Algal extracts with Anti-microbial activity for use on plant matter

Abstract

The present invention provides compositions comprising extracts from green algae to prevent microbial infestation, including the reduction of mold and fungal growth on plants or post-harvest fruits and vegetables. The present invention further provides compositions comprising environmentally safe aqueous extracts of Ulva Lactuca as antifungal agents that may be used in various forms including in conjunction with coatings for postharvest produce in order to prolong shelf-life, prevent shrinkage and preserve the appearance and gloss of fruits and vegetables.

Description

[0001] ALGAL EXTRACTS WITH ANTI MICROBIAL ACTIVITY FOR USE ON PLANT

[0002] MATTER

[0003] FIELD OF THE INVENTION

[0004] The present invention provides compositions comprising algal extracts having antimicrobial activity useful for application to plant matter. The composition can be used in various forms including in conjunction with various coatings to prevent mold and fungal growth on plants or postharvest fruits and vegetables as an environmentally acceptable alternative to synthetic fungicides.

[0005] BACKGROUND OF THE INVENTION

[0006] Fruits and vegetables are coated during postharvest as a way to extend their shelf life and improve their quality. Postharvest coatings form a physical protective barrier around the fruit or vegetable, which slows down the physiological processes and reduces the exposure to physical, biological, and environmental stresses. Coatings offer multiple benefits, including improved produce quality for consumers, decreased food waste, and greater food security in regions affected by food insecurity.

[0007] Spoilage of postharvest produce is caused by various factors including microbial growth, fungal growth, respiration, water loss, physical damage and chemical reactions, such as oxidation and enzymatic browning of the produce. A variety of coatings have been developed to prevent postharvest produce spoilage including edible coatings, film-forming coatings, breathable coatings, and antimicrobial coatings.

[0008] Edible coatings are primarily made from materials such as waxes, starches, and proteins, and are designed to provide a physical barrier against moisture, gases, and microorganisms. Film-forming coatings form a continuous film over the surface of the produce which helps to reduce water loss and slow down respiratory processes. Breathable coatings are utilized to allow the exchange of gases, but limit the exchange of moisture, which helps to maintain the quality of the fruit or vegetable. Antimicrobial coatings contain antimicrobial agents to help reduce the growth of microorganisms on produce which is more susceptible to microbial spoilage.

[0009] The postharvest coatings currently used to extend the shelf life of produce have certain limitations. First, some coatings are not suitable for certain types of fruits and vegetables, or they may affect their natural appearance or flavor. Wax-based coatings, for instance, are often used to provide a barrier against moisture and gas exchange, but they can affect the texture, natural flavor and appearance of delicate fruits. Additionally, the coatings may not effectively prevent spoilage caused by microorganisms and may also be permeable to gases and moisture. The use of coatings is also increasingly controversial among consumers due to environmental and health concerns, particularly regarding coatings with non-natural origins. A major limitation of the currently available coatings is their poor ability to protect against fungal infestations, which can reduce consumer appeal, shorten produce shelf-life, and require stricter storage requirements to maintain their quality. Furthermore, the use of synthetic fungicides for the postharvest coatings is undesirable due to potential health concerns.

[0010] US 9,648,890 discloses compositions of postharvest coatings and methods thereof for reducing the weight loss and preserving the natural gloss of post-harvest edible plant matter comprising: the step of applying to the surface of the plant matter a composition comprising: a. an edible wax having a melting temperature lower than about 70°C; b. a hydrocolloid polymer comprising a non-gelling hydrocolloid polymer; c. a fatty acid; d. an emulsifier; and e. water.

[0011] WO 2016 / 084094 discloses a composition for coating a postharvest plant matter, the composition comprising: a hydrocolloid polymer; an edible wax; a fatty acid; an edible alkaline component essentially free of morpholine and / or ammonia; and water, wherein the edible alkaline component allows formation of a homogeneous emulsion without the need for any additional emulsifier.

[0012] US 6,103,768 discloses the use of fatty acids and their derivatives to eradicate existing fungal and bacterial infection in plants, as well as the use of fatty acid to enhance the activity of fungicides, bactericides, and biological control agents.

[0013] Due to the limitations of existing coatings, new types of postharvest coatings are being developed. Algae and algae derivative compositions have shown potential as coating agents owing to their multiple benefits. First, algae based coatings provide a natural and safe alternative to synthetic chemical-based coatings, which are increasingly less desirable among consumers. Additionally, algae-based coatings are effective in maintaining nutrient quality, freshness, and shelf-life of fresh produce and are biodegradable, reducing their environmental impact, a key concern among consumers. Al- Alam et al. disclose a method for inhibiting P. digitatum growth on fresh oranges using 25, 50, 100 and 200 g / L solutions of Ulva Lactuca, a green alga, for the control of post-harvest citrus green mold. Further disclosed is the efficacy of said method on the inhibition of in vitro conidia germination and tube elongation of mold on potato dextrose broth (PDB) (Al-Alam et al., 2022. Arabian Journal of Medicinal & Aromatic Plants, S(l), 155-170).

[0014] Salim et al. disclose emulsions comprising an aqueous extract derived from Ulva Lactuca (8.3%) for the prevention of fungal growth in oranges. In vitro microbial assays assessed antifungal activities of the aqueous extracts and emulsions thereof against Penicillium digitatum. Further disclosed are in vivo assays to evaluate the use of the aqueous extract and emulsions thereof in inhibiting the growth of P. digitatum on orange peer surfaces (Salim et al., 2022. Sustainable Chemistry and Pharmacy, 25, 100583).

[0015] Ultrasonic-assisted extraction (UAE), as a type of plant extract, has emerged as a promising technology in the food industry due to its benefits of preventing taste loss, providing homogeneity, conserving energy, and being environmentally friendly. To optimize the UAE process and maximize product quality, several parameters including ultrasound power, geometry, kinetic studies, and control must be taken into account. (Ranjha et al., 2021. Processes; 9(S ,1406).

[0016] The molecular weight of ulvan, extracted from Ulva ohnoi, was studied for its effects on cytotoxicity and immunomodulatory activity. Results showed that the interaction of ulvan molecular weight and concentration had an impact on cytotoxicity and release of signaling molecules. Low molecular weight ulvan enhanced cell proliferation with little to no immunomodulatory activity, while high molecular weight ulvan was found to be more bioactive. (Kidgell et al., 2020. International Journal of Biological Macromolecules, 150, 839- 848).

[0017] There remains an unmet need for a natural and eco-friendly coating that can overcome the limitations of current coatings. Such a coating should be inexpensive, nontoxic, and possess broad-spectrum antimicrobial properties while being economically formulated. Additionally, it should also provide an extended shelf-life, while preserving the organoleptic properties of the postharvest produce. SUMMARY OF THE INVENTION

[0018] The present invention provides compositions comprising aqueous extracts of green seaweed incorporated into coatings based on natural, edible and generally recognized as safe ingredients for use on postharvest produce. The major active agent of the seaweed extracts are sulfated polysaccharides of seaweeds including the galactans (e.g., agarans and carrageenans), ulvans, and fucans.

[0019] As disclosed herein for the first time, an extract of Ulva Lactuca is compatible with a wide variety of coatings useful for extending the shelf life of postharvest produce. The ulvan extract may be incorporated into coatings that include combinations of hydrocolloids and lipids such as a wax; the coatings include monoacylglycerides similar to those that constitute the cutin layer of the plant cuticle; or coatings that are based on carboxylic acids.

[0020] The present invention is based, in part, on an unexpected finding that it is possible to significantly improve the antifungal activity of Ulva Lactuca seaweed extract by decreasing the size of the sulfated polysaccharides and / or increasing the content of the biologically active substance, ulvan, thereby improving the biological activity of the extract and subsequently the efficacy of the coating composition in which it is present. Unexpectedly, as exemplified hereinbelow, the efficacy of the extract is significantly improved by decreasing the particle size of the extracted matter. Without wishing to be bound by any theory or mechanism of action, it is contemplated that the decreased size of the extracted matter increases both the surface area of the particles in contact with the surface being coated and the ability to incorporate the extracted matter in a uniform manner in the coating composition.

[0021] The present invention provides a composition for coating a plant matter comprising an aqueous algal extract of Ulva Lactuca seaweed comprising ulvan or ulvan particles extracted in an aqueous solution, or an aqueous suspension, respectively, that is subsequently subjected to sonication or enzymatic degradation to decrease the average size of the polymer. The present invention further provides a fungicidal composition for application to plant matter which can be used in various forms including as a powder, as part of the packaging materials and in conjunction with various coatings. Further provided are antifungal coatings for plant matter, comprising a combination of an algal sulfated polysaccharide, a base and a carboxylic acid having between 10 and 35 carbons. The base interacts with the acidic sulfated polysaccharides to form salts which may be ammonium, magnesium, calcium, sodium, or potassium.

[0022] According to a first aspect, there is provided an antimicrobial composition for treating or preventing fungal or bacterial infestation of a plant matter, wherein the antimicrobial composition comprises an aqueous extract of green seaweed comprising a sulfated polysaccharide having an average particle size in the range of about 50 nm to about 2000 nm as determined by dynamic light scattering, including each value within the specified range.

[0023] In some embodiments, the algal sulfated polysaccharide is selected from the group consisting of ulvans, fucans, galactans, agarans, and carrageenans. Each possibility represents a separate embodiment. In other embodiments, the algal sulfated polysaccharide is extracted from the seaweed species selected from the group consisting of Rhodophyta (red), Phaeophyta (brown), and Chlorophyta (green) macroalgae. Each possibility represents a separate embodiment. In some embodiments, the algal sulfated polysaccharide is ulvan. In further embodiments, the algal sulfated polysaccharide is ulvan extracted from seaweed of the genus Ulva. In some particular embodiments, the species within the genus Ulva is Ulva Lactuca.

[0024] According another aspect, there is provided an antimicrobial composition for treating or preventing fungal or bacterial infestation of a plant matter, wherein the antimicrobial composition comprises an aqueous extract of green seaweed comprising an antimicrobial effective amount of ulvan having an average particle size in the range of about 50 nm to about 2000 nm as determined by dynamic light scattering, including each value within the specified range.

[0025] In certain embodiments, the composition is a dry composition. In other embodiments, the composition is in a form of an aqueous dispersion. In further embodiments, the aqueous dispersion further comprises an agricultural or food-grade acceptable carrier.

[0026] In various embodiments, the antimicrobial effective amount of the ulvan is between about 0.2% (w / w) to about 5% (w / w) of the total weight of the composition, including each value within the specified range.

[0027] In some embodiments, the ulvan is fragmented by a method selected from enzymatic digestion, exposure to chemical degradation, and sonication. Each possibility represents a separate embodiment. In some embodiments, the ulvan extract undergoes sonication to reduce the molecular weight. In alternative embodiments, the sonication disrupts the physical size of the particles of ulvan in the extract without significantly reducing the molecular weight. Unexpectedly, the efficacy of the sonicated extract is superior to the native extract even when the molecular weights are not reduced.

[0028] In some embodiments, the ulvan extract undergoes sonication to reduce the average particle size. According to currently preferred embodiments, the sonication is performed at high power. According to specific embodiments, the sonication is performed while utilizing an ultrasonic power of at least 200 Watts. According to some embodiments, the sonication is performed at about 400 Watts. According to some embodiments, the sonication comprises contacting the ulvan extract with the sonication horn / probe. In further embodiments, the sonication is performed in an acidic environment. In alternative embodiments, the sonication is performed in an alkaline environment. According to certain embodiments, the composition is prepared by providing an aqueous extract of an Ulva species comprising ulvan, preferably by extracting seaweed biomass of the Ulva species in an aqueous solution; and exposing the ulvan to conditions suitable for decreasing its average particle size. In specific embodiments, extracting seaweed biomass of the Ulva species in an aqueous solution comprises heating the seaweed biomass in distilled water at a temperature of about 85°C to about 100°C, including each value within the specified range. In some embodiments, exposing the ulvan to conditions suitable for decreasing its average particle size is performed by: (i) exposing the ulvan to ultra-sonic waves; (ii) exposing the ulvan to a peroxide; or (iii) exposing the ulvan to an enzyme capable of degrading it. Each possibility represents a separate embodiment.

[0029] According to the principles of the present invention, it is disclosed herein for the first time that the antifungal activity and efficacy of the ulvan extract after sonication is superior to the suspension comprising particles that comprise native ulvan (i.e., non-sonicated) polysaccharide molecules.

[0030] In the sonicated ulvan extract, the particle size is decreased to an average particle size in the range of about 50 nm to about 2000 nm, including each value within the specified range. According to some embodiments, the average particle size after sonication is in the range of about 100 nm to about 1200 nm, including each value within the specified range. In further embodiments, the average particle size after sonication is in the range of about 100 nm to about 1000 nm, including each value within the specified range. In additional embodiments, the average particle size after sonication is in the range of about 100 nm to about 800 nm, including each value within the specified range. In other embodiments, the average particle size after sonication is in the range of about 50 nm to about 600 nm, including each value within the specified range. In yet other embodiments, the average particle size after sonication is in the range of about 50 nm to about 400nm, including each value within the specified range. Typically, the sonicated extract will contain a fraction with molecular weights below 1200 nm, below 1000 nm, below 900 nm, below 800 nm, below 700 nm, below 600 nm, below 500 nm, below 400 nm, below 300 nm, or below 200 nm. Each possibility represents a separate embodiment.

[0031] According to various embodiments, the particles in the native ulvan extract prior to sonication have an average particle size of above 2000 nm and up to about 6000 nm, including each value within the specified range. According to various embodiments, the particles in the sonicated ulvan extract have an average particle size in the range of about 50 to about 2000 nm, including each value within the specified range. According to other embodiments, the particles in the sonicated ulvan extract have an average particle size in the range of about 100 to about 1200 nm, including each value within the specified range. According to other embodiments, the particles in the sonicated ulvan extract have an average particle size in the range of about 200 to about 1000 nm, including each value within the specified range. According to yet other embodiments, the particles in the sonicated ulvan extract have an average particle size in the range of about 100 to about 800 nm, including each value within the specified range.

[0032] According to some specific embodiments, sonication is performed using the horn / probe method and not in a sonication bath. During sonication, a high cavitation field is generated by an adjustable combination of horn / probe dimensions. According to some specific embodiments, the sonication is performed using a cylindrical tip with base dimensions of 1.5-4 cm2and a surface area of 6-10 cm2, including each value within the specified ranges. According to some embodiments, the power used is in the range 250-1000 Watts, including each value within the specified range. According to some specific embodiments, the sonication is performed using a vibrational mode with 80% of additional vibrational force and 80% of maximal peak-to-peak amplitude of the device. These settings yield a sonicator output in the range of 18-1600 Ws / ml, including each value within the specified range. In other embodiments, the ulvan extract undergoes enzymatic digestion to reduce the molecular weight. In additional embodiments, the enzymes utilized for the enzymatic digestion of the ulvan extract comprise a ulvan lyase. In other embodiments, the ulvan extract undergoes a chemical reaction to reduce the molecular weight. In certain embodiments, the chemical reaction for the reduction of the ulvan extract molecular weight is an alkaline or acidic thermochemical degradation.

[0033] In other embodiments, the ulvan extract undergoes enzymatic digestion to reduce the particle size. In additional embodiments, the enzymes utilized for the enzymatic digestion of the ulvan extract comprise an ulvan lyase. In other embodiments, the ulvan extract undergoes a chemical reaction to reduce the particle size. In certain embodiments, the chemical reaction for the reduction of the ulvan extract particle size is an alkaline or acidic thermochemical degradation.

[0034] In some embodiments, the extraction process is extraction in heated water. In additional embodiments, the aqueous extract is dried and further exposed to ethanol. In other embodiments, the extraction is selected from a microwave-assisted extraction, a pressurized liquid extraction, an enzymatic-assisted extraction, and a pulse-electric field extraction. Each possibility represents a separate embodiment. In some embodiments, the extract is an aqueous suspension comprising particles comprising ulvan.

[0035] According to another aspect, there is provided an aqueous composition comprising: (a) an extract of Ulva seaweed comprising fragmented ulvan characterized by an average particle size of 100 nm to 1000 nm; (b) a carboxylic acid having 10-35 carbon atoms; (c) a base; (d) an edible wax or wax substitute; and (e) water. In certain embodiments, the fragmented ulvan is present in the composition at a concentration of at least about 0.01% (w / w) of the total weight of the composition.

[0036] In some embodiments, the carboxylic acid is selected from the group consisting of saturated and unsaturated, straight chain or branched chain aliphatic acids having 10-35 carbons. In some embodiments, the carboxylic acid of the fungicidal composition is characterized by having 10-18 carbons. In other embodiments, the carboxylic acid comprises 19-24 carbons. In other embodiments, the carboxylic acid comprises 25-35 carbons. In other embodiments, the carboxylic acid of the fungicidal composition has 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 carbons. Each possibility represents a separate embodiment. In some embodiments, the base is selected from the group consisting of ammonium, magnesium, sodium, and potassium hydroxide. Each possibility represents a separate embodiment. In some embodiments, the composition is characterized by a basic pH. In some particular embodiments, the composition has a pH ranging between 8 and 10, for example between 8 and 9.5, including each value within the specified ranges.

[0037] In some embodiments, the edible was or wax substitute is beeswax. In one embodiment, the composition comprises beeswax in the range of 1% to 25% w / w, including each value within the specified range. In some specific embodiments, the composition comprises about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20 of beeswax. Each possibility represents a separate embodiment.

[0038] According to yet another aspect, there is provided an aqueous composition comprising: i. 0.01% (w / w) - 15% (w / w) of an extract of Ulva seaweed comprising fragmented ulvan characterized by an average particle size of 100 nm to 1000 nm; ii. 1% (w / w) - 50% (w / w) of an edible wax or wax substitute; iii. 0.25% (w / w) - 5% (w / w) of an emulsifier; and iv. water to 100% of the total weight of the composition, including each value within the specified ranges. In one embodiment, the emulsifier is an anionic surfactant.

[0039] In additional embodiments, the composition comprises at least one additive. In some embodiments, the additive is selected from the group consisting of a monoglyceride, a diglyceride, a sorbitan ester, a polysorbate, a sucrose ester, a fatty acid, a sucroglyceride, and a combination thereof. Each possibility represents a separate embodiment. In specific embodiments, the monoglyceride additive is selected from the group consisting of short and long chain carboxylic acids. In exemplary embodiments, the diglyceride additive is selected from the group consisting of short and long chain carboxylic acids.

[0040] In further embodiments, the composition further comprises an edible hydrocolloid polymer selected from the group consisting of locust bean gum (LBG), guar gum, xanthan gum, lambda-carrageenan, and combinations thereof. Each possibility represents a separate embodiment. In particular embodiments, the edible hydrocolloid polymer is present in a weight percent ranging from about 0.1% (w / w) to about 1.5% (w / w) of the total weight of the composition, including each value within the specified range.

[0041] In some embodiments, the composition is characterized by a viscosity of 1-500 cP, including each value within the specified range. The compositions disclosed herein are useful for application to plant matter, for example fruits and vegetables as set forth in detail hereinbelow. In some embodiments, the composition is formulated for application to plant matter. In various embodiments, the application is selected from postharvest application and pre-harvest application. In additional embodiments, the application is performed by any one of dipping, coating, immersing, spraying, rubbing or pouring. Each possibility represents a separate embodiment. In particular embodiments, the composition decreases fungal infestation. In further embodiments, the fungal infestation is derived from a fungus selected from the group consisting of penicillium digitatum, penicillium Italicum, geotrichum citri-aurantii, and alternaria alternata. Each possibility represents a separate embodiment. In additional embodiments, the composition reduces weight loss of a plant matter coated with the composition by at least 20%.

[0042] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 is a graph depicting the decreased weight loss of P. digitatum inoculated oranges treated with Algal formulation A, Algal formulation B, Suf 3.5, and a control of non-coated oranges over 26 days at 20°C.

[0045] Figure 2 is a bar graph depicting the average firmness of P. digitatum inoculated oranges treated with Algal formulation A, Algal formulation B, Suf 3.5, and a control after 5, 12, 19, and 26 days at 20°C. Firmness index scale: 0 = "hard", 1= "starts softening", 2 = "soft", 3 = "very soft", and 4 = "over soft / ripe". n=30 oranges per treatment.

[0046] Figure 3 is a bar graph depicting the infected decay (%) of P. digitatum inoculated oranges treated with Algal formulation A (SufUlv-A), Algal formulation B (SufUlv-B), algal extract (C-Algex), Control + IMZ + TBZ (D-SufFung), and a negative control over 1, 2 and 3 weeks at 20°C. n=24 oranges per treatment. Figure 4 is a bar graph depicting the total number of infected spots counted on lemons that had previously been coated with different coating formulations and inoculated with 106spores / mL of P. digitatum. For the formulations, Ulva lactuca was obtained from two suppliers (Seakura (S) or VIET D.E.L.T.A. INDUSTRIAL CO., LTD (I)), different concentrations (I 5%, 12.5%, I 1.25 % w / w and S 5%, S 2.5%, S 1.25 % w / w) of extracts of the seaweed were mixed with Sufresca beeswax formulation 3.5% w / w. Negative control of untreated / uncoated oranges (Control) and positive control of Beeswax with imazalil (1000 ppm) and thiabendazole (2000 ppm) (Control +IMZ TBZ) are depicted as well.

[0047] Figure 5 is a graph depicting the decay (%) of P. digitatum inoculated oranges treated with the Sufresca Algal-formulation 3.75% and a control over 4 weeks at 20°C.

[0048] Figure 6 is a bar graph depicting the decay (%) of P. digitatum inoculated oranges treated with Algal-I (5%), Algal-IW (5%), an untreated negative control (white), and a positive control (imazalil 1,000 ppm and thiabendazole 500 ppm) (black) over 3, 4 and 5 days at 20°C.

[0049] Figure 7 is a bar graph depicting the infected decay (%) of P. digitatum inoculated oranges treated with Algal-IW (5%), Algal-IWS, an uncoated negative control (white), and a positive control (imazalil 1,000 ppm, thiabendazole 500 ppm) (black) over 3, 4 and 5 days at 20°C.

[0050] Figures 8A-8C are photographs of oranges coated with coating formulations comprising sonicated or unsonicated ulvan extract on Day 5 after the inoculation with P. digitatum. (8A) Uncoated oranges, negative control; (8B) oranges coated with Algal-IW (5%); and (8C) oranges coated with Algal-IWS (5%).

[0051] Figure 9 is a bar graph depicting the infected decay (%) of P. digitatum inoculated oranges treated with Suf 10% (grey), Algal-IWS Suf 10% (checkered), an uncoated negative control (white), and a positive control (imazalil 1,000 ppm, thiabendazole 500 ppm) (black) over 3, 4 and 5 days at 20°C.

[0052] Figures 10A-10C depict P. digitatum growth in wells with Potato Dextrose Broth (PDB) on Day 2 after treatment with different formulations. Figure 10A, P. digitatum growth following treatment with water (negative control); Figure 10B, P. digitatum growth following treatment with coating formulation comprising unsonicated ulvan extract, Algal IW (1%); and Figure 10C, P. digitatum growth following treatment with coating formulation comprising sonicated ulvan extract, Algal IWS (1%).

[0053] Figure 11 shows particle size distributions (nm) of 12 measurements of ulvan extracts: a sonicated extract (grey) and a reference extract of ulvan extract prior to sonication (black).

[0054] Figure 12 depicts zeta potential distributions (mV) of 12 measurements of ulvan extracts: a sonicated extract (grey) and a reference extract of ulvan extract prior to sonication (black).

[0055] Figure 13A is a photograph of an emulsion coating formulation comprising 1% ulvan extract prior to sonication formulated with 10% beeswax, 0.9% oleic acid, 0.25% ammonium hydroxide, and <0.1% hydrocolloid, that was left to sit for 24hrs at room temperature.

[0056] Figure 13B is a photograph of a coating formulation comprising 1% sonicated ulvan extract formulated with 10% beeswax, 0.9% oleic acid, 0.25% ammonium hydroxide and <0.1% hydrocolloid, that was left to sit for 24hrs at room temperature.

[0057] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0058] The present invention provides improved compositions for coating a plant matter based on Ulva species algal extract in order to prolong shelf-life, improve appearance, reduce weight loss and decrease susceptibility to fungal infestation using only eco-friendly ingredients. It is now disclosed that the algal extracts are effective fungicides that are compatible with a wide range of types of coatings. Accordingly, they can be incorporated into coatings based on edible waxes, hydrocolloids, carboxylic acids and monoacylglycerides .

[0059] Problems that need to be overcome to achieve a viable, effective, and reproducible product include variability of the amount of the ulvan fraction within the algal biomass. The different bio-chemical constituents within the algal biomass vary according to the different algal species, seasonality, or aquaculture methods. Besides the variability of the amounts of the ulvan within the algae, there might be a significant variability in the structure of the polysaccharide and its level of sulfation, which has a direct effect on the bioactivity of the ulvan. Additionally, the content of other elements within the algae such as proteins, starch, minerals and bioactive moieties vary. According to certain embodiments, the degree of sulfation is at least 2%. According to other embodiments, the degree of sulfation is 2-20%. According to various embodiments, the degree of sulfation is 5-10%.

[0060] One of the options that can be used to overcome the variability of the extraction process is to minimize the variability of the biomass. This could be achieved by precise aquaculture methods such as the use of photo-bioreactors and aquaculture in closed systems. However, these methods are highly costly. Alternative strategies that may be used to improve the reproducibility of the product include various pretreatment of the bulk seaweed. These pretreatments may be selected from the group consisting of enzymatic treatment, fragmentation protocols, sonication and use of possible solvents.

[0061] The main biologically active compounds in an extract of Ulva seaweed species comprise sulfated polysaccharides referred to collectively as ulvan, and various additional components such as proteins, starch, minerals, oligo-sugars and other bioactive moieties. However, the amounts of the ulvan or the additional compounds within the algae may vary, as well as the structure of the ulvan and its sulfation, consequently, resulting in a direct effect on the bioactivity of the ulvan. According to some embodiments, the extract of Ulva seaweed species is an aqueous suspension comprising particles comprising ulvan, namely polysulfated polysaccharide molecules.

[0062] The present invention is based in part on an unexpected finding that it is possible to significantly improve the activity and reproducibility of the Ulva Lactuca seaweed extract by reducing the size of the particles comprising ulvan, decreasing the molecular weight of the sulfated polysaccharides, and / or increasing the fraction of the biologically active substance, ulvan.

[0063] According to one aspect, the present invention provides a fungicidal composition for coating plant matter, comprising a combination of an algal sulfated polysaccharide, a base and a carboxylic acid having between 10 and 35 carbons. The base interacts with the acidic sulfated polysaccharides to form salts. According to various embodiments, the salts may be selected from the group consisting of ammonium, magnesium, sodium, potassium, and calcium. According to a specific embodiment, the present disclosure provides compositions for coating a plant matter comprising an aqueous extract of Ulva species, a carboxylic acid having 10-35 carbons and a base. The composition can be advantageously applied in the form of an emulsion.

[0064] The native ulvan extract can be incorporated into a fungicidal composition including, but not limited to, an emulsion. However, these emulsions were not stable for more than a day or two at most. Notably, the sonicated ulvan extract was able to support stable emulsion compositions for up to six months.

[0065] According to alternative embodiments, as exemplified hereinbelow, the combination of a wax-based coating composition and the ulvan containing aqueous extract of Ulva species is stable and can avoid the necessity for use of synthetic fungicides. Unexpectedly, it is disclosed herein for the first time that the extract of Ulva species is compatible with coating composition that is wax based. According to certain embodiments, the Ulva species seaweed is fresh. The term "fresh" as used herein refers to a seaweed having at least 80% (w / w) water, out of the total weight of the seaweed. According to alternative embodiments, the Ulva species seaweed is dried. The term "dried" as used herein refers to a seaweed that has been dried such that it contains less than 10% (w / w) water, of the total weight of the seaweed.

[0066] The terms “algal extract”, "ulvan extract", "aqueous extract of Ulva species", "aqueous algal extract of Ulva Lactuca seaweed", "aqueous algal extract", “Ulva Lactuca seaweed extract", "extract of Ulva Lactuca", and "aqueous extracts of green seaweed" as used herein refer to the separated aqueous phase of the Ulva species seaweed biomass following an aqueous extraction with exposure to heated water with or without ethanol procedure, comprising ulvan, or particles comprising ulvan. In addition to the ulvan, the extract may comprise additional components from the algae selected from proteins, starch, minerals, oligo-sugars, and bioactive moieties.

[0067] The terms “bioactive compound” or "bioactive moiety" may be defined as agents that are present in small amounts in plants and certain foods (such as fruits, vegetables, nuts, oils, and whole grains). These agents are extra-nutritional constituents that provide health benefits beyond the basic nutritional value of the product exerting beneficial physiological, behavioral, and immunological effects. Exemplary bioactive compounds include, but are not limited to, carotenoids, flavonoids, carnitine, choline, coenzyme Q, dithiolthiones, phytosterols, phytoestrogens, glucosinolates, polyphenols, and taurine. Since vitamins and minerals elicit pharmacological effects, they can be categorized as bioactive compounds as well.

[0068] According to some embodiments, the fungicidal or antimicrobial composition disclosed herein comprises an aqueous extract of green seaweed comprising an antimicrobial effective amount of ulvan. The term “an antimicrobial effective amount” as used herein refers to the amount of a ulvan that is sufficient to inhibit the proliferation and / or growth of a yeast, mold or bacteria strain as defined herein. In certain embodiments, the extract comprises ulvan in a weight percent of at least about 1% (w / w) from the total weight of the algal extract. In further embodiments, the extract comprises ulvan in a weight percent of at least about 5% (w / w), at least about 10% (w / w), at least about 15% (w / w), of the total weight of the algal extract. Each possibility represents a separate embodiment of the invention. According to some embodiments, the extract comprises ulvan in the range of about 0.01 to about 12% (w / w) of the total weight of the algal extract, including any range therebetween. According to further embodiments, the extract comprises ulvan in the range of about 0.01 to about 10% (w / w) of the total weight of the algal extract, including any range therebetween. According to other embodiments, the extract comprises ulvan in the range of about 0.01 to about 5% (w / w) of the total weight of the algal extract, including any range therebetween. According to other embodiments, the extract comprises ulvan in the range of about 0.01 to about 3% (w / w) of the total weight of the algal extract, including any range therebetween. Each possibility represents a separate embodiment of the invention.

[0069] According to some embodiments, the algal extract further comprises at least one of a protein, a mineral and a bioactive moiety.

[0070] According to some embodiments, the algal extract comprises a protein in a weight percent of up to about 0.1% (w / w), of the total weight of the extract. According to further embodiments, the algal extract comprises protein in a weight percent of up to about 0.5% (w / w), or up to about 1% (w / w), of the total weight of the extract. Each possibility represents a separate embodiment of the invention.

[0071] According to some embodiments, the algal extract comprises starch in a weight percent of up to about 0.1% (w / w), of the total weight of the extract. According to further embodiments, the algal extract comprises starch in a weight percent of up to about 0.5% (w / w), or up to about 1% (w / w), of the total weight of the extract. Each possibility represents a separate embodiment of the invention.

[0072] According to some embodiments, the algal extract comprises a mineral in a weight percent of up to about 0.1% (w / w), of the total weight of the extract. According to further embodiments, the algal extract comprises minerals in a weight percent of up to about 0.5% (w / w), or up to about 1% (w / w), of the total weight of the extract. Each possibility represents a separate embodiment of the invention.

[0073] According to some embodiments, the extract comprises a mono-sugar in a weight percent of up to about 0.01% (w / w), of the total weight of the extract. According to further embodiments, the extract comprises a mono-sugar in a weight percent of up to about 0.05% (w / w), or up to about 0.1% (w / w), of the total weight of the extract. Each possibility represents a separate embodiment of the invention.

[0074] According to some embodiments, the extract comprises a bioactive moiety in a weight percent of up to about 1% (w / w), of the total weight of the extract. According to further embodiments, the extract comprises a bioactive moiety in a weight percent of up to about 10% (w / w), or up to about 20% (w / w), of the total weight of the extract. Each possibility represents a separate embodiment of the invention.

[0075] According to some embodiments, the algal extract comprises ulvan in a weight percent of at least 2% and at least one of: a protein in a weight percent of up to about 0.5% (w / w), starch in a weight percent of up to about 0.1% (w / w), a mineral in a weight percent of up to about 0.2% (w / w), a mono-sugar in a weight percent of up to about 0.05% (w / w), and a bioactive moiety in a weight percent of up to about 2% (w / w), of the total weight of the extract. According to further embodiments, the extract comprises ulvan in a weight percent of at least 1% and at least one of: a protein in a weight percent of up to about 0.1% (w / w), starch in a weight percent of up to about 0.1% (w / w), a mineral in a weight percent of up to about 0.1% (w / w), a mono-sugar in a weight percent of up to about 0.01% (w / w), and a bioactive moiety in a weight percent of up to about 1% (w / w), of the total weight of the extract. According to yet further embodiments, the extract comprises ulvan in a weight percent of at least 10% and at least one of: a protein in a weight percent of up to about 0.5% (w / w), starch in a weight percent of up to about 0.5% (w / w), a mineral in a weight percent of up to about 0.5% (w / w), a mono-sugar in a weight percent of up to about 0.05% (w / w), and a bioactive moiety in a weight percent of up to about 2% (w / w), of the total weight of the extract.

[0076] As stated above, the variability in the structure of the polysaccharide and its sulfation has a direct effect on the bioactivity of the algal extract. In order to minimize this variability and reach a uniform chain length, the extracted ulvan was further fragmented. Alternatively, the size of the particles comprising ulvan within the ulvan extract was reduced. Non-limiting examples of suitable techniques for ulvan fragmentation or reduction of particle size include ultra-sonic agitation, chemical fragmentation and enzymatic digestion by ulvanolytic enzymes e.g., Ulvan lyase. Additional techniques include ultrasonic fragmentation in acidic or alkaline environments.

[0077] According to some embodiments, the aqueous extract is in a weight percent ranging from about 0.01% (w / w) to about 15% (w / w) of the total weight of the coating composition. According to further embodiments, the extract is in a weight percent ranging from about 0.5% (w / w) to about 10% (w / w) of the total weight of the composition. In yet further embodiments, the extract is in a weight percent ranging from about 1% (w / w) to about 7.5% (w / w) of the total weight of the composition. The composition for coating usually consists of 75-90% of the extract. The term "extract" as used herein refers to the aqueous solution containing ulvan, or an aqueous dispersion comprising particles comprising ulvan. The concentration of the ulvan in this extract is usually 0.5-1.5%. The terms "sonicated extract" or “fragmented extract” refers to the aqueous suspension containing ulvan, or an aqueous suspension of particles comprising ulvan that underwent sonication to reduce the size of ulvan molecules or ulvan particles. The concentration of the ulvan in such extract is usually 0.5-1.5%.

[0078] According to some embodiments, the aqueous extract is a suspension comprising particles comprising ulvan. According to some embodiments, the size of the particles suspended within the aqueous extract is further reduced. Particle size reduction may be performed using any method known in the art, for example, sonication, enzymatic digestion, or chemical fragmentation.

[0079] Thus, according to the principles of the present invention, following size reduction, particles in the ulvan extract are characterized by a mean particle size in the range of about 100 to about 1200 nm, including any range therebetween. Within the scope of the present invention are mean particle sizes in the range of about 100 to about 1000 nm, about 100 to about 800 nm, about 100 to about 600 nm, about 100 to about 500 nm, or about 100 to about 400 nm, each possibility represents a separate embodiment.

[0080] The terms “average particle size” or “mean particle size” as used herein refer to a statistical average particle size (diameter) in a population of particles. The diameter of a substantially spherical particle can refer to a physical or hydrodynamic diameter. When non- spherical particles are characterized, the size of each particle typically refers to the largest linear distance between two points on the surface of the particle. Average particle sizes can be determined using various techniques known in the art including, but not limited to, laser diffraction, light scattering, sedimentation field flow fractionation, photon correlation spectroscopy, disc centrifugation, the Coulter Counter method, and the like. Each possibility represents a separate embodiment. In some embodiments, the terms “average particle size” or “mean particle size” refer to the mean diameter of an ulvan extract particles derived from particle size distribution based on a number distribution model. In other embodiments, said terms refer to the mean diameter of an ulvan extract particles derived from particle size distribution based on a volume distribution model. In additional embodiments, said terms refer to the mean diameter of an ulvan extract particles derived from particle size distribution based on a surface area distribution model. In currently preferred embodiments, the average particle size is determined by dynamic light scattering.

[0081] As disclosed herein for the first time, the sonicated extract of Ulva Lactuca is compatible with a wide variety of coatings useful for extending the shelf life of postharvest produce. According to various embodiments, the ulvan may be incorporated into coatings that include combinations of hydrocolloids and lipids such as waxes. According to additional embodiments, the extract containing sulfated polysaccharides may be incorporated into coatings containing monoacylglycerides, which are similar to those that also constitute the cutin layer of the plant cuticle. According to yet further embodiments, the ulvan may be incorporated into coatings that include medium chain or long chain carboxylic acids and bases.

[0082] According to certain embodiments, the composition comprises the algal extract, a carboxylic acid having 10-35 carbon atoms, and a base.

[0083] According to some embodiments, the composition comprises the algal extract, an edible wax, and an emulsifier. According to additional embodiments, the composition comprises the algal extract, an edible wax, a hydrocolloid, and an emulsifier. According to alternative embodiments, the composition comprises the algal extract and monoacylglycerides .

[0084] The term “carboxylic acid” as used herein refers to a carboxylic acid group ( — COOH), which is connected to a straight or branched aliphatic chain containing 10-35 carbon atoms. The carboxylic acid may be saturated or unsaturated with each possibility representing a separate embodiment. In some embodiments, the carboxylic acid contains 10- 18 carbon atoms. In other embodiments, the carboxylic acid contains 19-24 carbon atoms. In further embodiments, the carboxylic acid contains 25-35 carbon atoms.

[0085] According to some embodiments, the carboxylic acid is present in a weight percent ranging from about 0.5% (w / w) to about 10% (w / w) of the total weight of the composition. According to further embodiments, the carboxylic acid is present in a weight percent ranging from about 1% to about 5% of the total weight of the composition.

[0086] The term “base” as used herein refers to an alkaline compound such as hydroxides, carbonates, and bicarbonates. Suitable bases include, but are not limited to, ammonium, magnesium, calcium, sodium, or potassium hydroxide. The bases may form a salt with the carboxylic acids or with the polysaccharides in the composition.

[0087] According to some embodiments, the base is present in a weight percent ranging from about 0.1% (w / w) to about 5% (w / w) of the total weight of the composition. In certain embodiments, the base is present in a weight percent of 0.1% (w / w) or more of the total weight of the composition. In certain embodiments, the base is present in a weight percent of 5% (w / w) or less of the total weight of the composition, although the appropriate percentage of the base will be determined for the actual base used, as is well known to one of skill in the art.

[0088] Ulvan bioactivity is known to relate to its conformation, and in turn, polysaccharide configuration is strongly related to the pH of the medium in which it is maintained. Ulvan is usually extracted and maintained at neutral to acidic pH (2-6). However, the inventors of the present application have surprisingly found that the bioactivity of ulvan was increased in a coating composition having a basic pH. According to some embodiments, the composition has a pH ranging from about 8.0 to about 10.0, including each value within the specified range.

[0089] Wax based formulations The term “edible wax” as used herein refers to synthetic waxes suitable for human consumption, such as food-grade petroleum products, and to natural waxes obtained from plants, insects (honeybees or others) or animals.

[0090] According to some embodiments, the edible wax is selected from the group consisting of beeswax, carnauba wax, candelilla wax, alpha wax, rice-bran wax, Japan wax, polyethylene wax, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. In a specific embodiment, the edible wax is beeswax.

[0091] According to some embodiments, the edible wax is present in a weight percent ranging from about 1% (w / w) to about 25% (w / w) of the total weight of the composition. According to further embodiments, the edible wax is present in a weight percent ranging from about 1.5% to about 20% of the total weight of the composition. According to yet further embodiments, the edible wax is present in a weight percent ranging from about 2% to about 20% of the total weight of the composition.

[0092] Additional components that may be incorporated in the compositions and coatings disclosed herein are emulsifiers. The term “emulsifier” as used herein refers to an amphiphilic molecule that is a surface-active agent and that stabilizes emulsions by reducing the interfacial tension.

[0093] According to some embodiments, the emulsifier is an edible emulsifier selected from the group consisting of lecithin, polysorbate, sorbitan ester, sucrose ester, fatty acid, sucroglyceride of fatty acids, ethylene glycol monostearate, ammonium lauryl sulfate, sodium stearoyl-2-lactylate, potassium oleate, ammonium oleate, propylene glycol monostearate, sodium alkyl sulfate, oleic acid and polyethylene glycol (PEG) and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some exemplary embodiments, the emulsifier is ammonium oleate.

[0094] According to some embodiments, the emulsifier is present in a weight percent ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the composition. According to further embodiments, the emulsifier is present in a weight percent ranging from about 0.5% (w / w) to 4% (w / w) of the total weight of the composition. In yet further embodiments, the emulsifier is present in a weight percent ranging from about 1% (w / w) to 3% (w / w) of the total weight of the composition. According to some embodiments, the composition for coating fruit or vegetables is a wax based emulsion formulation comprising an edible wax, surfactants, a pH regulator and the algal extract.

[0095] In exemplary wax based formulations, beeswax constitutes 1.0%-25.0% of the composition. Without being bound by theory, it is contemplated that the lipophilic component of the wax forms a semi-permeable barrier on the surface of the fruit upon coating. Surfactants are typically included in amounts that constitute 0.5%-5.0% of the composition. Without being bound by theory, it is contemplated that the surfactant, typically an anionic surfactant, stabilizes the lipophilic colloids. Suitable surfactants within the scope of the present invention include, but are not limited to, fatty acids selected from the group consisting of stearic acid, palmitic acid, oleic acid, lauric acid, and myristic acid. According to certain aspects and embodiments, a pH regulator constituting 0.2%-2.0% of the wax based formulations is added. Without being bound by theory, it is contemplated that the pH regulator negatively charges the surfactant, thereby generating a stabilizing electrostatic repulsion between the colloids in the liquid formulation, suitable pH regulators within the scope of the present invention include, but are not limited to, KOH, NaOH, and NH4OH (ammonia). As is known to those skilled in the art, ammonia can evaporate during the manufacturing of the formulation. The medium of the emulsion is preferably water, for example, double distilled water (DDW) which constitute 70% - 97% of the total weight when applied to the fruit.

[0096] According to some embodiments, the coating composition with the algal extract comprises: from about 0.01% to about 15% (w / w) of Ulvan, from about 1% to about 25% (w / w) of an edible wax, from about 0.5% to about 5% (w / w) of an emulsifier, and water to 100% of the total weight of the composition.

[0097] According to other embodiments, the composition comprises: from about 0.5% to about 12% (w / w) of Ulvan, from about 5% to about 25% (w / w) of a carboxylic acid, from about 0.2% to about 2% (w / w) of a base, and water to 100% of the total weight of the composition. According to some alternative embodiments, the compositions comprise 75- 90% of the extract where "extract" refers to the aqueous composition containing ulvan, or aqueous suspension containing particles comprising ulvan. The concentration of the ulvan in this extract is usually 0.5-1.5%. formulations with beeswax substitutes

[0098] For vegans or for use in countries that do not approve the use of beeswax, the beeswax may be replaced by a wax or a combination of several waxes of plant origin. The amounts of the beeswax substitutes may be calculated based on the contents of the long- chained alkanes, such as heptacosane, that are present in various quantities in the different waxes. Suitable plant-based waxes include, but are not limited to, rice bran, candelilla wax and carnauba wax.

[0099] The coating composition may be a colloidal emulsion containing algal extract. The coating before addition of algal extract may comprise: beeswax substitutes comprising 1.0%- 25.0% of the formulation; surfactants: 0.5%-5.0% of the formulation; pH regulator: 0.2%- 2.0% of the formulation.

[0100] The surfactants may be both non-ionic and anionic. Suitable surfactants include, but are not limited to, sorbitan esters (also known as Spans), ascorbyl palmitate, ascorbyl stearate, stearic acid, palmitic acid, and other fatty acids.

[0101] A pH regulator is a stabilizing agent that negatively charges the surfactant, hence generating a stabilizing electrostatic repulsion between the colloids in the liquid formulation. The pH regulator may be evaporated during the manufacturing of the formulation. Suitable pH regulators include, but are not limited to, KOH, NaOH, and NH4OH. When non-ionic surfactants are used without the anionic surfactants, the use of pH regulators may be avoided.

[0102] Additional optional components of the emulsion formulations include hydrocolloids in an amount of 0.01 to 1.0% to result in a colloidal emulsion. The hydrocolloids may be selected from tara gum, cassia gum, guar gum, and locust bean gum.

[0103] Additional optional components include a chemical stabilizer that regulates the viscosity of the formulation.

[0104] The medium of the emulsion is typically water, for example, double distilled water (DDW) which constitute 70% - 97% of the total weight when applied to the plant matter.

[0105] Wax-less formulations

[0106] Alternatives to the wax based formulations include wax-less formulations. Beeswax substitutes comprise 1.0%-25.0% of the formulation. The beeswax may be replaced by plant-based lipophilic ingredients such as fatty acids, fats, long-chained esters and long-chained alcohols.

[0107] The beeswax may be replaced by completely saponified beeswax or individual beeswax ingredients, such as long-chained esters, long-chained alcohols, long chained aliphatic compounds, short-chained alcohols and short-chained ester salts.

[0108] The complete saponification can be performed with an alkaline solution selected from KOH, NaOH, NH4OH, among others.

[0109] The replacement of beeswax with the wax-less lipophilic ingredients can be performed to afford a similar Hydrophilic-Lipophilic Balance (HLB number) of the ingredients.

[0110] The surfactants may be both non-ionic and anionic. Suitable surfactants may include, but are not limited to, sorbitan esters (also known as Spans), ascorbyl palmitate, ascorbyl stearate, stearic acid, palmitic acid, and other fatty acids. Surfactants typically constitute 0.5%-10.0% of the formulation.

[0111] A pH regulator is a stabilizing agent that negatively charges the surfactant, hence generating a stabilizing electrostatic repulsion between the colloids in the liquid formulation. The pH regulator may be evaporated during the manufacturing of the formulation. Suitable pH regulators include, but are not limited to, KOH, NaOH, NH4OH. The pH regulator typically constitutes 0.2%-2.0% of the composition. When non-ionic surfactants are used without the anionic surfactants, the presence use of pH stabilizers regulators may be avoided.

[0112] Additional optional components of the emulsion formulations include hydrocolloids in an amount of 0.01 to 1.0% to result in a colloidal emulsion. The hydrocolloids may be selected from tara gum, cassia gum, guar gum, and locust bean gum.

[0113] Additional optional components include a chemical stabilizer that regulates the viscosity of the formulation.

[0114] The medium of the emulsion is typically water, for example, double distilled water (DDW) which constitute 70% - 97% of the total wet weight when applied to the fruit.

[0115] The term “hydrocolloid polymer” as used herein refers to a water-soluble polymer, of biotic, e.g., vegetable, animal, microbial, fossil, or abiotic, i.e., synthetic origin, that generally contains hydroxyl, carboxyl, amine and / or amide groups and is capable of increasing the viscosity of the composition.

[0116] According to some embodiments, the composition further comprises an edible hydrocolloid polymer selected from the group consisting of locust bean gum (LBG), guar gum, xanthan gum, lambda-carrageenan and derivatives, and combinations thereof. Each possibility represents a separate embodiment of the present invention. In a specific embodiment, the hydrocolloid is LBG.

[0117] According to some embodiments, the hydrocolloid polymer is present in a weight percent ranging from about 0.1% (w / w) to about 5% (w / w) of the total weight of the composition. In certain embodiments, the hydrocolloid polymer is present in a weight percent of 0.1% (w / w) or more of the total weight of the composition. In certain embodiments, the hydrocolloid polymer is present in a weight percent of 5% (w / w) or less of the total weight of the composition, although the appropriate percentage of the hydrocolloid polymer will be determined for the actual hydrocolloid polymer used, as is well known to one of skill in the art.

[0118] The composition of the present invention may further contain additional substances selected from the group consisting of antifoaming agents, preservative agents, adhesive agents, cross-linking agents, plasticizers, and surface-tension reducing agents. Each possibility represents a separate embodiment of the present invention. Exemplary additives include, but are not limited to potassium carbonate, sodium bisulfite, sodium benzoate, sodium propionate, calcium propionate, potassium sorbate, glycerol, propylene glycol, sorbitol, mannitol, among others. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the additives are FDA approved, GRAS (generally recognized as safe), biodegradable and non-toxic.

[0119] According to the principles of the present invention, the inclusion of additional substances into the composition is performed in order to obtain a composition having desired properties such as, but not limited to, desired viscosity, plasticity and elasticity, hydrophobicity, permeability, smoothness, glossiness, strength and resistance to shearing forces, pH, and the like.

[0120] The terms “plant matter” and “plant” as used interchangeably herein, refer to a plant organ or a plant tissue. Preferably, the coatings of the present invention are used to protect post-harvest fruits and vegetables. According to alternative embodiments, the plant matter is selected from the group consisting of a seed, seedling, plantlet, bulb, rhizome, tree, herb, shrub, creeper, legume, mushroom, flower, and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some specific embodiments, the plant matter is a citrus fruit. In other specific embodiments, the plant matter is other than a citrus fruit.

[0121] According to some embodiments, the composition is applied to the plant matter by means of dipping, immersing, spraying, rubbing or pouring, possibly when the plant matter is moving on a conveyor belt. Each possibility represents a separate embodiment of the present invention.

[0122] The composition according to the principles of the present invention, is for use in agricultural applications to decrease fungal infestation in plant tissues, while increasing the shelf life of the fruit with a wax-based coating. The term "decrease fungal infestation" as used herein, refers to the growth inhibition of or the killing of a certain percentage of a fungus or a mold.

[0123] According to some embodiments, the fungal infestation is induced by a fungus selected from the group consisting of penicillium digitatum, penicillium italicum, geotrichum citri-aurantii, alternaria alternata. Each possibility represents a separate embodiment of the present invention.

[0124] As exemplified hereinbelow, the compositions of the present invention were effective against penicillium digitatum both as dry films and as a coating with no fungal growth upon films produced from said composition and less than about 25% mold infection upon substrates coated with said composition compared to non-coated fruit. Moreover, the compositions of the present invention have demonstrated an equal or superior antifungal activity as compared to the same compositions supplemented with known synthetic fungicides, exemplified by thiabendazole (TBZ) and imazalil (IMZ) at concentrations of about 0.6% (w / w) and 0.2% (w / w), respectively. The antifungal properties of the plant coating comprising an algal extraction, were quantified using various assays known to a person skilled in the art.

[0125] According to some embodiments, the composition decreases fungal infestation of plant matter coated with said composition by at least 10% as compared to an uncoated plant matter. In certain embodiments, the composition decreases fungal infestation by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70% or by at least 80% as compared to an uncoated plant matter. Each possibility represents a separate embodiment of the present invention.

[0126] According to some embodiments, the composition decreases the weight loss of a plant coated with said composition by at least about 20%. The term "decrease weight loss" as used herein, refers to the reduction in the plant's postharvest water loss or dry matter loss due to respiration. In further embodiments, the weight loss of a plant coated with the composition of the invention is reduced by at least about 30%, about 40%, or about 50% as compared to an uncoated plant matter under the same storage conditions. Each possibility represents a separate embodiment of the present invention.

[0127] In another aspect, the invention provides a method for the preparation of an aqueous algal extract of Ulva Lactuca seaweed comprising ulvan with a decreased particle size or a reduced molecular weight compared to that of the original algal biomass, the method comprising the steps of: a) extracting the Ulva Lactuca seaweed biomass in hot water; b) optionally drying the aqueous extract and performing an additional extraction in ethanol; c) filtering the extract to remove large remnants; and d) fragmenting the extract to decrease the size of the particles comprising sulfated polysaccharides; to obtain an algal extract having at least one of decreased particle size or a reduced molecular weight.

[0128] In another aspect, the invention provides a method for the preparation of an aqueous algal extract of Ulva Lactuca seaweed comprising ulvan with a decreased particle size or a reduced molecular weight compared to that of the original algal biomass, the method comprising the steps of: a) extracting the Ulva Lactuca seaweed biomass in hot water; b) optionally drying the aqueous extract and performing an additional extraction in ethanol; c) filtering the extract to remove large remnants; and d) fragmenting the extract to decrease the size of the particles comprising sulfated polysaccharides; to obtain an algal extract comprising ulvan at a weight percent of at least 0.2% (w / w). According to various embodiments, fragmenting the ulvan fraction can be performed after extraction either by sonication, thermochemical or by enzymatic digestion.

[0129] Advantageously, the emulsion of a wax-based coating composition and the sonicated aqueous extract of Ulva species is stable for long periods, which entails prolonged shelf life. According to certain embodiments, the combination of a wax-based coating composition and sonicated ulvan containing aqueous extract of Ulva species is stable for at least 3 months, at least 6 months, or at least 9 months.

[0130] According to various embodiments, the sonicated ulvan containing aqueous extract of Ulva species has reduced viscosity compared to the unsonicated extract. According to certain embodiments, sonicated extract has a viscosity of about 4cP to about 6cP, including each value within the specified range. Viscosity can be measured as is known in the art using a suitable viscometer in a setup that is compatible for aqueous media having low viscosity. For example, viscosity can be measured using a viscometer such as, but not limited to, a Brookfield Viscometer or an Anton Paar Rheoplus viscometer with an appropriate setup. In one embodiment, viscosity of the sonicated extract can be measured using a Brookfield RTV viscometer with a LV-4 (64), a LV-5 (65), a RV / HA / HB-6 or a TE type spindle at 0.3-60 rpm. Each possibility represents a separate embodiment. In another embodiment, viscosity of the extract can be measured using a Brookfield DV-E viscometer with a LV-4 (64), a LV- 5 (65), or a RV / HA / HB-6 type spindle at 0.3-60 rpm. Each possibility represents a separate embodiment.

[0131] According to various embodiments, impurities can be removed through the use of ethanol washing.

[0132] In another aspect, the invention provides a method for the preparation of a composition for coating a plant matter, wherein the composition comprises an aqueous algal extract of Ulva Lactuca seaweed, one or more carboxylic acid having 10-35 carbons, and a base.

[0133] According to some embodiments, the Ulva Lactuca seaweed biomass is pretreated before being extracted in step (a). The term "pre-treated" as used herein, refers to the biomass being washed and ground into a powder. According to some embodiments, extracting the Ulva Lactuca seaweed in step (a) comprises heating the biomass in distilled water at a temperature of about 85°C to about 100°C.

[0134] According to some embodiments, ulvan is fragmented in order to minimize the variability of the extract and reach a decreased chain length. According to some embodiments, fragmenting the ulvan fraction in step (d) is performed by ultra-sonic agitation at 400 Watts.

[0135] In certain embodiments, the at least one enzyme is selected from the group consisting of ulvan lyases.

[0136] The inventors of the present invention have surprisingly discovered that minimizing impurities in the Ulva Lactuca seaweed and increasing the fraction of the biologically active substance, ulvan, improves the biological activity of the extract and subsequently the composition for coating comprising same.

[0137] According to some embodiments, removing impurities comprises the removal of at least some of the phytoconstituents selected from the group consisting of proteins, starch, minerals, oligo-sugars, and bioactive moieties.

[0138] According to some embodiments, the removal of proteins is performed by ethanol washing.

[0139] According to some embodiments, the removal of proteins comprises decreasing the concentration of the protein in the extract by about 10-80%. According to certain embodiments, the removal of proteins comprises decreasing the concentration of the protein in the extract by up to about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. Each possibility represents a separate embodiment of the present invention.

[0140] According to some embodiments, the removal of starch is performed by digestion of starch granules using amylase.

[0141] According to some embodiments, the removal of starch comprises decreasing the concentration of the starch in the extract by about 50-90%. According to certain embodiments, the removal of starch comprises decreasing the concentration of the starch in the extract by up to about 60%, about 70%, or about 80%. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the removal of minerals is performed by washing the biomass in distilled water.

[0142] According to some embodiments, the removal of minerals comprises decreasing the concentration of the minerals in the extract by about 50-95%. According to certain embodiments, the removal of minerals comprises decreasing the concentration of the minerals in the extract by up to about 50%, about 60%, about 70%, about 80%, or about 90%. Each possibility represents a separate embodiment of the present invention.

[0143] According to some embodiments, the removal of monosaccharides or oligosaccharides is performed by dialysis or fermentation. According to some embodiments, the removal of mono-sugars comprises decreasing the concentration of the mono-sugars in the extract by up to about 50-95%. According to certain embodiments, the removal of monosugars comprises decreasing the concentration of mono-sugars in the extract by up to about 50%, about 60%, about 70%, about 80%, or about 90%. Each possibility represents a separate embodiment of the present invention.

[0144] According to some embodiments, the ulvan in the extract obtained is in a weight percent ranging from about 0.01% (w / w) to about 5% (w / w) of the total weight of the composition. According to further embodiments, the extract obtained in step (d) comprises ulvan in a weight percent ranging from about 0.25% (w / w) to about 2.5% (w / w) of the total weight of the composition. According to yet further embodiments, the extract obtained in step (d) contains ulvan in a weight percent ranging from about 0.5% to about 1.5% of the total weight of the composition. According to certain embodiments, the ulvan in the extract obtained has a beneficial activity even at a weight percent as low as 0.01%.

[0145] According to some embodiments, the edible wax is selected from the group consisting of beeswax, carnauba wax, candelilla wax, alpha wax, rice-bran wax, Japan wax and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0146] According to some embodiments, the edible wax is present in a weight percent ranging from about 1% (w / w) to about 25% (w / w) of the total weight of the composition. According to further embodiments, the edible wax is present in a weight percent ranging from about 2% (w / w) to about 20% (w / w) of the total weight of the composition. According to some embodiments, the emulsifier is an edible emulsifier selected from the group consisting of lecithin, polysorbate, ethylene glycol monostearate, ammonium lauryl sulfate, sodium stearoyl-2-lactylate, potassium oleate, propylene glycol monostearate, sodium alkyl sulfate, oleic acid and polyethylene glycol (PEG) and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0147] According to some embodiments, the emulsifier is present in a weight percent ranging from about 0.25% (w / w) to about 5% (w / w) of the total weight of the composition. According to further embodiments, the emulsifier is present in a weight percent ranging from about 0.75% (w / w) to 4% (w / w) of the total weight of the composition. According to yet further embodiments, the emulsifier is present in a weight percent ranging from about 1% (w / w) to 3% (w / w) of the total weight of the composition.

[0148] According to some embodiments, the method further comprises the step of adding an edible hydrocolloid polymer to the mixture obtained at step (e).

[0149] According to some embodiments, the edible hydrocolloid polymer is selected from the group consisting of locust bean gum (LBG), guar gum, xanthan gum, lambda- carrageenan and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to some exemplary embodiments, the edible hydrocolloid polymer is LBG.

[0150] According to some embodiments, the hydrocolloid polymer is present in a weight percent ranging from about 0.1% (w / w) to about 5% (w / w) of the total weight of the composition. In certain embodiments, the hydrocolloid polymer is present in a weight percent of 0.1% (w / w) or more of the total weight of the composition. In certain embodiments, the hydrocolloid polymer is present in a weight percent of 5% (w / w) or less of the total weight of the composition.

[0151] As used herein, the term "about", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods

[0152] As used herein and in the appended claims the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. It should be noted that the term “and” or the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0153] The following Examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0154] EXAMPLES

[0155] Example 1: Preparation of compositions comprising Ulva seaweed an neons extract

[0156] The following process was followed to prepare aqueous extracts and dilutions of Ulva seaweed obtained from Nature Ltd.:

[0157] Deionized water wash

[0158] Fresh Ulva was washed in tap water and strained thrice (stones, insects, or any large debris were manually removed). Next, the washed Ulva was dried at 50°C for 12-18 hrs. Then, 50 g of the dried Ulva were blended in 1.5L of deionized water (Ninja max blender) after which water was strained through a fine strainer.

[0159] The water-strained Ulva was then stirred with ~1L of deionized water, for about 10 min, this step was repeated three times.

[0160] The remained Ulva was added with 200 ml of deionized water and crushed (Ninja max blender) until the mixture achieved a slurry, creamy, and homogenized consistency. Next, the slurry was dried at 50°C for 12-18 hrs, after which the dried Ulva matter was ground to powder.

[0161] Ethanol wash

[0162] The ground powder (30-40 gr) was then mixed with 200 ml of ethanol, the mixture was stirred while kept in a water bath at 80°C until the mixture reached 80°C. Once the mixture reached 80°C, it was removed from the water bath and stirred for additional 60 min. To remove the liquids, the mixture was strained through a strainer.

[0163] Ethanol extraction

[0164] The retained Ulva matter was then mixed with deionized water to achieve a total mass of approximately 1000 g, the mixture was stirred at - 80-90°C for 1 hr. After being left to cool, the cooled mixture was centrifuged at 3500 RPM, 10°C, for 15min.

[0165] The supernatant was stirred and heated in a water bath to evaporate some of the liquids. When ~100ml of the supernatant remained, 200ml of ethanol was added, and following 1 min stirring, the supernatant ethanol solution was strained through a fine strainer and centrifuged at 3500 RPM for 15 min.

[0166] Ulvan extract powder

[0167] The supernatant of the ethanol wash was collected and dried for -20-26 hrs at a 50°C. The dried matter was then ground to powder (Ninja max blender) and stored in a sealed container for future use.

[0168] Alternatively, dry Ulva was used for the preparation of compositions comprising Ulva seaweed aqueous extract. In that case, the dry seaweed was ground to powder after which all steps of the procedure were followed starting with the ethanol wash, or as detailed herein below.

[0169] Dry algae (Nature Ltd., Israel) (50g) were first ground to powder (Ninja max blender) over 60 seconds. The dry powder was stirred in 2L of distilled water for 30 minutes to obtain an Ulva seaweed suspension which was subsequently strained through a fine strainer and then heated at 95°C for 1 hour to obtain a 10% v / w Ulva extract composition.

[0170] The 10% Ulva extract composition was allowed to cool, forming a slurry which was subsequently filtered through a cloth strainer and then centrifuged at 3000 RPM for 20 minutes at room temperature. The supernatant formed following the centrifugation was collected and evaporated to obtain an Ulva seaweed dried extract.

[0171] The Ulva seaweed dried extract was diluted in water to form 2.5% and 5% aqueous extract compositions (as shown in Table 1).

[0172] Additional aqueous compositions were formulated by combining a 3.5% beeswax formulation (also referred to as “Suf 3.5” or “Sufresca beeswax formulation”), which comprises 3.5% beeswax, 0.9% oleic acid, 0.25% ammonium hydroxide, and <0.1% hydrocolloid, with the 2.5% and 5% Ulva aqueous compositions. This resulted in the formation of the “SufUlv-A” and “SufUlv-B” algal formulations, as depicted in Table 1.

[0173] Table 1: The of the Ulva seaweed 1

[0174] Example 2: Coating of oranges inoculated with P. digitatum with Ulva seaweed aqueous extract compositions

[0175] 120 harvested oranges (Valencia oranges supplied by Hapardesan) were inoculated with an aqueous inoculated suspension of P. digitatum at a concentration of 106spores / mL. Two punctures were made in each orange and subsequently the orange was inoculated with lOpl at each of the two punctures.

[0176] Groups of 24 inoculated oranges were coated with the compositions of Example 1 (Table 1). In addition, groups of 30 inoculated oranges were treated with Algal formulation A, Algal formulation B, Suf 3.5%, and a non-inoculated control. The treated oranges were stored at 20°C and fungal infection was evaluated over 4 weeks. Additional 30 uninfected oranges were coated as a control.

[0177] Example 3: Evaluation of weight loss (%), firmness and fruit decay (%) in oranges treated / coated with Ulva seaweed aqueous extract compositions The inoculated oranges treated with Ulva seaweed compositions as per Example 2 were evaluated for weight loss (%), firmness and fruit decay (%) caused by mold growth. The weight loss (%) evaluation, was performed over 26 days at 20°C, showed that all of the coating treatments significantly reduced weight loss compared to the control (Figure 1). Algal formulation A and Algal formulation B demonstrated a similar weight loss of 3.5%. The Suf 3.5% formulation, comprising 3.5% beeswax, demonstrated a reduced weight loss (%) with retention of 97.5% of the original weight.

[0178] A firmness test was conducted to assess the degree of softening of the inoculated oranges every few days over a 26-day period. A firmness index score was used. The results of the firmness test showed a comparable score for Algal Formulation A, Algal formulation B, and Suf 3.5. Over the 26-day period, the treated oranges had a firmness score of roughly 2 compared to 2.7 for the control (Figure 2).

[0179] The inoculated oranges treated with SufUlv-A (Algal formulation A), SufUlv-B (Algal formulation B), C-Algex (aqueous algal extract), D-SufFung (Suf 3.5, IMZ lOOOppm and TBZ 2000ppm), and a control, were evaluated for decay as a percentage of the fruit covered in mold over 1, 2 and 3 weeks (Figure 3). The results of the infected decay (%) evaluation showed that the algal-based coatings exhibited significantly less decay compared to the control. By week 3, the algal-based formulations displayed similar mold decay of approximately 50%, compared to 75% decay in the control. The D-SufFung positive control, which consisted of Sufresca 3.5% formulation combined with the antifungal agents imazalil (lOOOppm) and thiabendazole (2000ppm), showed a superior decay (%) prevention of 35% by week 3.

[0180] Example 4: Coatins of lemons inoculated with P. disitatum with Ulva seaweed au neons extract compositions

[0181] Ulva seaweed aqueous extracts comprising dry algae (supplied by Seakura (S) or VIET D.E.L.T.A INDUSTRIAL CO., LTD (I)) (60 g) were prepared according to the procedures of Example 1, to form 2.5%, 3.75% and 5% diluted compositions comprising 3.5% beeswax.

[0182] 480 harvested lemons were inoculated with an aqueous suspension of P. digitatum at a concentration of 106spores / mL. Two punctures were made in each lemon and subsequently the lemon was inoculated at each of the two punctures. Twenty-four hours (24h) after inoculation, groups of 60 lemons were coated with each of the compositions (Table 3). Additionally, groups of 30 treated lemons were stored at room temperature where they were monitored for 8 weeks during which the total number of infected spots was recorded.

[0183] Table 2: The composition of the Ulva seaweed coatings of Example 4

[0184] Figure 4 presents the results of the total number of infected spots for each formulation counted on Days 3 to Day 6.

[0185] As can be seen in Figure 4, formulations based on algae supplied by I at any of the tested concentrations (i.e., 5%, 2.5%, and 1.25%) exhibited higher antifungal activity than the respective formulations based on algae supplied by S. I 1.25% showed the highest reduction of the development of infected spots on days 3-5 after inoculation compared to all I and S based formulae. On day 6, 1 1.25% and I 2.5% antifungal activity were comparable. Infected fruits treated by I 1.25% or I 2.5% formula reached -33% decay 6 days after inoculation while the untreated control group reached > 90%.

[0186] Example 5: Preparation of Sufresca Algal-formulation 3.75%

[0187] The following steps were followed to prepare a 3.75% Ulva extract based formulation (hereinafter referred to as Sufresca Algal-formulation 3.75%): Ulva seaweed (S and I) (100g) was grinded to powder using a Ninja max blender for 60 seconds. The resulting powder was stirred in 2L of distilled water for 30 minutes to obtain a suspension. The suspension was strained through a fine strainer and then heated at 95°C for 1 hour to obtain a 10% composition.

[0188] After cooling, the resulting slurry was filtered through a cloth strainer and centrifuged at 3000 RPM for 20 minutes at room temperature. The supernatant produced after centrifugation was collected and evaporated to obtain a dry extract pellet.

[0189] The Ulva seaweed dried extract was diluted in distilled water to create 5% and 2.5% Ulva seaweed diluted compositions, which were then combined in equal parts to obtain the Sufresca Algal-formulation 3.75%.

[0190] Example 6: Evaluation of fruit decay (%) in oranges treated with the Sufresca Algal- formulation 3.75 %

[0191] 80kg harvested oranges (Taburi oranges) were first dipped in an aqueous chlorine suspension (150 ppm), and then washed and dried. The oranges were then inoculated with P. digitatum according to the procedures of Example 2. A total of 40 kg oranges were spray- coated with the Sufresca Algal-formulation 3.75% using spinning brushes. Another batch of 40 kg oranges was used as an uncoated control. The treated and untreated oranges were stored at room temperature and monitored over 8 weeks during which the total number of infected spots were recorded.

[0192] The inoculated oranges treated were evaluated for infected decay as a percentage of the fruit covered in mold over 4 weeks (Figure 5). The results of the infected decay (%) evaluation showed that the oranges treated with the 3.75% Sufresca Algal-formulation exhibited 5% fruit decay (%) over 4 weeks compared to a 25% decay seen in the control group.

[0193] Example 7: Preparation of Algal-I, Algal-IW and Algal-IWS extracts

[0194] The following steps were followed to prepare two Ulva seaweed algal compositions: first, fresh algae (I) (50 g) were mixed with deionized water (IL) and heated for 10 minutes at 50°C. The algae composition was then cooled and filtered 3 times through a fine metal mesh.

[0195] The strained composition was blended three consecutive times (maximal setting on a Ninja blender) to obtain an algae slurry. The slurry was heated at 40°C overnight and then combined with ethanol (100 ml). This was then heated in a water bath for 60 minutes at 50°C to evaporate the ethanol content, any residual ethanol was discharged. Deionized water was added to the composition to reach a total mass of 500 g and the solution was heated to 90°C and stirred for 2.5 hours to obtain a slurry.

[0196] After cooling, the slurry was filtered through a cloth strainer and centrifuged at 3000 RPM for 20 minutes at room temperature. The supernatant produced following the centrifugation was collected and evaporated to obtain the Algal-I extract (50g). Another extract, Algal-IW, was prepared by introducing an additional washing (ethanol 200 ml) and filtration step to the Algal-I composition.

[0197] A sonicated Algal-IWS extract was prepared by subjecting the Algal-IW extract to sonication at 1500 W, 200 KHz and 60 amplitude for 10 minutes.

[0198] Example 8: Evaluation of impact of Algal- and Algal-IW coatings on inoculated oranges

[0199] To evaluate the Algal-I and Algal-IW coatings, 120 harvested oranges (Taburi oranges) were inoculated with P. digitatum according to the procedures of Example 2. 24 hours after inoculation, the oranges were coated by dipping for 30 seconds with Algal-I (5%), Algal IW (5%) diluted compositions and a positive control composition comprising imazalil (lOOOppm) and thiabendazole (500ppm), as well as an uncoated control. The treated oranges were stored at room temperature and monitored over 8 weeks during which the total number of infected spots were recorded.

[0200] The inoculated oranges treated were evaluated for infected decay as a percentage of the fruit covered in mold over 5 days (Figure 6). The results of the infected decay (%) evaluation showed that the Algal-IW (5%) brought the best reduction of fruit decay, wherein after 4 days oranges treated with Algal-IW (5%) showed 42% rot spots, compared to 78% in the control group.

[0201] To evaluate the Algal-IWS extract, harvested oranges (Taburi oranges) were inoculated with P. digitatum according to the procedures of Example 2. 24 hours after inoculation, 120 oranges were coated by dipping for 30 seconds with 5% diluted Algal-IWS. 60 further oranges as a positive control were coated by dipping for 30 seconds with a suspension of imazalil and thiabendazole, and another 60 oranges were left untreated as a negative control. The inoculated oranges were evaluated for decay (%) as a percentage of the fruit covered in mold over 5 days (Figure 7). As can be seen in Figure 7, the sonicated Algal-IWS extract exhibited a superior antifungal activity and significantly reduced fruit decay (%) profile compared to the unsonicated extract of approximately 40% decay after 5 days from inoculation. The unsonicated extract resulted in decay 60%, while the positive control, comprising imazalil and thiabendazole, exhibited a 30% decay.

[0202] Figures 8A-8C are pictures of oranges on Day 5 after the inoculation with P. digitatum. These pictures demonstrate the surprisingly increased antifungal effect of the sonicated Algal-IWS coating compared with the unsonicated Algal-IW coating (Figures 8C vs. 8B, respectively).

[0203] Example 9: Evaluation of fruit decay (%) in oranges treated with 5% beeswax Algal- IWS formulation

[0204] The sonicated Algal-IW (IWS) extract of Example 8 was combined with the Sufresca beeswax formulation of Example 1, to form a 10% beeswax Algal-IWS formulation. A 5% dilution of the Algal-IWS extract was made with distilled water.

[0205] 300 harvested oranges (Taburi oranges) were inoculated with P. digitatum according to the procedures of Example 2. Twenty -four hours after inoculation, 60 oranges were coated by dipping for 30 seconds with the 10% beeswax Algal-IWS formulation (Suf 10% IWS 10%), while another batch of 60 oranges was treated with a 10% Sufresca beeswax formulation. Additional 60 oranges were utilized as a positive control and were coated by in a suspension of imazalil and thiabendazole. Additionally, 60 oranges were left untreated and utilized as a negative control.

[0206] The inoculated oranges were evaluated for infected decay as a percentage of the fruit covered in mold over 5 days (Figure 9). The results of the infected decay (%) evaluation showed that the Algal-IWS (10%) formulation, which comprises Algal-IWS (10%) formulated with beeswax, unexpectedly demonstrated an enhanced decay (%) profile over 5 days of as low as 40%. This suggests that the sonicated Algal extract and beeswax act synergistically to enhance the antimicrobial profile when coated on oranges. The 10% beeswax Algal-IWS formulation surprisingly showed a comparable decay (%) over 5 days when compared to the positive control, which comprises IMZ and TBZ, supporting its potential use as a postharvest coating.

[0207] Example 10: Evaluation of P. digitatum growth in glucose and Potato dextrose broth treated with the Algal-IWS formulation A spore suspension of Penicillium digitatum at a concentration of 107per ml was mixed with PDB (Potato Dextrose Broth) to a concentration of 20%. lOgr glucose were added into 50ml double distilled water vortexed and passed through a syringe filter 0.2pm. Then lOOpl of the PDB spore suspension was transferred into wells. The wells were treated with 900pl of Algal IWS 5% and Algal IWS 0.5%, a positive control (imazalil 500 ppm), and a negative control (water) (Table 3) reaching a spore suspension of 106spores in 2% PDB.

[0208] Over the course of 5 days, the P. digitatum growth was monitored.

[0209] Minor sprouting and mycelial growth were observed in the glucose and PDB wells treated with a negative control. Unexpectedly, the wells treated with Algal-IWS 5% or 0.5% did not exhibit sprouting or mycelial growth after 1 day. After 5 days, the Algal-IWS 0.5% treated wells showed some mycelia and sprouting, but to a lesser extent than the untreated control groups. Surprisingly, the Algal-IWS 5% treated agar plates or wells did not exhibit mycelia or sprouting after 5 days.

[0210] Table 3: Treatments applied to glucose and PDB agar plates

[0211] Example 11: The antifungal effect of formulations comprising sonicated or unsonicated ulvan extract on P. digitatum growth in glucose and Potato dextrose broth

[0212] To compare the antifungal effect of formulations comprising sonicated or unsonicated ulvan extract on P. digitatum growth, 106P. digitatum spores in 2% PDB and glucose were prepared as detailed in Example 10. Then lOOpl of the PDB spore suspension was transferred into wells. The wells were treated with 900pl of Algal IWS (1%) and Algal IW (1%), a positive control (imazalil 500 ppm), and a negative control (water). Results of the P. digitatum growth on Day 2 after treatment are presented in Figures 10A-10C. Figure 10A, 106P. digitatum growth following treatment with water (negative control). P. digitatum growth in the different wells was monitored for two days. Figure 10B, P. digitatum growth following treatment with coating formulation comprising unsonicated ulvan extract, Algal IW (1%). Figure 10C, P. digitatum growth following treatment with coating formulation comprising sonicated ulvan extract, Algal IWS (1%).

[0213] As can be seen in Figures 10A-10C, the formulation comprising sonicated ulvan extract exhibited superior activity against P. digitatum, compared to the formulation comprising unsonicated ulvan extract. Nevertheless, both the formulations exhibited antifungal activity compared to the negative control.

[0214] Example 12: Characterization of Ulvan extract particles after extract sonication

[0215] Size distribution

[0216] The effect of sonication on particle size distributions of the ulvan extract was determined by Dynamic Eight Scattering (DLS). To that end, ulvan extract was prepared according to Example 1 and sonication was performed according to Example 7.

[0217] Next, the ulvan extracts sonicated or non-sonicated (reference) were measured using a laser diffraction particle sizer to determine the DLS (Malvern Instruments, U.K) and analyzed using a refractive index of 1.5, absorption of 0.1, dispersant RI 1.33, and viscosity (cP) of 0.8872. Figure 11 presents the DLS results for the size distribution of particles within the sonicated ulvan extract (grey), and the reference unsonicated ulvan extract (black). To further analyze the effect of sonication on ulvan extract particles according to embodiments of the present invention the zeta potential distribution of the sonicated ulvan extracts was determined and compared to the zeta potential of the reference extract (not sonicated). Briefly, measurements at 25°C were performed using a Zetasizer (Version 7.13; Serial no: MAL 1029220; Malvern Instruments). Figure 12 present the zeta potential distribution (mV) of 12 measurements of the sonicated ulvan extract (grey) and the reference, unsonicated ulvan extract (black).

[0218] Example 13: The effect of sonication on the viscosity of Ulvan extract and coating formulation comprising ulvan extract

[0219] To determine the viscosity of ulvan extract and coating formulation comprising ulvan extract before sonication (unsonicated) and after sonication, ulvan was extracted as described in Example 1. Next, "Suf 10" coating formulations were prepared with 1% ulvan extract similar to Example 1. Briefly, 1% of sonicated or unsonicated ulvan extract was formulated with a 10% beeswax formulation, which comprises 10% beeswax, 0.9% oleic acid, 0.25% ammonium hydroxide, and <0.1% hydrocolloid. Viscosity was measured by an IKA ROTAVISC viscometer, the results are summarized in Table 4.

[0220] Table 4: Viscosity (cP) of ulvan extract and coating formulation comprising ulvan extract before and after sonication

[0221] The sonication reduced the viscosity of the Ulvan extract and accordingly the viscosity of the coating formulation. Lower viscosity allows for better mixing of the ulvan extract with the Suf 10 formulation and results in lower viscosity of the Suf 10 +1% ulvan coating formulation. This may be advantageous for the application of the coating formulation to a plant matter.

[0222] Example 14: The effect of sonication of Ulvan extract on the stability of a coating formulation comprising ulvan extract

[0223] Suf 10 and 1% ulvan coating formulation were prepared as described in Examples 1 and 13. 1% of sonicated or unsonicated ulvan extract was formulated with a 10% beeswax formulation, which comprises 10% beeswax, 0.9% oleic acid, 0.25% ammonium hydroxide, and <0.1% hydrocolloid. The coating formulations with sonicated or unsonicated ulvan extract were left to sit for 24hrs at room temperature. Figure 13A shows a picture of Suf 10 and 1% unsonicated ulvan extract coating formulation. Figure 13B shows a picture of Suf 10 and 1% sonicated ulvan extract coating formulation.

[0224] As can be seen in Figure 13 A, the coating formulation comprising unsonicated ulvan underwent phase separation within 24hrs, having a clear liquid at the upper layer and a white substance at the bottom layer. In contrast, the coating formulation comprising unsonicated ulvan depicted in Figure 13B, maintained its stability and remained intact.

[0225] Remarkably, coating formulations that comprise sonicated ulvan extract remained stable for over 6 months, while coating formulations that comprise unsonicated ulvan extract were unstable and disintegrated, curdle and / or separated to phases within 24hrs. A coating formulation that maintains its stability for over 6 months is advantageous for prolonged shelf life of the formulation.

[0226] Although the invention is described in conjunction with specific embodiments thereof, it is evident that numerous alternatives, modifications and variations that are apparent to those skilled in the art may exist. It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and / or methods set forth herein. Other embodiments may be practiced, and an embodiment may be carried out in various ways. Accordingly, the invention embraces all such alternatives, modifications and variations that fall within the scope of the appended claims.

Claims

CLAIMS1. An antimicrobial composition for treating or preventing fungal or bacterial infestation of a plant matter, wherein the antimicrobial composition comprises an aqueous extract of green seaweed comprising an antimicrobial effective amount of ulvan having an average particle size in the range of about 50 nm to about 2000 nm as determined by dynamic light scattering.

2. The antimicrobial composition according to claim 1, wherein the composition is a dry composition.

3. The antimicrobial composition according to claim 1, wherein the composition is in a form of an aqueous dispersion.

4. The antimicrobial composition of claim 3, wherein the aqueous dispersion further comprises an agricultural or food-grade acceptable carrier.

5. The antimicrobial composition of any one of claims 1 to 4, wherein said antimicrobial effective amount is between about 0.2% (w / w) to about 5% (w / w) of said ulvan of the total weight of the composition.

6. The antimicrobial composition of any one of claims 1 to 5, wherein said ulvan is derived from a biomass of an Ulva species.

7. The antimicrobial composition of any one of claims 1 to 6. wherein the average particle size of said ulvan is between lOOnm to lOOOnm.

8. The antimicrobial composition of any one of claims Ito 7, wherein said ulvan is fragmented by a method selected from enzymatic digestion, exposure to chemical degradation, and sonication.

9. The antimicrobial composition of claim 8, wherein said ulvan is fragmented by sonication.

10. The antimicrobial composition of claim 9, wherein the sonication is performed in an acidic environment.

11. The antimicrobial composition of claim 9, wherein the sonication is performed in an alkaline environment.

12. An aqueous composition comprising:(a) an extract of Ulva seaweed comprising fragmented ulvan characterized by an average particle size of 100 nm to 1000 nm;(b) a carboxylic acid having 10-35 carbon atoms;(c) a base;(d) an edible wax or wax substitute; and(e) water.

13. The aqueous composition according to claim 12, wherein the fragmented ulvan is present in said composition at a concentration of at least about 0.01% (w / w), of the total weight of the composition.

14. An aqueous composition comprising: i. 0.01% (w / w) - 15% (w / w) of an extract of Ulva seaweed comprising fragmented ulvan characterized by an average particle size of 100 nm to 1000 nm; ii. 1% (w / w) - 50% (w / w) of an edible wax or wax substitute; iii. 0.25% (w / w) - 5% (w / w) of an emulsifier; and iv. water to 100% of the total weight of the composition.

15. The aqueous composition according to claim 14, wherein said emulsifier is an anionic surfactant.

16. The aqueous composition according to any one of claims 12 to 15, wherein the composition further comprises an edible hydrocolloid polymer selected from the group consisting of locust bean gum (LBG), guar gum, xanthan gum, lambda- carrageenan and combinations thereof.

17. The aqueous composition according to claim 16, wherein the edible hydrocolloid polymer is present in a weight percent ranging from about 0.1% (w / w) to about 1.5% (w / w) of the total weight of the composition.

18. The aqueous composition according to any one of claims 12 to 17, wherein the composition has a pH ranging from about 8.0 to about 10.0.

19. The aqueous composition according to any one of claims 12 to 18, wherein the composition is characterized by a viscosity of 1-500 cP.

20. The aqueous composition according to any one of claims 12 to 19, wherein said composition is formulated for application to plant matter.

21. The aqueous composition according to claim 20, wherein the application is selected from postharvest application and pre-harvest application.

22. The aqueous composition according to claim 20, wherein said application is performed by any one of dipping, coating, immersing, spraying, rubbing or pouring.

23. The composition according to any one of claims 1 to 22, wherein the composition (i) decreases fungal infestation; and / or (ii) reduces weight loss of a plant matter coated with said composition by at least 20%.

24. The composition according to any one of claims 1 to 23, wherein the fungal infestation is derived from a fungus selected from the group consisting of penicillium digitatum, penicillium Italicum, geotrichum citri-aurantii, and alternaria alternata.

25. A method for the preparation of the composition of any one of claims 1 to 24 comprising: a) providing an aqueous extract of an Ulva species comprising ulvan; and b) exposing said ulvan to conditions suitable for decreasing its average particle size.

26. The method according to claim 25, further comprising a step of extracting seaweed biomass of said Ulva species in an aqueous solution prior to step (a).

27. The method according to claim 26, wherein said extracting comprises heating the seaweed biomass in distilled water at a temperature of about 85°C to about 100°C.

28. The method according to any one of claims 25 to 27, wherein step (b) is performed by: (i) exposing said ulvan to ultra-sonic waves; (ii) exposing said ulvan to a peroxide; or (iii) exposing said ulvan to an enzyme capable of degrading said ulvan.

29. The method according to claim 28, wherein exposing said ulvan to ultra-sonic waves comprises utilizing an ultrasonic power of at least 200 Watts.

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