not shown) were aligned to a public non-redundant protein database available at GenBank using BlastP (Camacho et al., 2009), and in both cases results were filtered using an e-threshold of 1e-5.

Example 2

Production of Lactococcus lactis 16QF-e for food sanitation and preservation, and for pesticide and composting purposes

Thaw one frozen aliquot of Lactococcus lactis isolate 16QF-e; • Quality-control the chosen frozen stock by streaking out its content onto an agar plate, and by sub-culturing in commercial liquid media followed by the streaking out of the new culture onto an agar plate;

• Inspect the colonies obtained for satisfactory growth, and for the presence of polymorphic colonies and/or contamination. Select a single colony for further sub-culturing;

• Grow the bacterial cells using growth medium composed of a fluidic portion, sugars, amino acids and lipids at 30°C pH 6-8 within a bioreactor, a fermentation tank and/or laboratory shakers;

• Stop growth of the bacterial culture, and conduct quality-control and cellular quantification using microbiology, molecular biology, chemistry and/or biochemistry methods;

• The quality-controlled culture may be utilised alone to constitute the final product, or mixed with an extra portion of water, growth medium, acetic acid, agar, gelatine, collagen, vitamins, salts, organic compounds, other living microbes, microbial extracts and/or a combination of all mentioned portions for scaled-up sub-culturing, constituting a secondary culture;

• The quality-controlled secondary culture may be dried, dehydrated, spray-dried, freeze- dried, vacuum-dried and/or lyophilised, and utilised as a powder ingredient which can be employed alone to constitute the final product, or mixed with the aforementioned ingredients, others and/or a combination of all mentioned portions either in the liquid or powder form;

• Dispense the final ingredient into bottles, drums, buckets, bags, packs and/or boxes to constitute the final product.

Example 3

Growth kinetics of Lactococcus lactis 16QF-e

The present example refers to FIG. 3, which demonstrates the growth performance of isolate 16QF-e under different manufacturing media, and whereby F3 is made of powder milk ranging from 3 to 5% supplemented with sucrose ranging from 0.25 to 5%, and F4 contains powder milk ranging from 3 to 5% supplemented with a mix of beef heart, sodium chloride and tryptose ranging from 0.05 to 0.5%.

Example 4

Pre-harvest application of Lactococcus lactis 16QF-e as a sanitiser and/or antioxidant

• If the final product supplied is ready-to-use, it is poured into a sprayer equipment and applied onto loaded trees, bush, crawling crops, vines, herbs, flowers and/or leaves at a ratio ranging between 10L/150 Kg and 10L/10,000 Kg of produce;

• If the final product supplied is a concentrate, it is poured into a sprayer equipment, mixed with water to a specified dilution of 1 : 100 or 1 :1 ,000 or 1 : 10,000, as suggested by the manufacturer, and using water that contains no bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties, and applied onto loaded trees, bush, crawling crops, roots, vines, herbs, flowers and/or leaves at a ratio ranging between 10L/150 Kg and 10L/10,000 Kg of produce;

• The final product is applied within reasonable time-frame to allow air-drying prior to or after rains, irrigation, wind and/or gusts, or whenever the targeted produce is dry and suitable to retain the product on the produce surface;

• The final product should be re-applied within at least monthly and ideally weekly intervals for better results;

• The final product should not be co-applied with bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties;

• Produce treated with the final product can be harvested and stored as per usual, although MAP packaging is recommended for better results.

Example 5 Application of Lactococcus lactis 16QF-e for food preservation as a post-harvest and/or a processing sanitiser

• If the final product supplied is ready-to-use liquid, it is poured into a sprayer equipment and applied onto harvested produce at a ratio ranging between 10L/150 Kg and 10L/75 Kg of produce, or mixed up to dips and/or sauces at a ratio ranging between 1ml/100g and 1 ml/1 Kg, or if the final product is supplied as a ready-to-use powder it is resuspended in food-grade water, and applied as aforementioned at the specified ratio range;

• Alternatively, the liquid product may be poured into a dip tank and/or a bath tank and used at the aforementioned ratio range;

· If the final product is supplied as a concentrate it may be poured into a sprayer equipment, a dip tank and/or a bath tank, mixed with water to a specified dilution of 1 :100 or 1 : 1 ,000 or 1 : 10,000 using food-grade water that contains no bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties (as suggested by the manufacturer), and applied as aforementioned;

· The final product should be applied within reasonable time-frame to allow its air-drying prior to food packaging. In other cases, the treated food can be immediately packed, wrapped, packaged and/or boxed.

• The final product should not be co-applied with bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties. · Produce treated with the final preservative product can be stored as usual, although MAP packaging is recommended for better results.

Example 6

Improved shelf-life of strawberries treated with Lactococcus lactis 16QF-e The viability of strawberries treated post-harvest with L lactis 16QF-e was compared to untreated controls. As shown in FIG. 4, the treated strawberries displayed at least 80% viability after 18 days, whereas untreated control strawberries were completely mold-infested by day 12.

Example 7

Improved shelf-life of raspberries treated with Lactococcus lactis 16QF-e

The viability of raspberries treated post-harvest with L. lactis 16QF-e was compared to untreated controls. As shown in FIG. 4, the treated raspberries displayed at least 80% viability after 15 days, whereas untreated control strawberries were completely mold-infested by day 9. Example 8

Improved shelf-life of habanero chillies treated with Lactococcus lactis 16QF-e

The viability of habanero chillies treated post-harvest with L. lactis 16QF-e was compared to untreated controls. As shown in FIG. 4, the treated habanero chillies displayed at least 80% viability after 24 days, whereas untreated control strawberries were completely mold-infested by day 20.

Example 9

Protection of canola against rancidity by Lactococcus lactis 16QF-e

FIG. 5 provides the results of the assessment of the antioxidant properties of L lactis 16QF-e during a Rancimat test. In this example, non-organic Canola oil was mixed 1 : 1 with cultures of L lactis isolates 1 (parental 16QF) and 2 (16QF-e) grown in different media, and subjected to 120°C for as many hours as necessary for rancidity to be observed. Isolate 1 corresponds to an initial Lactococcus lactis parental isolate that was sub-cultured in culture media straight after obtaining, and isolate 2 corresponds to Lactococcus lactis 16QF-e after its serial sub-culturing within membrane bags in growth media supplemented with a multidrug resistant isolate of Staphylococcus aureus, aimed to create specific selection pressure that led to adaptation and prompt response of isolate 16QF-e to Gram-positive pathogens (and potentially Gram-negative pathogens) (see FIG. 1 Step 3). As a negative control canola oil was used alone, and as a positive control canola oil was mixed with the commercial antioxidant Ethoxyquin (Novus International). It is also proposed that L lactis 16QF-e can protect oils and fats in biological systems, forming the basis for the development of cosmetics, ointments, medications, sprays and/or skincare products.

Example 10

Mechanisms and properties underlying sanitation and shelf-life extension by Lactococcus lactis

16QF-e

FIG. 6A-B provides a mechanistic illustration of the antimicrobial and antioxidant properties of L. lactis 16QF-e. It is proposed that L lactis 16QF-e forms a biofilm that inhibits, kills and/or repels bacteria and/or fungi that potentially contaminate and/or spoil food items (e.g as shown in FIG. 6C-F). It is also proposed that 1) the biofilm formed by L lactis 16QF-e prevents and/or reduces oxygen penetration in a fruit and/or any other food type by creating a physical barrier, which in turn limits contact of the pulp with oxygen, and/or 2) the L lactis cells incorporate part of, or all the oxygen existing within a packaging system into their own metabolism (either a box, a bag, a wrap or any other), contributing towards reduced food oxidation rate (FIG. 6B). In the same example, the sanitation levels of chicken and beef meats are significantly improved upon treatment with L lactis 16QF-e (FIG. 6D-F) were compared to untreated controls. Although not show in FIG. 6 due to its colorimetric nature, the treated foods displayed absence of mold contamination and oxidation/colour change for a longer than untreated controls, which were in fact deemed unmarketable significantly earlier.

Example 1 1

Health-promoting probiotic properties of Lactococcus lactis 16QF-e

Rats were fed with fruits that were treated with Lactococcus lactis isolate 16QF-e, which not only led to produce sanitation and preservation but promoted significant improvement of rat gut biology as seen by histopathology (despite those rats having a diet rich in gluten, sugars and fats for 30 days). Of note, control rats who received bad food only presented defective, shorter and sparse villi. Throughout this specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All computer programs, algorithms, patent and scientific literature referred to herein is incorporated by reference.

Example 12

Application of Lactococcus lactis 16QF-e as a pesticide

• The final product supplied is a concentrate to be poured into a sprayer and/or fertigation equipment, mixed with water to a specified dilution of 1 :100 or 1 :1 ,000 or 1 :10,000 as suggested by the manufacturer, and using water that contains no bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties, and applied onto fruit-loaded, vegetable-loaded, and/or cereal-loaded plants, trees, bush, crops and/or parts of plants that can be susceptible to insects, either pre- or post-harvest at a ratio ranging between 1 L/10,000 Kg and 1 L/1 ,000 Kg of produce;

• The final product is applied within reasonable time-frame to allow air-drying prior to or after rains, irrigation, wind and/or gusts, or whenever the target produce, crop, plant and/or plant material is dry and suitable to retain the product on the produce surface;

• The final product should be re-applied within at least fortnightly and ideally weekly intervals for better results;

• The final product should not be co-applied with bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties;

• Produce treated with the final product can be harvested and stored as usual.

Example 13

Effect of Lactococcus lactis 16QF-e on the biology of QLD fruit fly life stages, and protection of fruits and vegetables

16QF-e was sprayed onto organic fruits at the same concentration later tested during field trials, and the treated produce were placed into cages containing 50-60 adult QLD fruit flies separated per gender. Control cages received organic fruits that had not been treated with 16QF-e. Survival rates were monitored for 7 days and death was determined every 24h, revealing that 16QF-e is able to kill fruit flies under a controlled environment (FIG. 7A). In addition, 16QF-e was also tested as a product to delay or stop the fruit fly life-cycle when directly applied on the insect life stages, aiming to make it susceptible to environmental challenges and/or predators. Herein it was revealed its ability to significantly delay pupation when compared to untreated controls (FIG. 7B). Furthermore, field trials were conducted to demonstrate protective efficacy against QLD fruit flies in realistic situations (rural and domestic settings), and to compare untreated vs treated groups side-by-side (FIG. 7C), confirming and validating all laboratory findings.

Example 14

Application of Lactococcus lactis 16QF-e as a composting inoculant

• The final product supplied is a concentrate to be mixed with water to a specified dilution of 1 :100 or 1 :1 ,000 or 1 :10,000 as suggested by the manufacturer, and using water that contains no bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties, and applied onto foods, food waste, food scraps, paper, paper waste, cellulose, cellulose waste, gardening waste, agricultural waste and/or any other waste characterised as organic or green, including when this comes into contact with chemicals and/or synthetic compounds;

• Application ratio ranges between 1 L/100 Kg and 1 L/10,000 Kg of material to be digested, composted, degraded, deteriorated, reduced, recycled, converted, transformed, broken down and/or pasteurised;

• The final product is applied into an unit or system whereby internal temperature reaches at least 50°C, but it can also range between 30°C and 40°C or between 50°C and 80°C on occasions, ultimately leading to and/or contributing towards an outcome, by-product and/or end- product of the referred processes, while promoting, contributing to and/or assisting achieve sanitation;

• Within a reasonable time-frame a nutrient-rich resource, soil conditioner and/or fertiliser will be generated, and which can be safely used in agriculture, horticulture, gardening, farming and/or similar applications;

• The 16QF-e inoculant should be re-applied within at least fortnightly and ideally weekly intervals for better composting results;

• The final product should not be co-applied with bactericides, antibiotics, bacteriostatic products and/or any compounds and/or molecules that confer the aforementioned properties. Example 15

Use of Lactococcus lactis 16QF-e as an inoculant to convert food waste to a safe and nutrient- rich fertiliser

16QF-e was diluted and added to a jacketed composting machine that maintains internal temperature constantly at 50°C, while rotating and aerating the introduced waste material. Two weeks after introduction of the inoculant, food waste had been completely converted to a nutrient- rich fertiliser with a fine powder texture. The 16QF-e inoculant was applied only once during week 1 , and the composting process was monitored twice a week for four weeks. This trial confirmed that the ideal application rate is weekly, aiming to ensure the consistent obtaining of an outcome similar to week 2: absence of contamination, 100% conversion rates, and ideal texture for further distribution (FIG. 8).

Technical Field

Table 1

Table 1 continued - DNA sequences corresponding to L lactis 16QF-e unique genome