WO2020225237A1

WO2020225237A1 - A vegetal association as a functional ingredient for aquaculture feed

Info

Publication number: WO2020225237A1
Application number: PCT/EP2020/062383
Authority: WO
Inventors: Giuliano LEONARDI; Vito MACCHIA
Original assignee: Green Innovation Gmbh
Priority date: 2019-05-07
Filing date: 2020-05-05
Publication date: 2020-11-12
Also published as: EP3965563A1; IT201900006617A1

Abstract

The use of a vegetal association as a functional ingredient for aquaculture feed, and particularly as an antibacterial ingredient against aquaculture pathogens, as well as aquaculture feed and aquaculture feed supplements comprising the same, are disclosed. The vegetal association of the invention surprisingly and unexpectedly showed a synergistic effect that can be used in aquaculture feed against aquaculture pathogens, thus achieving great results in terms of effectiveness even at very low concentrations, while avoiding the use of antibiotics, and at the same time preserving the human and animal health.

Description

“A VEGETAL ASSOCIATION AS A FUNCTIONAL INGREDIENT FOR

AQUACULTURE FEED”

DESCRIPTION

FIELD OF THE INVENTION

The present invention concerns a vegetal association as a functional ingredient for aquaculture feed, as well as aquaculture feed supplements comprising the same.

STATE OF THE ART

Fishes are known for their exceptional health benefits, particularly against cardiovascular diseases and for infant brain development due to their long-chain polyunsaturated fatty acids (PUFAs) content. Despite such nutritional benefits and culinary fondness, infection of fishes by pathogenic microorganisms and other contaminants is a major concern for fish consumers. Fish contaminated with bacterial pathogens could cause severe foodbome illness and offset the health benefits of PUFAs. The primary sources of microbial pathogens in fish are anthropogenic activities that generate point and non-point pollution in coastal waters. There are also naturally occurring waterborne pathogens like vibrio that could cause human illness by way of food consumption.

Similar concerns are for fishes grown in aquacultures. Recent studies have demonstrated that aquaculture pathogenic bacteria in live feed can be controlled by probiotics and that mortality of infected fish larvae can be reduced significantly by probiotic bacteria. However, the successful management of the aquaculture microbiota is currently hampered by our lack of knowledge of relevant microbial interactions and the overall ecology of these systems. Therefore, it is still felt the need to apply antibiotics in aquaculture in order to counteract the bacteria proliferation, even if this means that also good microorganisms are sacrificed and antibiotic resistance is promoted.

Antimicrobial resistance (AMR) is the ability of a microbe to resist the effects of medication previously used to treat them. The term includes the more specific "antibiotic resistance", which applies only to bacteria becoming resistant to antibiotics. Resistant microbes are more difficult to treat, requiring alternative medications or higher doses, both of which may be more expensive or more toxic.

The World Health Organization affirmed that inappropriate use of antibiotics in animal husbandry is an underlying contributor to the emergence and spread of antibiotic- resistant germs, and that the use of antibiotics as growth promoters in animal feeds should be restricted. The World Organisation for Animal Health has added to the Terrestrial Animal Health Code a series of guidelines with recommendations to its members for the creation and harmonization of national antimicrobial resistance surveillance and monitoring programs, monitoring of the quantities of antibiotics used in animal husbandry, and recommendations to ensure the proper and prudent use of antibiotic substances. Another guideline is to implement methodologies that help to establish associated risk factors and assess the risk of antibiotic resistance.

It is therefore felt the need to effectively counteract bad microorganisms in aquacultures while avoiding the use of antibiotics, and at the same time preserving the human and animal health.

SUMMARY OF THE INVENTION

The above object has been achieved by a vegetal association of a lignin fraction and a resin acid, as claimed in claim 1.

In a further aspect, the present invention also concerns the use of said vegetal association as a functional ingredient for aquaculture feed.

In this regard, the present invention also concerns an aquaculture feed or an aquaculture feed supplement comprising said association and suitable feed carriers.

In another aspect, the present invention concerns the use of the vegetal association for the antibacterial treatment of aquaculture tanks and devices.

Aquaculture, also known as aquafarming, is the farming of fish, crustaceans, molluscs, aquatic plants, algae, and other aquatic organisms. Aquaculture involves cultivating freshwater and saltwater populations under controlled conditions, and can be contrasted with commercial fishing, which is the harvesting of wild fish. Mariculture refers to aquaculture practiced in marine environments and in underwater habitats. Farming implies some forms of intervention in the rearing process to enhance production, such as regular stocking, feeding, protection from predators, etc. Preferably, the term “aquaculture” includes fish farming, molluscs farming, crustaceans farming, mariculture, algaculture (such as seaweed farming), and the cultivation of ornamental fishes. Particular methods include aquaponics and integrated multi-trophic aquaculture, both of which integrate aquatic animal farming and aquatic plant farming. The characteristics and the advantages of the present invention will become apparent from the following detailed description and the working examples provided for illustrative and non-limiting purposes.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the invention therefore is a vegetal association of a lignin fraction and a resin acid, wherein the lignin fraction comprises fragments having a weight average molecular weight up to 10,000 Daltons, as measured by Size-Exclusion Chromatography, said fragments comprising up to 55 phenylpropane units on weight average, and wherein the lignin fraction is in an weight percent higher than the resin acid, as based on the weight of the vegetal association.

As it will be widely discussed and demonstrated in the Examples below, the vegetal association of the invention surprisingly and unexpectedly showed a synergistic effect that can be used in aquaculture feed against aquaculture pathogens, thus achieving great results in terms of effectiveness even at very low concentrations, while avoiding the use of antibiotics, and at the same time preserving the human and animal health.

Similar advantageous results can be achieved by placing the vegetal association in contact with aquaculture tanks and devices, where significantly highly satisfying results are attained in disinfecting surfaces, apparatus and filtering elements composing the same, at the same time without damaging them, so as to suitably prevent aquatic animal infections and inflammatory states.

Lignin is a class of complex organic polymers that form important structural materials in the support tissues of some algae, vascular plants, included their bark, and herbaceous plants, such as wood (i.e. softwood and hardwood), straw of all cereals, cane bagasse, grass, linen, jute, hemp, or cotton. Lignin can also have mineral source, such as peat, leonardite and coal.

Chemically, lignin is a very irregular, randomly cross-linked polymer of phenylpropane units joined by many different linkages, with a weight average molecular weight of 20,000 Daltons or higher. A representative and illustrative lignin fragment (I) containing the most important bonding patterns is shown herein below:

Said polymer is the result of an enzyme-mediated dehydrogenative polymerization of three phenylpropanoid monomer precursors:

coumaryl alcohol coniferyl alcohol

which result in the following moieties, respectively:

hydroxyphenyl (H) guaiacyl (G) syringyl (S)

Coniferyl alcohol occurs in all species and is the dominant monomer in conifers (softwoods). Deciduous (hardwood) species contain up to 40% synapyl alcohol units while grasses and agricultural crops may also contain coumaryl alcohol units. Lignin can be categorized to softwood and hardwood lignins according to their raw biomass sources.

Raw biomass sources that can be suitable starting materials for obtaining the relevant lignin fraction are any lignin including essentially pure lignin as well as kraft lignin, biomass originating lignin, lignin from alkaline pulping process, lignin from soda process, lignin from organosolv pulping and any combination thereof.

By the expression“essentially pure lignin”, it should be understood as at least 90% pure lignin on a dry raw biomass basis, preferably at least 95% pure lignin, the remainder being extractives and carbohydrates such as hemicelluloses as well as inorganic matter. By the expression“kraft lignin”, it is to be understood lignin that originates from kraft black liquor. Black liquor is an alkaline aqueous solution of lignin residues, hemicellulose, and inorganic chemicals used in a kraft pulping process. The black liquor from the pulping process comprises components originating from different softwood and hardwood species in various proportions. Lignin can be separated from the black liquor by different techniques including e.g. precipitation and filtration. Lignin usually begins precipitating at pH values below 11 - 12. Different pH values can be used in order to precipitate lignin fractions with different properties. These lignin fractions may differ from each other by molecular weight distribution, e.g. M_w and M_n, polydispersity, hemicellulose and extractive contents, contents of inorganic material. The precipitated lignin can be purified from inorganic impurities, hemicellulose and wood extractives using acidic washing steps. Further purification can be achieved by filtration.

Alternatively, the lignin is separated from pure biomass. The separation process can begin with liquidizing the biomass with strong alkali followed by a neutralization process. After the alkali treatment, the lignin can be precipitated in a similar manner as presented above.

Alternatively, the separation of lignin from biomass comprises a step of enzyme treatment. The enzyme treatment modifies the lignin to be extracted from biomass. Lignin separated from pure biomass is essentially sulphur-free (sulphur content less than 3%) and thus valuable in further processing.

Preferably, the lignin so separated is also subjected to a depolymerization process in order to further reduce the weight average molecular weight of fragments.

Preferably, the lignin so separated is also subjected to a depolymerization process in order to further reduce the weight and number average molecular weights of fragments. Suitable depolymerization processes include base-catalyzed depolymerization, acid- catalyzed depolymerization, metallic catalyzed depolymerization, ionic liquids -assisted depolymerization, and supercritical fluids-assisted lignin depolymerization.

In preferred embodiments, said lignin fraction is obtained by base-catalyzed depolymerization .

Preferably, said lignin fraction is obtained by subjecting the separated lignin to a base- catalyzed depolymerization at a temperature lower than 300°C and a pressure lower than 30 MPa.

The pH is set between 11 and 14, by adding a base such as NaOH, KOH, Ca(OH)2, LiOH, K2CO3, or a mixture thereof.

For the purposes of the present invention, the weight average molecular weight (M_w) of fragments in the lignin fraction is measured by Size-Exclusion Chromatography (or ‘SEC’). SEC employs a stagnant liquid present in the pores of beads as the stationary phase, and a flowing liquid as the mobile phase. The mobile phase can therefore flow between the beads and also in and out of the pores in the beads. The separation mechanism is based on the size of the polymer molecules in solution. Bigger molecules will elute first. Small molecules that can enter many pores in the beads take a long time to pass through the column and therefore exit the column slowly. To determine the molecular weights of the components of a polymer sample, a calibration with standard polymers of known weight must be performed. Values from the unknown sample are then compared with the calibration graph. The retention times depends on the used column material, eluent and how similar the used standards are compared to the samples. In the present invention, the eluent is preferably 0.1 M NaOH.

The association of the invention also comprises a resin acid.

Preferably, said resin acid is abietic acid, dehydroabietic acid, palustric acid, neoabietic acid, pimaric acid, isopimaric acid, sandaropimaric acid, or an ester thereof, or an ether thereof, or an alkali or alkaline-earth salt thereof, or a hydrogenated form thereof, or a mixture thereof.

Suitable esters include esters of glycerol.

Suitable alkali or alkaline-earth salts include salts of Na, K, Ca, Mg, NH4, and combinations thereof. Suitable hydrogenated forms of resin acids include dihydro- and tetrahydro resin acids, such as dihydropimaric acid, dihydro- and tetrahydroabietic acid.

Resin acids are present in coniferous trees, and there are three main species of resin acid products, namely Tall Oil Rosin (TOR), Wood Rosin and GUM Rosin. TOR is the resin acid fraction separated by vacuum distillation from Crude Tall Oil (CTO) which is produced by the preparation of pulp. CTO is obtained via acidulation of Crude Tall Oil Soap or Crude Sulphate Soap (TOS). TOS is separated from cooking liquid in pulp mill often called black liqueur during pulping process. Wood Rosin is the fraction separated by steam distillation or other means from dead trees, tree stumps, branches etc. and GUM Rosin is the resin fraction that has been steam distilled or separated by other means from resin harvested often called tapping from a living tree.

Substances containing resin acids and obtained by vacuum distillation from crude tall oil include Distilled Tall Oil (DTO), Tall Oil Fatty Acid (TOFA) and Tall Oil Pitch (TOP). DTO contains 10- 40% of resin acids. CTO typically contains 15-70% of resin acids, and the lowest resin acid contents are generally provided by the cooking of mixed wood pulp.

The term "Tall Oil Rosin" or "TOR" should be understood as referring to a composition obtained by distillation of crude tall oil and further refinement of distilled tall oil. TOR typically comprises 60-99% (w/w) resin acids.

The term "Wood Rosin" should be understood as referring to a composition obtained by distillation or other means from dead trees, tree stumps, branches etc. Wood Rosin typically comprises 50-99% (w/w) resin acids.

The term "GUM Rosin" should be understood as referring to a composition obtained by distillation or separated by other means from resin harvested from a living tree. GUM Rosin typically comprises 50-99% (w/w) resin acids.

The term "Distilled Tall Oil" or "DTO" should be understood as referring to a composition obtained by distillation of crude tall oil and further refinement of distilled tall oil. DTO typically comprises 10-60% (w/w) resin acids.

The resin acid based composition TOR, Wood Rosin, GUM Rosin, CTO, TOS and DTO can also be produced by mixing one or more resin acid compositions and one or more fatty acid compositions in form of oils or fats. Produced resin acid derivatives are for example esters, ethers or alkali metal salts. Resin acids are known to show many properties, such as antibacterial, anti inflammatory, and antioxidant properties. However, resin acids are poorly stable over time, especially in solid forms, as they are subjected to oxidation, spontaneous ignition and packing.

It has been surprisingly found that when a resin acid is mixed with the lignin fraction according to the present invention, the resulting association, either solid or liquid, is very stable over time and without oxidation, so that it is possible to fully take benefit of the properties of resin acids. This means that, in the resulting association, not only resin acids are preserved from degradation and oxidation, but also a synergistic anti-bacterial effect between lignin fraction and resin acids is observed against aquaculture pathogens. It should be reminded that wastewaters from different pulp-making processes contain various organic and inorganic compounds: wood-derived, process chemicals and compounds generated during the reactions between the chemicals and raw materials. Among all wood-derived compounds, resin acids are generally recognised as the main source of acute toxicity in pulp and paper industry effluents. A review of studies on toxicity of resin acids has shown that the acute lethal concentration of individual acids for fish (rainbow trout and salmon) is around 0.4-1 mg/1.

It is therefore surprising and unexpected that, once in admixture with the lignin fraction as above described, the resulting association with resin acid shows indeed a great effect on aquaculture pathogens without negatively affecting the overall fish health.

Moreover, without wishing to be bound by any theory, it is believed that the synergistic antibacterial effect is achieved also because the lignin fraction has proven to be able to break down bacterial biofilms, thus allowing resin acid to more effectively kill their targets. As a result, not only the vegetal association of the invention is effective against aquaculture pathogens, such as Gram-positive and Gram-negative bacteria, but also this effect is altogether boosted by the ability of the lignin fraction to tear down bacterial biofilms as well as preserve resin acids from oxidation, thus prolonging their activity over time.

In nature, bacteria are predominately associated with surfaces in highly structured communities called bio films. Biofilm bacteria usually have distinct characteristics compared to free-swimming bacteria of the same species, including a significantly increased resistance to antimicrobial treatments and immune system responses. In modem clinical microbiology, the biofilm mode of growth has become established as a pathogenicity trait in chronic infections. Another example of microbial community behaviour is quorum sensing, a regulatory mechanism involving cell-to-cell communication mediated by diffusible signal molecules. Quorum sensing is prevalent among both symbiotic and pathogenic bacteria associated with plants and animals. Recent findings suggest that quorum sensing regulates biofilm formation in the opportunistic pathogens. These findings suggest that therapy targeting quorum sensing may provide a means of treating many common and important chronic infections without the use of antibiotics that select for resistant organisms.

As said, in the aquaculture sector, so far, antibiotics and disinfectants have only had limited success in the prevention or cure of aquatic diseases caused by aquatic bacterial pathogens. Many of these pathogens are found to control virulence factor expression by a cell-to-cell communication system. Hence, disruption of bacterial quorum sensing has been proposed as a new anti-infective strategy and some techniques that could be used to disrupt quorum sensing have been investigated, such as (1) the inhibition of signal molecule biosynthesis, (2) the application of quorum sensing antagonists (including natural occurring as well as synthetic halogenated furanones, antagonistic quorum sensing molecules and undefined exudates of higher plants and algae), (3) the chemical inactivation of quorum sensing signals by oxidised halogen antimicrobials, (4) signal molecule biodegradation by bacterial lactonases and by bacterial and eukaryotic acylases and (5) the application of quorum sensing agonists. In view of the results achieved by the vegetal association of the instant invention, it is believed that the same can be considered a new approach and innovative technique to effectively address the bacterial quorum sensing, thus preventing and hindering the formation of bacterial biofilms in aquaculture.

Preferably, said lignin fraction comprises fragments having a weight average molecular weight up to 6,000 Daltons.

More preferably, said lignin fraction comprises fragments having a weight average molecular weight up to 5,500 Daltons.

Even more preferably, said lignin fraction comprises fragments having a weight average molecular weight up to 5,000 Daltons.

In some embodiments, said lignin fraction comprises fragments having a weight average molecular weight up to 2,000 Daltons.

In preferred embodiments, said lignin fraction comprises fragments having a weight average molecular weight up to 1,500 Daltons.

In other embodiments, said lignin fraction comprises fragments having a weight average molecular weight down to 150 Daltons.

In preferred embodiments, said lignin fraction comprises fragments having a weight average molecular weight of 150 Daltons to 6,000 Daltons, preferably having a weight average molecular weight of 250 Daltons to 5,000 Daltons, more preferably having a weight average molecular weight of 500 Daltons to 2,500 Daltons.

Preferably in these embodiments, said fragments comprise up to 30 phenylpropane units on weight average, more preferably, up to 27 phenylpropane units on weight average. The molecular weight of the three phenylpropanoid monomer precursors varies between 150 Da of coumaryl alcohol, 180 Da of coniferyl alcohol, and 210 Da of synapyl alcohol. The average weight is therefore 180 Da and this value has been used as “phenylpropane unit”. The M_w values have been divided by 180 Da, thus obtaining the phenylpropane unit numbers on weight average.

In other embodiments, the lignin fraction comprises fragments having a number average molecular weight (M_n) up to 2,000 Daltons.

For the purposes of the present invention, the number average molecular weight (M_n) of fragments in the lignin fraction is measured by Size-Exclusion Chromatography. Preferably, the lignin fraction comprises fragments having a number average molecular weight (M_n) up to 1,500 Daltons.

In preferred embodiments, said lignin fraction comprises fragments having a number average molecular weight of 150 Daltons to 1,300 Daltons.

Without wishing to be bound by any theory, it is believed that lower number average molecular weights mean more active molecules. This is put forward considering that lower molecular weights mean smaller fragments, and smaller fragments mean less crosslinked/shorter fragments, and less crosslinked/shorter fragments mean a higher number of free functional groups thereon, thus more reactive fragments.

Moreover, it is believed that smaller molecules can easily pass through the cell membrane of pathogens and diffuse therewithin, thus significantly increasing the overall effectiveness of the lignin fraction. Preferably in these embodiments, said fragments comprise up to 11 phenylpropane units on number average, more preferably, up to 8 phenylpropane units on number average. The molecular weight of the three phenylpropanoid monomer precursors varies between 150 Da of coumaryl alcohol, 180 Da of coniferyl alcohol, and 210 Da of synapyl alcohol. The average weight is therefore 180 Da and this value has been used as “phenylpropane unit”. The M_n values have been divided by 180 Da, thus obtaining the phenyl propane unit numbers on number average.

In preferred embodiments, said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 150 Daltons to 2,500 Daltons, and fragments having a number average molecular weight (M_n) up to 2,000 Daltons.

More preferably, said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 150 Daltons to 2,500 Daltons and 2 to 13 phenylpropane units on weight average, and fragments having a number average molecular weight (M_n) up to 2,000 Daltons and up to 11 phenylpropane units on number average.

In further embodiments, the lignin fraction has a polydispersity index (PD I) of 1.25 to 6. The polydispersity index (PDI) or heterogeneity index, or simply dispersity, is a measure of the distribution of molecular mass in a given polymer sample. PDI is the weight average molecular weight (M_w) divided by the number average molecular weight (M_n). It indicates the distribution of individual molecular masses in a batch of polymers.

Particularly preferred embodiments are those wherein said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 150 Daltons to 2,500 Daltons and 2 to 13 phenylpropane units on weight average, and wherein said lignin fraction has a polydispersity index of 1.25 to 6.

Particularly preferred embodiments are also those wherein said lignin fraction comprises fragments having a number average molecular weight (M_n) up to 2,000 Daltons and up to 11 phenylpropane units on number average, and wherein said lignin fraction has a polydispersity index of 1.25 to 6.

The most preferred embodiments are those wherein said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 150 Daltons to 2,500 Daltons and 2 to 13 phenylpropane units on weight average, a number average molecular weight (M_n) up to 2,000 Daltons and up to 11 phenylpropane units on number average, and wherein said lignin fraction has a polydispersity index of 1.25 to 6.

In particularly preferred embodiments of the present invention, said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 4,400 Daltons to 5,000 Daltons and 24-28 phenylpropane units on weight average, a number average molecular weight (M_n) of 1,200 to 1,300 Daltons and 6-7 phenylpropane units on number average.

In other particularly preferred embodiments of the present invention, said lignin fraction comprises fragments having a weight average molecular weight (M_w) of 800 Daltons to 1,500 Daltons and 4-8 phenylpropane units on weight average, a number average molecular weight (M_n) of 300 to 700 Daltons and 2-4 phenylpropane units on number average. In these particularly preferred embodiments, in said lignin fraction, the most abundant phenylpropane units are those from coniferyl alcohol, whereas the less abundant phenylpropane units are those from synapyl alcohol.

In preferred embodiments, said resin acid in the vegetal association of the invention is present in the form of GUM Rosin. In fact, differently from the other types of acid resin sources, GUM Rosin is produced by tapping living pine trees and collecting the oleoresin exudate. Oleoresin is exuded by the pine tree as a defense mechanism and can be collected until the tree is harvested. This means that the collected oleoresin is a naturally high-quality acid resin source, thus well suitable for feed applications.

Preferably, in the vegetal association of the invention, said lignin fraction and said resin acid are in a weight ratio of 20: 1 to 2: 1.

More preferably, in the vegetal association of the invention, said lignin fraction and said resin acid are in a weight ratio of 15:1 to 2:1.

Even more preferably, in the vegetal association of the invention, said lignin fraction and said resin acid are in a weight ratio of 10:1 to 3:1.

The vegetal association of the invention can be in a solid or liquid form.

When the vegetal association is in a solid form, said solid form can be tablet, mini tablet, micro-tablet, granule, micro-granule, pellet, multiparticulate, micronized particulate, or powder.

When the vegetal association is in a liquid form, said liquid form is a solvent solution. Suitable solvents are water, glycols, alcohols, polyalcohols, organic acids, and combinations thereof. Preferred solvents are water, methanol, ethanol, n-propanol, iso-propanol, n-butanol, isobutanol, allyl alcohol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-ethylene glycol, polyethylene glycol (PEG), glycerol, lactic acid, polylactic acid, and mixtures thereof.

More preferred solvents are water, 1,2-propylene glycol, 1,3-propylene glycol, 1,2- ethylene glycol, polyethylene glycol (PEG), and mixtures thereof.

In the most preferred embodiments, the solvent is water.

Preferably, when the vegetal association is in a liquid form, said liquid form has a pH of 8-11, more preferably 9.5-10.5.

In preferred embodiments, the vegetal association of the invention is in a solid form.

The vegetal association can also additionally comprise at least one alkali or alkaline- earth salt of fatty acid C12-C24, at least one fatty oil, or a mixture thereof. These fatty oil or salt allow a better solubilization of resin acids when liquid form of the vegetal association is preferred.

Preferably, said alkali or alkaline-earth salt is a salt of lithium, sodium, potassium, magnesium, calcium, or a mixture thereof.

Preferably, said fatty acid C12-C24 is lauric acid (C12), tridecylic acid (C13), myristic acid (C14), pentadecylic acid (C15), palmitic acid (C16), margaric acid (C17), stearic acid (C18), oleic acid (C18: l), linoleic acid (C18:2), a-linolenic acid (C18:3), g- linolenic acid (C18:3), nonadecylic acid (C19), arachidic acid (C20), heneicosanoic acid (C21), behenic acid (C22), tricosylic acid (C23), lignoceric acid (C24), stearidonic acid (C18:4), eicosapentaenoic acid (C20:5), docosahexaenoic acid (C22:6), dihomo-g- linolenic acid (C20:3), arachidonic acid (C20:4), adrenic acid (C22:4), palmitoleic acid (C16:l), vaccenic acid (C18:l), paullinic acid (C20:l), elaidic acid (Ctrans-18:l), gondoic acid (C20:l), erucic acid (C22:l), nervonic acid (C24:l), mead acid (20:3), or a mixture thereof.

In preferred embodiments, said at least one alkali or alkaline-earth salt of fatty acid C12- C24 is calcium palmitate, calcium laurate, calcium oleate, calcium soap of palm oil, or a mixture thereof.

Preferably, said at least one fatty oil is hemp oil, canola oil, sunflower oil, olive oil, com oil, palm oil, coconut oil, pine oil, cottonseed oil, wheat germ oil, soya oil, safflower oil, linseed oil, tung oil, castor oil, soybean oil, peanut oil, rapeseed oil, sesame seed oil, rice germ oil, fish oil, whale oil, marine oil, or a mixture thereof.

Preferably, the vegetal association comprises microspheres or drops or beads comprising the resin acid, and said at least one at least one alkali or alkaline-earth salt of fatty acid C12-C24, at least one fatty oil, or a mixture thereof. This allows the resin acid to be further protected from oxidation owing to the chemical and physical shielding effect of said at least one at least one alkali or alkaline-earth salt of fatty acid C12-C24, at least one fatty oil, or a mixture thereof.

In preferred embodiments, in said microspheres or drops or beads, the resin acid and the at least one at least one alkali or alkaline-earth salt of fatty acid C12-C24, at least one fatty oil, or a mixture thereof are in a weight ratio of 30:70 to 70:30, more preferably 40:60 to 60:40.

In a further aspect, the present invention also concerns the use of said vegetal association as a functional ingredient for aquaculture feed, and particularly as an antibacterial ingredient against aquaculture pathogens.

In fact, the vegetal association of the invention has unexpectedly and surprisingly proved to be very selective and effective against aquaculture pathogens, at the same time without negatively affecting the good microorganisms, as shown in the Examples provided below.

Aquaculture pathogens are Gram-positive and Gram-negative bacteria, such as those belonging to species Aerococcus viridans, Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida subsp. Achromogenes, Aeromonas salmonicida subsp. Salmonicida, Aeromonas schubertii, Aeromonas sobria, Arcobacter cryaerophilus ( Campylobacter cryearophila), Bacteriosis Pinnerum (Fin Rot), Burkholderia cepacian, Citrobacter braakii, Citrobacter freundii, Clostridium perfringens, Edwardsiella ictalurid, Edwardsiella tarda, Enterobacter cloacae, Erysiopelothrix rhusiopathiae, Escherichia vulneris, Flavobacterium branchiophilum, Flavobacterium columnare (Flexibacter columnaris, Cytophaga columnaris), Flavobacterium hydatis, Flavobacterium johnsoniae, Flavobacterium psychrophilum, Flexibacter Columnaris, Hafnia alvei, Ichthyophthirius multifiliis, Lactococcus garvieae, Listeria monocytogenes, Listonella anguillarum (Vibrio anguillarum), Mycobacterium chelonae, Mycobacterium chelonae marinumb, Mycobacterium chelonae montefiorense, Mycobacterium chelonae pseudoshottsii, Mycobacterium chelonae scrofulaceum, Mycobacterium chelonae shottsii, Mycobacterium chelonae similae, Mycobacterium chelonae szulgai, Mycobacterium chelonae triplex, Mycobacterium chelonae absscessus, Mycobacterium chelonae chesapeaki, Mycobacterium chelonae fortuitum, Mycobacterium chelonae interjectum, Nocardia asteroids, Pantoea agglomerans, Photobacterium damselae sub, Photobacterium damselae subsp. damselae (Vibrio damselae), Photobacterium damsella subsp. piscicida (Pasteurella piscicida), Piscicida, Aeromonas salmonicida sub Salmonicida, Pseudomonas aeroginasa, Pseudomonas anguilliseptica, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas luteola, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomoniasis (P. anguilliseptica), Salmonella typhimurium, Serratia liquefeciens, Shewanella putrefaciens grp, Staphylococcus cohnii subsp. Cohnii, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus iniae, Tenacibaculum maritinum, Vagococcus salmoninarum, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio ordalii, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia ruckeri.

Preferably, aquaculture pathogens are selected from those belonging to genera Vibrio, Salmonella, Clostridium, Streptococcus, Photobacterium, Pseudomonas, Aeromonas, Edwardsiella, Yersinia, Tenacibaculum, Listeria, Nocardia, and combinations thereof. Aquaculture feed comprises feed for fish, crustaceans, molluscs, and other aquatic organisms, as well as feed for aquatic plants, and algae.

In this regard, the present invention also concerns an aquaculture feed or an aquaculture feed supplement comprising said vegetal association and suitable feed carriers.

In preferred embodiments, the vegetal association is used in an amount so as to provide up to 10 kg of ‘lignin fraction+resin acid’ per ton of aquaculture feed or aquaculture feed supplement, more preferably 1-5 kg per ton.

Said aquaculture feed or aquaculture feed supplement can be in a solid or liquid form. When in a solid form, said solid form can be tablet, mini-tablet, micro-tablet, granule, micro-granule, pellet, multiparticulate, micronized particulate, or powder.

When in a solid form, said solid form comprises up to 99 wt% of vegetal association, preferably, 5-90 wt% of vegetal association.

When in a liquid form, said liquid form can be solution, emulsion, dispersion, suspension, gel, drops, or spray.

When in a liquid form, said liquid form comprises up to 50 wt% of vegetal association, preferably, 0.1-25 wt% of vegetal association. This means that the composition is a concentrate that can be suitably diluted in water or directly mixed with other aquaculture feed before use, if desired.

Suitable feed carriers are acidifying agents, acidity correctors, anti-agglomerants, antioxidants, fillers, resistance agents, gelling agents, coating agents, modified starches, sequestering agents, thickeners, sweeteners, thinners, solvents, disaggregating agents, glidants, dyes, binders, lubricants, stabilizers, adsorbents, preservatives, wetting agents, flavors, film-forming substances, emulsifiers, wetting agents, release retardants and mixtures thereof.

In fact, it has been surprisingly found that the vegetal association of the invention once placed in contact with aquaculture tanks and devices, significantly enables highly satisfying results to be attained in disinfecting surfaces, apparatus and filtering elements composing the same, at the same time without damaging them, so as to suitably prevent aquatic animal infections and inflammatory states.

Said surfaces and apparatus can be made of concrete, metals, steel, polymers, such as polyethylene and PVC, elastomers, or combinations thereof.

Similarly, said vegetal association can be suitably used as an antibacterial agent for disinfecting water for aquaculture before aquatic animals are housed therein. In this regard, the vegetal association can preferably be in a solid form, such as granules, in order to be easily removed from water after treatment (e.g. by mechanical filtration) and before aquatic animals are introduced, or alternatively the vegetal association can be directly a part of the water filter elements, as a coating or as a solid form embedded therein or as a solution impregnating said elements. The poor solubility in water of the vegetal association of the invention allows to obtain suitable persistent films on the treated surfaces and elements. This persistence durably avoids the development and proliferation of pathogens, as well as the formation of their biofilms.

It should be also understood that all the combinations of preferred aspects of the use of vegetal association of the invention, as well as of the aquaculture feed, aquaculture feed supplement, and uses of the same, as above reported, are to be deemed as hereby disclosed. All combinations of the preferred aspects of the use of vegetal association of the invention, aquaculture feed, aquaculture feed supplement, and the respective uses disclosed above are to be understood as herein described.

Below are working examples of the present invention provided for illustrative purposes. EXAMPLES

M_w and M_n in these Examples have been measured by Size-Exclusion Chromatography according to the following procedure.

Reagents and materials

- Eluent: 0.1 M NaOH, flow 0.5 ml/min

- Calibration for RI detector: Pullulan standards, M_p: 100,000 - 1,080 (six standards), where M_p is peak maximum molecular weight

- Calibration for UV-detector (280 nm): PSS standards, polystyrenesulfonate sodium salt, M_p 65,400 - 891 (six standards). Standards are dissolved into ultra-pure water, concentration should be approximately 5 mg/ml. Injection volume is 20 pi.

- Quality control samples: lignin with known M_w distribution is used.

Equipment and instruments

- Dionex Ultimate 3000 Autosampler, column compartment, and pump

- Dionex Ultimate 3000 Diode Array Detector

- Reflective Index detector: Shodex RI-101

- Columns: PSS MCX columns: precolumn and two analytical columns: 1000 A and 100 000 A, column material is sulfonated divinylbenzen copolymer matrix.

- Syringe filters 0,45 pm and glass sample bottles for STD samples. Sample filtration: Mini-Uniprep syringeless filter device PTFE or Nylon, 0,45 pm. For prefiltration 5 pm syringe filter if needed.

- Measuring bottles

Procedure

- Preparation of the eluent

Ideally, water used to prepare eluents should be high quality deionized water of low resistivity (18 MW*ah or better) that contains as little dissolved carbon dioxide as possible. The water must be free of biological contamination (e.g., bacteria and molds) and particulate matter.

- Needle washing with 10 % MeOH-water - Liquid samples

Strong alkaline liquor samples are diluted 1:100 and filtered with PTFE syringe filters (0,45 pm) to vials. Solid lignin samples are diluted and dissolved into 0.1 M NaOH and filtered with PTFE, 0,45 pm syringe filters. Ready samples are load into autosampler. Injection volume is 20 pi. After samples 1 M NaOH is injected as a sample to clean the column.

Instrument parameters:

- Flow rate 0.5 ml/min

- Eluent 0.1 M NaOH

- Column oven temperature 30°C

- Isocratic run

- Run time 48 minutes

- Solid samples

Solid samples (lignin) are dried overnight in an oven at 60°C, if needed. Approximately 10 mg is weighed into a 10-ml measuring bottle. Sample is dissolved and diluted into 0.1 M NaOH solution and filled into a mark. Sample is filtered with PTFE, 0,45 pm filters. If sample does not dissolve properly, it can be put in a ultrasound water bath or sample can be filtered through a 5 pm syringe filter.

- Standard samples for calibration

Approximately 50 mg of each standard is weighed into a 10-ml measuring bottle and ultrapure water is added and filled into a mark. Standards are filtered with PTFE 0,45 pm syringe filters. After running the calibration samples, calibration results are integrated and processed in the processing method and saved. Calibration is linear 1st order calibration.

- Quality control samples

For lignin samples, lignin with known M_w distribution is used as a quality control sample. Lignin is dissolved into 0.1 M NaOH and the concentration is approximately 1 mg/ml.

EXAMPLE 1.

Organosolv lignin obtained from Beech wood ( Fagus sylvatica ) was subjected to a base-catalysed depolymerization (‘BCD’). The BCD process is ran at 280°C and 250 bar for 8 minutes at pH 12-14. The resulting lignin product consisted of a liquid fraction and a solid fraction.

These fractions were then separated.

The liquid lignin fraction was an oil and had the following characteristics:

Single Species: Fagus sylvatica

M_w 100-300 Da (1-2 phenylpropane units)

phenols 0%

guaiacols 15-20%

syringols 50-60%

catechols and metoxycatecols 5-10%

oligomers/unknown 15-30%

The solid lignin fraction had the following characteristics:

Single Species: Fagus sylvatica

M_w 800-1,500 Da (4-8 phenylpropane units)

M_n 300-700 Da (2-4 phenylpropane units)

Structures of OH-groups:

aliphatic 0.2-0.4 mmol/g

carboxylic 0.3-0.5 mmol/g

condensated and syringyl 1.0-2.0 mmol/g

guaiacyl 0.4 mmol/g

catecholic and p-OH-phenyl 1.0- 1.8 mmol/g

Example la.

50 g of the oily lignin fraction (5%w/w) above has been mixed with 950 g of 1 ,3- propylene glycol, and warmed at 40-50°C.

The mixture has been cooled to room temperature, thus obtaining a viscous solution (shortly referred to as“LMW12”).

Example lb.

100 g of the solid lignin fraction (10%w/w) above has been hot-mixed with 800 g of 1,3-propylene glycol, and 100 g of NH₄OH (solution at 30%).

The mixture has been cooled to room temperature and then filtered off, thus obtaining a black solution (shortly referred to as“LMW11”).

Example lc.

100 g of the solid lignin fraction (10%w/w) above has been hot-mixed with 835 g of 1,3 -propylene glycol, and 65 g of KOH (solution at 20%).

The mixture has been cooled to room temperature and then filtered off, thus obtaining a black solution (shortly referred to as“LMW10”).

EXAMPLE 2.

The following lignin fraction has been extracted from Kraft black liquor, said lignin fraction having the following characteristics:

> 95% of total solids

Single Species: Southern Pine

M_w 4400-5000 Da (24-28 phenylpropane units)

M_n 1200-1300 Da (6-7 phenylpropane units)

Structures of OH-groups:

aliphatic 2.1 mmol/g

carboxylic 0.5 mmol/g

condensated and syringyl 1.7 mmol/g

guaiacyl 2.0 mmol/g

catecholic and p-OH-phenyl 4.0 mmol/g

Example 2a.

100 g of the lignin fraction (10%w/w) above has been hot-mixed with 840 g of 1,3- propylene glycol, and 60 g of NH₄OH (solution at 30%).

The mixture has been cooled to room temperature and then filtered off, thus obtaining a black solution (shortly referred to as“OX11”).

Example 2b.

100 g of the lignin fraction (10%w/w) above has been hot-mixed with 840 g of 1,3- propylene glycol, and 60 g of NaOH (solution at 30%).

The mixture has been cooled to room temperature and then filtered off, thus obtaining a black solution (shortly referred to as“0X10”).

EXAMPLE 3.

The lignin fraction 0X10 according to Example 2b has been mixed with GUM rosin in a weight ratio of about 4:1.

A vegetal association has been prepared in a liquid form having the following composition: 8wt% of lignin fraction, 2wt% of GUM rosin, 0.5wt% KOH, the remainder being propylene glycol. EXAMPLE 4.

The antibacterial activity of the vegetal association of Example 3 (shortly referred to as “GEM”) was evaluated. The Minimum Inhibitory Concentrations (MICs) were determined in multiwell plates, where Blank is 1,3-propylene glycol only.

The antibacterial activity was tested on a number of bacteria and the results are reported in the table below.

The synergy (%) has been calculated by using the following formulas:

MICteo= (MICoxio x 80%) + (MICGUM X 20%)

% Sinergy = [(MICteo - MlCoem) / MICteo] x 100 wherein:

MICoxio is the Minimum Inhibitory Concentration of 0X10,

MICGUM is the Minimum Inhibitory Concentration of GUM rosin,

MICteo is the calculated overall MIC, i.e. (MICoxio + MICGUM),

MIC_Gem is the experimentally observed overall MIC of Example 3.

As it can be immediately seen from the table above, the vegetal association of the invention surprisingly and unexpectedly allows to drastically reduce the overall amount of lignin fraction and especially resin acid that can be used in aquaculture feed against aquaculture pathogens, thus achieving great results in terms of effectiveness even at very low concentrations, while avoiding the use of antibiotics, and at the same time preserving the human and animal health.

Claims

1. A vegetal association of a lignin fraction and a resin acid, wherein the lignin fraction comprises fragments having a weight average molecular weight up to 10,000 Daltons, as measured by Size-Exclusion Chromatography, said fragments comprising up to 55 phenylpropane units on weight average, and wherein the lignin fraction is in an weight percent higher than the resin acid, as based on the weight of the vegetal association.

2. The vegetal association of claim 1, wherein said resin acid is abietic acid, dehydroabietic acid, palustric acid, neoabietic acid, pimaric acid, isopimaric acid, sandaropimaric acid, or an ester thereof, or an ether thereof, or an alkali or alkaline- earth salt thereof, or a hydrogenated form thereof, or a mixture thereof.

3. The vegetal association of claim 1 or 2, wherein said resin acid is present in the form of GUM Rosin.

4. The vegetal association of any one of claims 1-3, wherein said lignin fraction and said resin acid are in a weight ratio of 20:1 to 2:1, preferably 15:1 to 2:1, more preferably 10:1 to 3:1.

5. The vegetal association of any one of claims 1-4, wherein said lignin fraction comprises fragments having a weight average molecular weight of 150 Daltons to 6,000 Daltons.

6. The vegetal association of any one of claims 1-5, further comprising at least one alkali or alkaline-earth salt of fatty acid C12-C24, at least one fatty oil, or a mixture thereof.

7. The vegetal association of any one of claims 1-6, for use as an antibacterial ingredient against aquaculture pathogens in aquaculture feed.

8. An aquaculture feed or an aquaculture feed supplement comprising the vegetal association of any one of claims 1-6, and suitable feed carriers.

9. The aquaculture feed or the aquaculture feed supplement of claim 8, comprising the vegetal association in an amount so as to provide up to 10 kg of‘lignin fraction+resin acid’ per ton of aquaculture feed or aquaculture feed supplement, preferably 1-5 kg per ton.

10. Non- veterinary use of the vegetal association of any one of claims 1-6 as an antibacterial for disinfecting aquaculture tanks and devices, or for the antibacterial treatment of water for aquaculture before aquatic animals are housed therein.