# Patent

## Title
Use of a composition comprising an antimicrobial peptide as a food preservative

## Abstract

The inventors provide the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to prevent or inhibit spoilage of a foodstuff by a microorganism. Also provided is a method of preventing or inhibiting spoilage of a foodstuff by a microorganism comprising administering to a foodstuff in need thereof an effective amount of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof.

## Background
The present application is a continuation of pending application Ser. No. 13/879,162 filed 15 Jul. 2013 and claims priority to and the benefit of the following, to the extent allowable by law: U.S. application Ser. No. 13/879,162 filed 15 Jul. 2013, PCT/EP2011/067821, filed 12 Oct. 2011, and priority applications PT 105331 filed 12 Oct. 2010 and GB 1017283.1 filed 13 Oct. 2010, all of which, together with all references disclosed in this and all priority applications, are hereby incorporated by reference for all purposes.
A sequence listing text (.txt) file is submitted herewith under 37 CFR. 1.821(c) and hereby incorporated by reference in its entirely. The details of the file as required under 37 CFR. 1.52(e)(5) and 37 CFR 1.77(b)(5) are as follows: Name of file is P11336 WO_ST25.txt; date of creation is Friday, Jun. 26, 2015; size is 9.37 KB (9,605 bytes). The information recorded in electronic form (if any) submitted (under Rule 13ter if appropriate) with this application is identical to the sequence listing as contained in the application as filed.


FIELD OF THE INVENTION
The invention relates to the field of antimicrobial agents that target microorganisms that spoil food.
INTRODUCTION
Food preservation is the process of treating food to prevent or inhibit food spoilage caused by endogenous chemical/enzymatic degradation and/or caused or accelerated by a microorganism. A number of techniques exist for preserving food, some of which inhibit endogenous processes (e.g. antioxidants), some of which inhibit microbial processes (e.g. antimicrobials), and some of which inhibit both types of process (e.g. freezing). A compound that is used to inhibit food spoilage is commonly referred to as a preservative, which may be, for example an antioxidant or an antimicrobial.
Particular food preservation techniques include drying, heating, refrigerating or freezing, osmotic inhibition (e.g. use of syrups or salt), vacuum packing, canning and bottling, jellying, potting, jugging, ionising irradiation, pulsed electric field processing, high pressure food preservation, and ultra high water pressure food preservation, use of antioxidants, and/or use of antimicrobial preservatives (e.g. sulphur dioxide, carbon dioxide, ethanol, acetic acid, citric acid, lactic acid, sorbic acid, benzoates, nitrates and nitrites, sulphites, calcium propionate and methylchloroisothiazolinone).
Despite the relatively large number of food preservation techniques that are currently employed there is a need to develop new antimicrobial preservatives. This is because of the inadequacies of many pre-existing techniques to effectively target microorganisms and problems with efficacy and/or safety of many pre-existing antimicrobial preservatives in particular.
Many food preservation techniques that attempt to create non-favourable growth conditions for microorganisms are ineffective against organisms that survive in extreme conditions (e.g. Pseudomonas species can grow at very low temperatures; Bacillus coagulans is heat resistant and acid tolerant; many species of Aspergillus demonstrate oligotrophy; Zygosaccharomyces species have high xerotolerance).
Many pre-existing antimicrobial preservatives have moderate activity, especially against microorganisms with innate or acquired resistance, and/or narrow spectrum. For example, Zygosaccharomyces species have high tolerance to ethanol, acetic acid, sorbic acid, benzoic acid and sulphur dioxide. In addition, a number of pre-existing antimicrobial preservatives have been associated with various side-effects such as respiratory problems or ADD. In particular examples, sulphur dioxide is irritating to the bronchial tubes of asthmatics, nitrites are potentially carcinogenic, benzoates have been associated with various allergies, asthma, skin rashes and brain damage.
Furthermore, effective techniques for inhibiting microbial growth in food, such as low pH or low water activity, are often unacceptable to the consumer (e.g. give an acid taste) or have negative health implications (e.g. high salt or sugar).
It is among the objectives of the present invention to attempt a solution to these problems, and specifically for example to provide a new antimicrobial agent with potent and broad-spectrum activity against microorganisms whilst having low toxicity.
SUMMARY OF THE INVENTION
The inventors have surprisingly found that the Blad polypeptide from Lupinus shows potent antimicrobial activity against a large number of diverse bacterial and fungal organisms that cause food spoilage. The inventors have also found that the Blad polypeptide is non-toxic, therefore making Blad an excellent compound for use as an anti-microbial food preservative.
Accordingly, the inventors provide the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to prevent or inhibit spoilage of a foodstuff by a microorganism. Preferably said microorganism is a bacterium (preferably a food-spoiling species from the Pseudomonas or Bacillus genera) or a fungus (preferably a food-spoiling species from one of the following genera: Alternaria, Aspergillus, Fusarium, Botrytis, Colletotrichum, Saccharomyces, Kluyveromyces and Zygosaccharomyces).
In preferred embodiments the foodstuff is derived from, provides, or is, a fruit, a nut, a vegetable, a seed, a sugar, a dairy product, a liquid or paste food, meat, fish or bread.
In preferred embodiments the foodstuff is a strawberry, preferably wherein the microorganism is Botrytis cinerea or Colletotrichum acutatum, preferably Botrytis cinerea. 
In preferred embodiments said composition further comprises a chelating agent.
The inventors also provide a method of preventing or inhibiting spoilage of a foodstuff by a microorganism comprising administering to a foodstuff in need thereof an effective amount of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof.



DESCRIPTION OF THE DRAWINGS
The invention will be described with reference to the accompanying drawings, in which:
FIG. 1 shows the Lupinus albus β-conglutin precursor encoding sequence (SEQ ID NO: 1); and
FIG. 2 shows the internal fragment of the β-conglutin precursor encoding sequence that corresponds to Blad (SEQ ID NO: 3).



DETAILED DESCRIPTION OF THE INVENTION
Blad
Blad (“banda de Lupinus albus doce”—band from sweet L. albus) was the name given to a stable and intermediary breakdown product of β-conglutin, the major storage protein present in seeds of the Lupinus genus. It was characterised as a 20 kD polypeptide, composed of 173 amino acid residues, and encoded by an internal fragment (519 nucleotides, deposited in GenBank under the accession number ABB13526) of the gene encoding the precursor of β-conglutin from Lupinus (1791 nucleotides, published in GenBank, under the accession number AAS97865). When primers encoding Blad terminal sequences are used to amplify a sequence from genomic Lupinus DNA, a  ̃620 bp product is obtained, indicating the presence of an intron in the gene fragment encoding Blad. Naturally-occurring Blad is the main component of a 210 kD glycooligomer which accumulates exclusively (following intensive limited proteolysis of β-conglutin) in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Whilst said oligomer is glycosylated, naturally-occurring Blad is non-glycosylated. The Blad-containing glycooligomer is composed of several polypeptides, the major ones exhibiting molecular masses of 14, 17, 20, 32, 36, 48 and 50 kD. The 20 kD polypetide, Blad, is by far the most abundant polypeptide within the oligomer and appears to be the only one with lectin activity. Naturally-occurring Blad constitutes approximately 80% of the total cotyledonary protein in 8-day old plantlets.
The L. albus β-conglutin precursor encoding sequence (SEQ ID NO: 1) is given in FIG. 1. The β-conglutin parent subunit coding sequence is located at residues 70 to 1668. The encoded, 533 amino acid residue β-conglutin parent subunit (SEQ ID NO: 2) is:







MGKMRVRFPTLVLVLGIVFLMAVSIGIAYGEKDVLKSHERPEEREQEEW


 


QPRRQRPQSRREEREQEQEQGSPSYPRRQSGYERRQYHERSEQREEREQ


 


EQQQGSPSYSRRQRNPYHFSSQRFQTLYKNRNGKIRVLERFDQRTNRLE


 


NLQNYRIVEFQSKPNTLILPKHSDADYVLVVLNGRATITIVNPDRRQAY


 


NLEYGDALRIPAGSTSYILNPDDNQKLRVVKLAIPINNPGYFYDFYPSS


 


TKDQQSYFSGFSRNTLEATFNTRYEEIQRIILGNEDEQEYEEQRRGQEQ


 


SDQDEGVIVIVSKKQIQKLTKHAQSSSGKDKPSDSGPFNLRSNEPIYSN


 


KYGNFYEITPDRNPQVQDLNISLTYIKINEGALLLPHYNSKAIYVVVVD


 


EGEGNYELVGIRDQQRQQDEQEEKEEEVIRYSARLSEGDIFVIPAGYPI


 


SINASSNLRLLGFGINADENQRNFLAGSKDNVIRQLDRAVNELTFPGSA


 


EDIERLIKNQQQSYFANGQPQQQQQQQSEKEGRRGRRGSSLPF






The internal fragment of the β-conglutin precursor encoding sequence that corresponds to Blad (SEQ ID NO: 3) is given in FIG. 2. The Blad polypeptide (SEQ ID NO: 4) is:







RRQRNPYHFSSQRFQTLYKNRNGKIRVLERFDQRTNRLENLQNYRIVEF


 


QSKPNTLILPKHSDADYVLVVLNGRATITIVNPDRRQAYNLEYGDALRI


 


PAGSTSYILNPDDNQKLRVVKLAIPINNPGYFYDFYPSSTKDQQSYFSG


 


FSRNTLEATFNTRYEEIQRIILGNED






The invention relates to a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof. It therefore relates to a composition comprising an antimicrobial polypeptide comprising the polypeptide sequence of SEQ ID NO: 4 or an active variant thereof. In alternative embodiments, the composition consists essentially of an antimicrobial polypeptide comprising Blad or an active variant thereof and/or the antimicrobial polypeptide consists essentially of Blad or an active variant thereof. In further embodiments the antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof may be used in isolated form.
An active variant of Blad is a variant of Blad that retains the ability to act as an antimicrobial (i.e. has antimicrobial activity—see below for a description of the level of such activity and how to measure it). “An active variant of Blad” includes within its scope a fragment of SEQ ID NO: 4. In preferred embodiments, a fragment of SEQ ID NO: 4 is selected that is at least 10% of the length of SEQ NO: 4, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of SEQ NO: 4. Blad or a variant thereof generally has a length of at least 10 amino acid residues, such as at least 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160 or 173 amino acid residues.
“An active variant of Blad” also includes within its scope a polypeptide sequence that has homology with SEQ ID NO: 4, such as at least 40% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably at least 99% identity, for example over the full sequence or over a region of at least 20, preferably at least 30, preferably at least 40, preferably at least 50, preferably at least 60, preferably at least 80, preferably at least 100, preferably at least 120, preferably at least 140, and most preferably at least 160 or more contiguous amino acid residues. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as “hard homology”).
The homologous active Blad variant typically differs from the polypeptide sequence of SEQ ID NO: 4 by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably ‘conservative’, that is to say that an amino acid may be substituted with a similar amino acid, whereby similar amino acids share one of the following groups: aromatic residues (F/H/W/Y), non-polar aliphatic residues (G/A/P/I/L/V), polar-uncharged aliphatics (C/S/T/M/N/Q) and polar-charged aliphatics (D/E/K/R). Preferred sub-groups comprise: G/A/P; I/L/V; C/S/T/M; N/Q; D/E; and K/R.
An antimicrobial polypeptide comprising Blad or an active variant thereof (as described above) may consist of Blad or an active variant thereof with any number of amino acid residues added to the N-terminus and/or the C-terminus provided that the polypeptide retains antimicrobial activity (again, see below for a description of the level of such activity and how to measure it). Preferably, no more than 300 amino acid residues are added to either or both ends of Blad or an active variant thereof, more preferably no more than 200 amino acid residues, preferably no more than 150 amino acid residues, preferably no more than 100 amino acid residues, preferably no more than 80, 60 or 40 amino acid residues, most preferably no more than 20 amino acid residues.
An antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof (as described above) may be utilised in the invention in the form of a purified (e.g. removed from a plant, animal or microbial source) and/or recombinant protein. Production of a recombinant form enables the production of active variants of Blad.
Methods of purifying naturally-occurring Blad are already described in the art (e.g. Ramos et at (1997) Planta 203(1): 26-34 and Monteiro et at (2010) PLoS ONE 5(1): e8542). A suitable source of naturally-occurring Blad is a plant of the Lupinus genus, such as Lupinus albus, preferably a cotyledon of said plant, preferably harvested between about 4 to about 14 days after the onset of germination, more preferably harvested 6 to 12 days after the onset of germination (such as 8 days after the onset of germination). Methods are disclosed in the art for a total protein extraction leading to a crude extract comprising Blad, and for a protein purification of such an extract leading to a partially purified extract e.g. comprising the Blad-containing glycooligomer that comprises Blad.
To isolate Blad itself one can then use SDS-PAGE and/or, preferably, reverse phase (RP)-HPLC on a C-18 column.
An alternative way of obtaining a partially purified extract comprising the glycooligomer that comprises Blad is to utilise the chitin binding activity of Blad. The glycooligomer binds in a very strong manner to a chitin column as part of a chitin affinity chromatography purification, being eluted with 0.05 N HCl. Details of an example of this purification method are as follows:

    
    
        Cotyledons from eight-day old lupin plants were harvested and homogenized in Milli-Q plus water (pH adjusted to 8.0), containing 10 mM CaCl2 and 10 mM MgCl2. The homogenate was filtered through cheesecloth and centrifuged at 30,000 g for 1 h at 4° C. The pellet was subsequently suspended in 100 mM Tris-HCl buffer, pH 7.5, containing 10% (w/v) NaCl, 10 mM EDTA and 10 mM EGTA, agitated for 1 h at 4° C., and centrifuged at 30,000 g for 1 h at 4° C. The total globulin fraction, contained in the supernatant, was precipitated with ammonium sulphate (561 g/l), left stirring in the cold for 1 h and centrifuged at 30,000 g for 30 min at 4° C. The pellet obtained was dissolved in 50 mM Tris-HCl buffer, pH 7.5, desalted in PD-10 columns equilibrated in the same buffer and passed through a chitin-affinity chromatography column pre-equilibrated in the same buffer. The column was washed with 50 mM Tris-HCl buffer, pH 7.5, and the bound proteins eluted with 0.05 N HCl. The eluted fractions were immediately neutralized with 2 M Tris and the peak fractions pooled, lyophilized and analyzed by SDS-PAGE.
        For the preparation of the chitin column, crude chitin was obtained from Sigma and processed as follows: the chitin sample was washed extensively with Milli-Q plus water, followed by 0.05 N HCl. It was then washed with 1% (w/v) sodium carbonate and then with ethanol, until the absorbance of the wash was less than 0.05. Chitin was then packed into a pipette tip and equilibrated with 50 mM Tris-HCl buffer, pH 7.5.
    
    


Methods of producing recombinant proteins are well known in the art. Such methods as applied here will involve inserting the polynucleotide encoding a polypeptide comprising Blad or an active variant thereof into a suitable expression vector—enabling the juxtaposition of said polynucleotide with one or more promoters (e.g. an inducible promoter, such as T7lac) and with other polynucleotides or genes of interest—introducing the expression vector into a suitable cell or organism (e.g. Escherichia coli), expressing the polypeptide in the transformed cell or organism and removing the expressed recombinant polypeptide from that cell or organism. To assist such purification the expression vector may be constructed such that the polynucleotide additionally encodes, for example, a terminal tag that can assist purification: e.g., a tag of histidine residues for affinity purification. Once the recombinant polypeptide is purified, the purification tag may be removed from the polypeptide, e.g., by proteolytic cleavage.
In a composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof, said polypeptide is preferably in partially purified form, more preferably in purified form. Said polypeptide is partially purified when it is present in an environment lacking one or more other polypeptides with which it is naturally associated and/or is represented by at least about 10% of the total protein present. Said polypeptide is purified when it is present in an environment lacking all, or most, other polypeptides with which it is naturally associated. For example, purified Blad means that Blad represents at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the total protein in a composition.
In a composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof, the Lupinus protein content may consist essentially of the Blad-containing glycooligomer that comprises a polypeptide that comprises (or consist essentially of) Blad or an active variant thereof.
A composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad may also be a formulation comprising another compound(s) added to the composition by the skilled person.
Food Spoilage and Foodstuffs
Spoilage of a foodstuff by a microorganism means any alteration of a foodstuff by a microorganism that results in a change in e.g. the taste, odour or appearance (e.g. shape, colour, texture, firmness) that decreases its nutritional and/or commercial value. By foodstuff it is intended to mean any liquid or solid substance intended for consumption by a human or animal for nutritional or pleasurable reasons. The foodstuff may be consumed directly or indirectly (e.g. after cooking or processing, such as refinement of cereals). Where applicable, the foodstuff is preferably contemplated in a form isolated from its natural environment, such as harvested plant foodstuffs (e.g. fruit, vegetables, seeds) and products isolated from animals (e.g. meat, fish, milk).
The foodstuff may be derived from, may provide, or may be, a fruit, a nut, a vegetable, a seed, a sugar, a dairy product, a liquid or paste food, meat, fish or bread. Foodstuffs derived from fruit include wine and fruit juice. Plants providing seeds include cereals (e.g. maize, wheat, barley, sorghum, millet, rice, oats and rye) and legumes (e.g. beans, peas and lentils). Sugars, preferably sucrose, may be derived from sugar beet or sugar cane. Dairy products include milk, cream, cheese and yoghurt. Liquid or paste food includes soup, sauces, pickles, mayonnaise, salad cream and other salad dressings, preserves, syrup and baby food. The meat and/or fish foodstuffs contemplated may be processed or otherwise, and may be cooked or otherwise. Further particular foodstuffs that are contemplated may be found in the next section, which refers to example foodstuffs that may be spoiled by microorganisms which may be targeted in the uses and methods of the invention.
Foodstuffs also include pre-prepared composite foods such as sandwiches, pies, quiches etc, especially those currently designed for chilled storage.
Microbial Targets
The inventors provide the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to prevent or inhibit spoilage of a foodstuff by a microorganism. Microorganisms that may cause spoilage of a foodstuff—food-spoiling microorganisms—include, in particular, bacteria and fungi. In such use the antimicrobial polypeptide may be considered as an antimicrobial food preservative.
The antimicrobial polypeptide can be used to prevent or inhibit food spoilage by either Gram-positive or Gram-negative bacteria. Particularly preferred bacterial targets (with examples of foodstuffs that they may spoil given in brackets) include: food-spoiling Pseudomonas species, such as Pseudomonas aeruginosa (thale cress and lettuce), Pseudomonas syringae (various plant-derived foodstuffs such as beet, wheat and barley), Pseudomonas tolaasii (mushroom), Pseudomonas agarici (mushroom), Pseudomonas fragi (dairy products) and Pseudomonas lundensis (milk, cheese, meat and fish), most preferably P. aeruginosa; and food-spoiling Bacillus species such as Bacillus subtilis (tomato, potato, bread) and Bacillus coagulans (milk, tomato juice).
The antimicrobial polypeptide can be used to prevent or inhibit food spoilage by either unicellular (yeast) or multicellular (filamentous, mold) fungi. Particularly preferred yeast targets (with examples of foodstuffs that they may spoil given in brackets) include: food-spoiling Saccharomyces species, such as Saccharomyces cerevisiae (sugar, sugar syrups, wine, and soft drinks such as fruit juices); food-spoiling Kluyveromyces species, such as Kluyveromyces marxianus (cheese); and food-spoiling Zygosaccharomyces species, such as Zygosaccharomyces bailii (wine, fruit juice, salad dressings and tomato sauce) and Zygosaccharomyces rouxii (sugar syrups, fruit juices, jams and salad dressings). Particularly preferred mold targets (with examples of foodstuffs that they may spoil given in brackets) include: food-spoiling Alternaria species, such as Alternaria alternate (potato), Alternaria arborescens (tomato), Alternaria arbusti (Asian pear), Alternaria brassicae (vegetables), Alternaria brassicicola (cole crops), Alternaria carotiincultae (carrot), Alternaria conjuncta (parsnip), Alternaria dauci (carrot), Alternaria euphorbiicola (cole crops), Alternaria gaisen (pear), Alternaria infectoria (wheat), Alternaria japonica (cole crops), Alternaria petroselini (parsley), Alternaria selini (parsley), Alternaria solani (potato, tomato) and Alternaria smyrnii (alexanders, parsley); food-spoiling Aspergillus species, such as Aspergillus fumigatus (nuts, potato, rice and bread), Aspergillus niger (fruit and vegetables e.g. grapes and onions), and Aspergillus flavus (corn, peanut); food-spoiling Fusarium species, such as Fusarium oxysporum (fruit) and Fusarium graminearum (barley, wheat and maize); food-spoiling Botrytis species, such as Botrytis cinerea (strawberry, grape and tomato); and food-spoiling Colletotrichum species, such as Colletotrichum actuatum (strawberry, celery, apple, avacado, aubergine, coffee and guava), Colletotrichum coccodes (tomato, potato), Colletotrichum capsici (basil, chickpea, pepper), Colletotrichum crassipes (passion fruit), Colletotrichum gloeosporioides (vegetables and fruit e.g. quince and apple), Colletotrichum graminicola (cereals), Colletotrichum kahawae (coffee), Colletotrichum lindemuthianum (bean), Colletotrichum musae (banana), Colletotrichum nigrum (tomato), Colletotrichum orbiculare (melon, cucumber), Colletotrichum pisi (pea) and Colletotrichum sublineolum (rice).
In preferred embodiments the antimicrobial polypeptide is used to prevent or inhibit spoilage of a fruit by a microorganism, preferably a strawberry, and preferably wherein the microorganism is Botrytis cinerea or Colletotrichum acutatum, preferably Botrytis cinerea. 
The skilled person will be able to identify, through routine methods, a suitable concentration (i.e. an effective concentration) with which to use the antimicrobial polypeptide to prevent or inhibit spoilage in any particular setting. Preferably, for example, Blad is used at a concentration of at least 1 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 50 μg/ml, at least 100 μg/ml, or at least 150 μg/ml, and up to 350 μg/ml, up to 500 μg/ml, up to 600 μg/ml, up to 1 mg/ml, up to 2.5 mg/ml, up to 5 mg/ml or up to 10 mg/ml. Preferably the concentration of Blad selected is between 10 μg/ml and 5 mg/ml, more preferably between 50 μg/ml and 2.5 mg/ml, more preferably between 100 μg/ml and 1 mg/ml, and even more preferably between 150 μg/ml and 600 μg/ml (such as about 250 μg/ml). The inventors have provided evidence (see Examples 4 and 5) that Blad is non-toxic to animals to at least 400 μg/ml.
The inventors have surprisingly found that a combination of Blad with a chelating agent (e.g. EDTA) produces a synergistic antimicrobial effect. Therefore, preferably, a chelating agent is used to improve the antimicrobial activity of the antimicrobial polypeptide, and the use of such a chelating agent may decrease the concentration of the antimicrobial polypeptide required to achieve a particular level of prevention or inhibition of spoilage. A chelating agent (also known as a chelant, a chelator or a sequestering agent) is any compound that binds to a metal ion to form a non-covalent complex and reduces the ion's activity. Suitable chelating agents include polyamino carboxylates, such as EDTA (ethylenediaminetetraacetic acid) and EGTA (ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid). Preferably, EDTA is used as the chelating agent, preferably at a concentration of at least 10 μg/ml, at least 50 μg/ml, or at least 100 μg/ml, and up to 500 μg/ml, up to 1 mg/ml, up to 5 mg/ml, up to 10 mg/ml, or up to 20 mg/ml. Preferably, EDTA is used at a concentration of between 0.1 mg/ml and 1 mg/ml.
Outcomes
The antimicrobial polypeptide may be used to inhibit the growth of a food-spoiling microorganism (meaning that it has microbistatic activity) and/or to kill said microorganism (meaning that it has microbicidal activity) on a foodstuff such that spoilage of said foodstuff by said microorganism is prevented or inhibited. The skilled person will be able to identify a suitable dose and/or concentration to obtain a particularly desired growth inhibition or killing of the microorganism.
Preferably, when used as a microbistatic agent, the antimicrobial polypeptide reduces the rate of growth by 10%, more preferably by 50%, more preferably by 75%, more preferably by 90%, more preferably by 95%, more preferably by 98%, more preferably by 99%, and even more preferably by 99.9% in comparison to equivalent conditions where the antimicrobial polypeptide is not present. Most preferably the antimicrobial polypeptide prevents any growth of the microorganism.
Preferably, when used as a microbicidal agent, the antimicrobial polypeptide kills 10% of the population of the microorganims, more preferably 50% of said population, more preferably 75% of said population, more preferably 90% of said population, more preferably 95% of said population, more preferably 98% of said population, more preferably 99% of said population, and even more preferably by 99.9% of said population in comparison to equivalent conditions where the antimicrobial polypeptide is not present. Most preferably the antimicrobial polypeptide kills all of the population of the microorganism.
When used to prevent or inhibit spoilage of a foodstuff by a microorganism the antimicrobial polypeptide is preferably used in an effective amount, that is to say an amount that provides a level of growth inhibition and/or killing of a microorganism such that a detectable level of spoilage prevention or inhibition (e.g. a decrease in the rate of spoilage) is achieved, preferably in comparison to equivalent conditions where the antimicrobial polypeptide is not present. Preferably, the effective amount of the antimicrobial polypeptide is non-toxic to a human or animal.
Uses and Methods
The inventors provide the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to prevent or inhibit spoilage of a foodstuff by a microorganism. To this end they also provide a method of preventing or inhibiting spoilage of a foodstuff by a microorganism comprising administering to a foodstuff in need thereof an effective amount of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof. Preferably, the effective amount of the antimicrobial polypeptide is non-toxic to humans or animals. The prevention or inhibition of spoilage can occur during the storage, transport, handling, processing or display of the foodstuff.
A composition comprising the antimicrobial polypeptide can for example be mixed into the foodstuff or may for example be applied to the surface of the foodstuff (for example as a liquid film or a spray). The foodstuff may also be immersed in (and optionally maintained in) said composition. For any particular foodstuff the use of said composition as a preservative can be combined with any other well known food preservation technique, antimicrobial or otherwise, including drying, heating, refrigerating or freezing, osmotic inhibition (e.g. use of syrups or salt), vacuum packing, canning and bottling, jellying, potting, jugging, ionising irradiation, pulsed electric field processing, high pressure food preservation, and ultra high water pressure food preservation, use of antioxidants, and/or use of other antimicrobial preservatives (e.g. sulphur dioxide, carbon dioxide, ethanol, acetic acid, citric acid, lactic acid, sorbic acid, benzoates nitrates and nitrites, sulphites, calcium propionate and methylchloroisothiazolinone).
EXAMPLES
In the following Examples BLAD denotes the naturally-occurring Blad-containing glycooligomer comprising the 20 kD Blad polypeptide, purified as per Ramos et at (1997) Planta 203(1): 26-34: see “Plant material and growth conditions” and “Purification of proteins” parts of the Materials and Methods section of that document.
DEFINITIONS
MIC—Minimum Inhibitory Concentration: the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism.
MFC/MBC—Minimum Fungicidal/Bactericidal Concentration (or Minimal Lethal Concentration): the lowest concentration of an antimicrobial agent needed to kill 99.9% of the initial inoculum after 24 hours under a standardized set of conditions.
Example 1
Bactericidal Activity of BLAD
MIC and MBC of BLAD for Various Bacterial Species (Using Mueller-Hinton Medium):















Bacterial Species
MIC (μg/ml)
MBC (μg/ml)









Pseudomonas aeruginosa
32-256
128-256



Listeria monocytogenes
8
>512



Bacillus subtilis
4
>512



Staphylococcus aureus
8
>512



Salmonella thyphimurium
64
128










In particular, P. aeruginosa and B. subtilis can cause food spoilage. BLAD was found to be bacteriostatic at 100 μg/ml and bactericidal at 250 μg/ml against P. aeruginosa. Against P. aeruginosa BLAD at 50 μg/ml or EDTA at 1 mg/ml inhibits growth (i.e. both are bacteriostatic) but a combination of the two is bactericidal.
Example 2
Fungicidal Activity of BLAD
MIC and MFC of BLAD for Various Filamentous Fungi (Using RPMI Medium)















Fungal Species
MIC (μg/ml)
MFC (μg/ml)




















Alternaria sp.
64
>512



Aspergillus fumigatus
32
>512



Aspergillus niger
32-64
>512



Botrytis cinerea
128 
512



Colletotrichum acutatum
64
>512



Colletotrichum gloesporioides
64
>512



Fusarium oxysporum
64
>512










MIC of BLAD for Various Filamentous Fungi (Using Various Media):














MIC (μg/ml)










PDB












Strain
PDB
pH 7.5
AM3
RPMI


















Botrytis cinerea BM
128
(1)
4-8
(3)
8-32
(3)
128
(2)


B. cinerea BT
32-128
(3)
8
(3)
16-32
(3)
64-128
(3)











Colletotrichum
32
4
1-4 
64


kahawae


(from Kenya)


C. kahawae
16
4-8
4-16
64


(from Zimbabwe)









Inhibition Halo (Diameter) Data for BLAD Against Botrytis cinera BM on Potato Dextrose Agar (PDA) at 0.6% or 0.9% w/v (Incubation 3 Days at 25° C.):
















BLAD per
Average halo diameter
Average halo diameter



disk (μg)
on 0.6% agar (mm)
on 0.9% agar (mm)



















20
μg
0
0


50
μg
13
0


100
μg
25.5
11


200
μg
36
22









On 0.6% agar, growth of B. cinera was increasingly inhibited with increasing amounts of BLAD from 20 μgl to 200 μg. A less marked inhibition was seen at 5 mg/ml and 10 mg/ml on 0.9% agar.
Inhibition Halo (Diameter) Data for BLAD at 200 μg/Disk Against Various Yeasts on PDA with 0.9% (w/v) Agar (Incubation 3 to 5 Days Days at 25° C.):















Average inhibition



Yeast
halo diameter (mm)



















Saccharomyces cerevisiae
30



Kluyveromyces marxianus
28



Zygosaccharomyces bailii
>40



Zygosaccharomyces rouxii
40










BLAD at 200 μg/disk showed significant growth inhibition of all of these yeasts, all of which can cause food spoilage.
Example 3
Strawberry Preservation/Decontamination Assays
The susceptibility of strawberries to B. cinerea contamination following treatment with BLAD was investigated. Up to 38% of untreated strawberries supplied by a commercial supplier were found to be contaminated (sample size: ten 500 g boxes), underlining the problem of strawberry contamination.
Assay Protocol


    1. Prepare homogeneous and representative samples.
    2. Wash in water and decontaminate using 70% v/v ethanol.
    3. Rinse twice in water.
    4. Treat with BLAD at various concentrations (omitted for control): immerse in treatment solution for 1 minute before leaving to dry in incubation petri dishes (containing a wet sponge housing sterile water, filter paper and plastic mesh).
    6. Innoculate each strawberry with 10 μl of B. cinerea spore solution at 1-5×106 spores/ml.
    7. Incubate for 5 days under the following conditions:
    
        Temperature: 25° C. (+/−3° C.)
        Relative humidity: 80-90%
        Lighting: protected from direct sunlight
    
    
    8. At intervals, collect 1 g sample and homogenize using a vortex.
    9. Perform serial decimal dilution.
    10. Disperse on solid media and perform colony count.


All handling techniques were performed under sterile conditions, using autoclaved material.
The following data represent the percentage of strawberries infected with B. cinerea after inoculation with the B. cinerea spore solution:














Days post
BLAD concentration (mg/ml):














inoculation
0(control)
50
150
250
350


















0
0
0
0
0
0



1
44
33
4
0
0



2
63
63
15
6
5



3
93
77
46
50
40



4
100
100
56
70
60



5
100
100
73
70
64










BLAD at concentrations of 150 μg/ml to 350 μg/ml significantly delayed the onset of contamination with B. cinerea and reduced the total proportion of contaminated strawberries in comparison to the control.
Example 4
Dermal Toxicity Study of BLAD in Guinea Pigs
Confidential study carried out at the Faculty of Veterinary Medicine, Technical University of Lisbon, on behalf of Instituto Superior de Agronomia (Jul. 18, 2006-Aug. 1, 2006) using OECD Guideline for testing of chemicals, No. 402, Acute Dermal Toxicity. The study was conducted in accordance with good laboratory practice and animal welfare.
The acute dermal toxicity of BLAD was evaluated after single dose exposure in guinea pigs, which are widely accepted as suitable animals for dermal toxicity studies. BLAD was applied to the glabrous skin in two groups of 10 animals each, with dosing at 200 μg/ml and 400 μg/ml respectively. After exposure the animals were kept under observation for a period of 15 days, during which body mass, morbidity and mortality were recorded.
Materials and Methods


    1. Materials


Test item: BLAD was supplied at 5 mg/ml (yellowish opaque liquid, 0-4° C.) and stored at −80° C.
Animals: albino guinea pigs; strain: Dunkin Hartley (HsdPoc: DH) by Harlan Iberica, Barcelona.
Number of animals used: 30.
Body weight: 400-449 g.
Age: 6 weeks.
Lodging: the animals were individually placed in polyethylene boxes with sterilized wood shavings (Lignocel).
Ambient conditions:

    
    
        a) Photoperiod: cycles of light/dark for 12 h in 12 h.
        b) Controlled environment: an average temperature of 19/22° C. and average humidity of 60%.
    
    


Adaptation: the animals were kept under environmental conditions of the test for seven days before the start of the test.
Food: Global Diet 2014, Rodent Maintenance Diet supplied by Harlan Iberica, Barcelona; water ad libitum.

    2. Methods


Administration: animals were shaved 48 h before the test and only animals that had lesion-free skin were taken forward in the study. An aliquot of 1 ml (at either 200 μg/ml or 400 μg/ml) was applied to the shaved skin of each animal.
Study design: the 30 animals of the study were divided into four groups, two groups of ten animals each and two groups with five animals each. A group of ten animals was exposed to BLAD at 200 μg/ml (test group 1) and another group of ten animals was exposed to BLAD at 400 μg/ml (test group 2). The two groups of five animals served as controls: one group was exposed to water (1 ml aliquot) whilst another group was not subjected to any administration but handled as per all the other groups.
Outcomes: after exposure the animals were observed daily for 15 days to record any signs of morbidity or even death. In terms of morbidity particular attention was paid to possible appearance of skin lesions at the site of exposure and possible signs of general toxicity such as changes in normal behavior patterns. Body weight was individually assessed before exposure and at the end of test period.
Results
At neither concentration of BLAD were there signs of any physical changes in the dermal administration area or changes in drinking/feeding or general behavior. No adverse reactions or death occurred upon BLAD administration. Increase in body mass was similar in all groups (and was consistent with the increase expected from developing animals of such young age).
Conclusions
BLAD at concentrations up to 400 μg/ml (and possibly higher) does not show dermal toxicity.
Example 5
Oral Toxicity Study of BLAD in Albino Rats
Confidential study carried out at the Faculty of Veterinary Medicine, Technical University of Lisbon, on behalf of Instituto Superior de Agronomia, using OECD Guideline for testing of chemicals, No. 401, Acute Oral Toxicity. The study was conducted in accordance with good laboratory practice and animal welfare.
The acute oral toxicity of BLAD was evaluated after single dose exposure in rats, which are widely accepted as suitable animals for acute oral toxicity studies. BLAD was administered by gavage in two groups of 10 animals each, with dosing at 200 μg/ml and 400 μg/ml respectively. After exposure the animals were kept under observation for a period of 15 days, during which body mass, morbidity and mortality were recorded. After the observation period the animals were euthanized and underwent necropsy.
Materials and Methods


    1. Materials


Test item: BLAD was supplied at 5 mg/ml (yellowish opaque liquid, 0-4° C.) and stored at −80° C.
Animals: Rattus norvegicus, strain: Wistar Hannover, acquired by the vivarium of the Faculty of Veterinary Medicine of Lisbon from Harlan Iberica, Barcelona.
Number of animals used: 30.
Body weight: 250-300 g.
Age: 10 weeks.
Lodging: the animals were individually placed in polyethylene boxes with sterilized wood shavings (Lignocel).
Ambient conditions:

    
    
        a) Photoperiod: cycles of light/dark for 12 h in 12 h.
        b) Controlled environment: an average temperature of 19/22° C. and average humidity of 60%.
    
    


Adaptation: the animals were kept under environmental conditions of the test for seven days before the start of the test.
Food: Global Diet 2014, Rodent Maintenance Diet supplied by Harlan Iberica, Barcelona; water ad libitum.

    2. Methods


Administration: an aliquot of 1 ml (at either 200 μg/ml or 400 μg/ml) was applied to each animal by oral (oro-esophageal) intubation, commonly known as gavage. The administration was carried out with a metal probe appropriate to the species of animal used. The animals were subjected to fasting for 18 h prior to administration and fed 3 h following administration.
Study design: the 30 animals of the study were divided into four groups, two groups of ten animals each and two groups with five animals each. A group of ten animals was exposed to BLAD at 200 μg/ml (test group 1) and another group of ten animals was exposed to BLAD at 400 μg/ml (test group 2). The two groups of five animals served as controls: one group was exposed to water (1 ml aliquot) whilst another group was not subjected to any administration but handled as per all the other groups.
Outcomes: after administration the animals were observed daily for 15 days to record any signs of morbidity or even death. Body weight was individually assessed before exposure and at the end of test period. After the observation period the animals were euthanized (by asphyxiation in an atmosphere saturated with carbon dioxide) for subsequent post-mortem examination.
Results
At neither concentration of BLAD were there signs of any physical changes or changes in drinking/feeding or general behavior. No adverse reactions or death occurred upon BLAD administration. Increase in body mass was similar in all groups (and was consistent with the increase expected from developing animals of such young age). Necropsy/macroscopic observation of the organs of the thoracic and abdominal cavity revealed no changes thereto.
Conclusions
BLAD at concentrations up to 400 μg/ml (and possibly higher) does not show oral toxicity.

## Claims

1. A method of inhibiting spoilage of foodstuffs by a microorganism comprising administering to a foodstuff in need thereof an effective amount of a composition comprising an antimicrobial polypeptide comprising SEQ ID NO:4 or an active variant thereof which has antimicrobial activity which comprises a sequence which has at least 85% identity to either SEQ ID NO:4 or to a fragment of SEQ ID NO:4 which is at least 100 amino acids in length, and in the case where the foodstuff is a plant foodstuff it is a harvested foodstuff, and said inhibiting of spoilage occurs during storage, transport, handling, processing or display of the foodstuff.

2. The method according to claim 1 wherein the microorganism is a fungus.

3. The method according to claim 2 wherein the fungus is a food-spoiling species of a genera selected from the group consisting of: Alternaria, Aspergillus, Fusarium, Botrytis, Collectotrichum, Saccharomyces, Kluyveromyces and Zygosaccharomyces.

4. The method according to claim 1 wherein the foodstuff is derived from, provides, or is, a fruit, a nut, a vegetable, a seed, a sugar, a dairy product, a liquid or paste food, meat, fish or bread.

5. The method according to claim 1 wherein the foodstuff is a strawberry.

6. The method according to claim 1 wherein the microorganism is Botrytis cinerea or Colletotrichum acutatum.

7. The method according to claim 1 wherein said composition further comprises a chelating agent.