Multi-step or cascade extraction process for extracting saline, fresh green or lignified biomasses and uses thereof

Abstract

The present invention relates to multi-step or cascade extraction process for extracting bio-active compounds from non-food fractions of saline, fresh green or lignified biomasses such as halophytes and seaweed. The extraction processes can also be used for treating circular feedstocks, such as polyphenolic rich circular feedstocks.

Description

[0001] 82457PC01 1Multi-step or cascade extraction process for extracting saline, fresh greenor lignified biomasses and uses thereofTechnical field of the inventionThe present invention relates to multi-step or cascade extraction process forextracting bio-active compounds from non-food fractions of saline, fresh green orlignified biomasses, such as halophytes and seaweed. The present inventionfurther relates to bactericidal and bacteriostatic uses of the extracts produced by the multi-step or cascade extraction process. Background of the inventionExtraction procedures such as decoction, maceration, liquid-liquid extraction,infusion, percolation, hydro distillation, digestion, and continuous hot refluxextraction (Soxhlet) have been extensively studied for many different plantspecies and are considered conventional extraction techniques. Although theseeffectively extract certain chemicals, the bioactive compounds can be sensitive to extreme conditions.Moreover, these conditions and conventionally used organic solvents also makethe extraction processes unsafe, requiring tremendous capital when upscaled. More environmentally friendly techniques are developed in the green extraction context to improve the extraction yield and diminish the extraction time and solvent used. Ultrasound-assisted extraction or pulsed electric fields assistedextraction, microwave irradiation or pressurized liquid extraction (PLE) areexamples of these novel techniques. A key challenge in the extraction techniques is that they were traditionally developed to deal with a single compound family, like polyphenols.Ko et al., (2020) discloses dynamic subcritical water extraction (DSWE) methodsfor extracting bioactive compounds, e.g. flavonoids and phenolic constituents,such as gallic acid from Orostachys japonicus (rock pine) at temperatures of 110-260°C using only water. 82457PC01 2CN101642632A (2009) discloses separated ultrasound assisted extraction andstatic subcritical liquid extraction methods to extract active ingredients of variousnatural products, such as green thorn fruit, using subcritical organic solvents.CN101642632A also envisages the combination of a subcritical liquid extractiontechnology and an ultrasonic extraction technology in order to increase extraction rate and reduce extraction time.Correia et al., (2022) discloses that Salicornia ramosissima extracts, such aspolyphenols for use as cosmetics, were obtained by static subcritical waterextraction (SSCE) at different temperatures (110, 120, 140, 160 and 180°C). Ultrasound-assisted extraction (UAE) is also envisaged.Carreira Casais et al., (2021) discloses that ultrasound assisted extraction (UAE)may be performed on a wide range of different marina algae for the recovery of various polyphenols. Water is reported to be a useful solvent for the UAEextraction. Carreira Casais et al. also discloses that UAE may be combined with arange of both new and conventional extraction techniques, such as pressurizedliquid extraction (PLE), also known as subcritical liquid extraction, for thedetermination of isoflavones in sea algae.Topuz et al., (2015) discloses ultrasound assisted extraction (UAE) was performedon red seaweed (Laurencia obtusa) in an ultrasonic bath at 250 W, 40 kHz and seaweed sample with the extracting solvent and sonicated for different times at the required temperature. After the extraction the red seaweed extracts were filtered and solvent was evaporated for the determination of the total phenolic content. FR2987756A1 discloses a method of extracting fragrant compounds from e.g. cocoa (Theobroma cacao) by using subcritical water extraction, e.g. combinedwith ultrasound. FR2987756A1 further discloses that ultrasound was used toimprove the extraction of the raw material in accelerant and / or improve the bursting of cells of the raw material. 82457PC01 3Radovanovic K., et al., (2023) discloses extraction of e.g. polyphenols andflavonoids from different species of yarrows (Achillea spp.) of the Asteraceaefamily by use of UAE and static subcritical water extraction (SSCE)) techniques.Hulkko L. S. S. et al., (2022) discloses that salt-tolerant halophytes have shownpotential for biorefinery when harvested green but not food-grade, andfractionated to green juice and fibre residue. The harvested aerial parts ofhalophyte biomass were fractionated to green juice and fibre residue by using a horizontal single-auger juicer and the species Salicornia europaea, Tripoliumpannonicum and Crithmum maritimum were fractionated.Hence, at least four main technical challenges relating to (i) the use of organicsolvents, (ii) upscaling, (iii) recovery of several ingredients in a cascade approachwhere extraction of one compound group does not destroy the potential to extractother compounds / compound groups, and (iv) in-situ concentration ofproducts / removal of solvent / water, enabling water recycling and energy saving indrying of the extracted products down-stream (see details below) need to beovercome to realize the invention. Organic solvents Extraction solvents are often toxic and dangerous for the user and environment. Conventional industrial extraction solvents are organics volatiles such as methanol, n-hexane, sulfolane, and benzene, produced from fossil resources and are often harmful to human health and the environment. As an example, n- hexane, despite being easy to obtain and recycle and offering good extractives, is produced from fossil resources and has recently been classified as CMR 3, i.e. it is suspected to be reprotoxic. In Canada alone, over 4.000 tons of n-hexane is emitted into the atmosphere, with about a third being from oilseed processing.Recovery of such a solvent is also energy-intensive on a large scale and carriesmany inherent safety concerns. Moreover, there are severe restrictions in the EU for the use of solvents in the extraction of food ingredients, which disqualifies many of the commonly used industrial extraction fluids solvents. There is a need for novel, user-friendly and environmentally sound solvents that are effective during extraction. 82457PC01 4 Upscaling Today's industrial extraction processes are considered unsafe and expensive in upscaling, mostly due to volatile organic solvents and the high temperature and pressure applied. These factors demand high safety measures and capital tosecure safe facilities for the people and environment. In the European Union, anyprocess using flammable or explosive materials needs to be approved by the ATEX214 "equipment" directive, involving requirements related to ventilation, escaperoutes, dedicated areas around the equipment, certificated components, etc. (Directive 2014 / 34 / EU of the European Parliament and of the Council, vol. OJ L.2014). Furthermore, although effective at small laboratory scales, extractionmethods using such volatile solvents demand huge capital investments on industrial scales. So, for extraction methods that are effective on a laboratory scale to reach industrial scales, the costs need to be kept low during upscaling by keeping the processes safe and simple on a laboratory scale. Recovery of several ingredients Conventional extraction processes that yield only one specific bioactive compound often do not preserve other bioactive ingredients. Attempts to extract several sensitive compounds from biomass for enhanced valorization are manually operated today by experience, limiting their efficiency. Advanced andautomatically supervised cascaded extraction technologies have not been realizedto the same extent as other chemical unit operations. Removal of solvent / water from the extracted compoundsIn traditional extraction processes the product to solvent ratio is often very low.The concentration of specific bioactive compounds / compound groups in the feedstocks are often low and significant amounts of solvent is needed to extract these high-value compounds from the feedstock biomasses. Hence, the endresults are often very dilute extracts, which require large amounts of energy (e.g.solvent evaporation) to achieve pure compounds. The end products are usuallyrequired by the end-users in a powder form for stability and optimal product formulation (e.g. as ingredient in food and feed products). Finally, currently applied extraction processes are often developed to yield and focus on only one product, whereas large fractions of the initial raw material are 82457PC01 5 considered waste. More complex multi-product biorefinery systems are required to achieve the maximum valorization of the raw material. It is an objective of the present invention to overcome these drawbacks in the art. Summary of the inventionThus, one aspect of the invention relates to a multi-step method of extracting bio-active compounds from non-food fractions of saline biomass having a dry matter content (DMC) of 70-100%, wherein the method comprises the following steps: (Step 1a) pretreatment of the biomass by subjecting said biomass to shredding, sieving, and cold-water wash (10-20°C), followed by (Step 2a) one or more extraction steps, followed by (Step 3a) separation of fibres and extracts of the slurry resulting from step 2a and 2b, followed by (Step 4a) concentration of the extracts by membrane filtration resulting from step 3a, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4a using permeate water as solvent or by using the extract obtained from 3a for extraction with possible auto-catalytic reactions.Another aspect of the present invention relates to a multi-step method ofextracting bio-active compounds from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugal juiced, saline biomass having a dry matter content (DMC) of 20-50%, wherein the method comprises the following steps: (Step 1b) drying of the biomass, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b usingwater as the sole extraction solvent, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b, followed by(Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds. Hence, the problems solved by the present invention are e.g. 82457PC01 6 ^High energy consumption and low throughput in traditional extraction ofbioactive compounds. ^High energy consumption of traditional lignocellulose pretreatment.^ Harsh chemicals are used in biomass processing, which otherwise couldend up (and give undesired side effects) in the end products. ^Use of flammable and explosive solvents used in traditional extractions thatincrease CAPEX due to ATEX-approval. ^High cost of pure bioactive compounds not yet produced by biosynthesis.^ Handling large volumes for extraction, as traditional extractors are made insteel or glass, which is not practical for large volumes and salinity. ^Handling waste saline biomass is unsuitable for traditional biorefineryprocesses, such as pyrolysis and gasification, or biowaste disposal due to its high salt content. ^Sugar degradation and production of toxic furans (e.g. furfural and 5-hydroxymethylfurfural) from severe pretreatment conditions used for lignocellulose biomass. ^Large solvent / water consumption in the extraction process.^ Low concentration of the bioactive compounds in the produced extracts, i.e.high cost for downstream purification and water / solvent removal. ^Low total yield of the bioactive compounds due to “one-pot” processapproach where the extraction of one compounds target group will destroy the possibility to extract another compound group with different properties. The present invention solves the above-outlined problems in the art by e.g. ^Combining static sub-critical extractor, ultra-sound and Soxhlet systemthat uses water as the sole solvent. ^Enabling several consecutive steps / cycles of the extractions until a targetconcentration of the product (compounds) is achieved without expensive continued distillation by applying pressing and nanofiltration separation / concentration of the compounds from the water between theextraction steps and the novel percolation reactor set-up. ^Using a multi-step system with flexible parameter settings to avoid veryhigh temperatures, toxic / expensive / flammable solvents, and long operation times. ^Using in-line filters and membranes for easy separation of fractions.^ Flexible use of water fraction (intermediate extract). 82457PC01 7 ^Simple reactor vessels in non-corrosive materials.^ Low-severity hydrothermal pretreatment and extraction to remove the lastextractive compounds (secondary extraction) and open the lignocellulosic structure. ^Utilizing the good ability of water to pre-treat the biomass using ultrasoundand extract bioactive compounds. Brief description of the figuresFigure 1 shows an overview of the multistep cascade extraction (for details, seeexample 4). Figure 2 shows the detailed drawing of the washing step design to remove sand, sea creatures such as e.g., snails, and surface salt from the halophyte biomass.Figure 3 shows the total amount of extractives fraction (figure 3a) obtained in therefence and cascade extractions as well as total amount of phenolics (figure 3b),flavonoids (figure 3c), and antioxidant capacity (figure 3d) of the obtainedextracts (for details, see example 4).Figure 4 shows the total amount of extractives fraction (figure 4a), total amountof phenolics (figure 4b), flavonoids (figure 4c), and antioxidant capacity (figure4d) obtained after one to four cycles of Soxhlet on: raw fibres (S1-S4), fibres after subcritical water extraction (SWE+S), ultrasound pretreated fibres (US), andfibres after ultrasound and subcritical water extractions cascade (USWE+S) (fordetails, see example 4).Figure 5 shows total phenolics (figure 5a) and total flavonoids (figure 5b) insoaking extracts (EXT1 and 2), in drained liquids after soaking in EXT1, 2, and water (cold extraction), and in extracts produces with biomass soaked in EXT1, 2,and water. SWE is tradition Subcritial Water Extraction for reference (for details,see example 4). 82457PC01 8Figures 6a-b show the chemical composition of pre-soaking liquid EXT1 (fordetails, see example 4).Figures 7a-b show the results of temperature influence in subcritical waterextractions (for details, see example 4).Figures 8a-b show (figure 8a) comparison of the total amount of flavonoidsextracted from Salicornia ramosissima biomass in subcritical water extraction withand without presoaking in natural biomass derived solvent at 40^C (NS1), 60^C(NS2), and 80^C (NS3) and (figure 8b) shows comparison of the total amount ofphenolic compounds extracted from Salicornia ramosissima biomass in subcriticalwater extraction with and without pre-soaking in natural biomass derived solvent at 40°C (NS1), 60°C (NS2), and 80°C (NS3) Figure 9 shows comparison of the enzymatic convertibility of residual fibres ofSalicornia ramosissima biomass after subcritical water extraction with and withoutpre-soaking in natural biomass derived solvent at 40°C (NS1), 60°C (NS2), and 80°C (NS3). Figure 10 shows bactericidal activity of extracts produced according to the methods of the present invention at different concentration, againstTenacibaculum maritimum (A), Photobacterium damselae subsp. piscicida (B),Vibrio anguillarum (C), Vibrio parahaemolyticus (D), Edwardsiella tarda (E),Yersinia ruckeri (F) and Aeromonas hydrophila (G).Figures 11 (a)-(c) show the bacteriostatic activity of the extracts againstTenacabaculum maritimum at extract concentrations of 0.5 mg / ml extract (figure11 (a)), 0.25 mg / ml extract (figure 11 (b)) and 0.125 mg / ml extract (figure 11(c)).Figures 12 (a)-(c) show the bacteriostatic activity of the extracts against Vibrioanguillarum at extract concentrations of 0.5 mg / ml extract (figure 12 (a)), 0.25mg / ml extract (figure 12 (b)) and 0.125 mg / ml extract (figure 12 (c)). 82457PC01 9Figures 13 (a)-(c) show the bacteriostatic activity of the extracts against Vibrioparahaemolyticus at extract concentrations of 0.5 mg / ml extract (figure 13 (a)),0.25 mg / ml extract (figure 13 (b)) and 0.125 mg / ml extract (figure 13 (c)).Figures 14 (a)-(c) show the bacteriostatic activity of the extracts against Vibrioharveyi at extract concentrations of 0.5 mg / ml extract (figure 14 (a)), 0.25 mg / mlextract (figure 14 (b)) and 0.125 mg / ml extract (figure 14 (c)).Figures 15 (a)-(c) show the bacteriostatic activity of the extracts againstEdwardsiella tarda at extract concentrations of 0.5 mg / ml extract (figure 15 (a)),0.25 mg / ml extract (figure 15 (b)) and 0.125 mg / ml extract (figure 15 (c)).Figures 16 (a)-(c) show the bacteriostatic activity of the extracts againstPhotobacterium damselae subsp. piscicida at extract concentrations of 0.5 mg / mlextract (figure 16 (a)), 0.25 mg / ml extract (figure 16 (b)) and 0.125 mg / mlextract (figure 16 (c)).Figures 17 (a)-(c) show the bacteriostatic activity of the extracts against Yersiniaruckeri at extract concentrations of 0.5 mg / ml extract (figure 17 (a)), 0.25 mg / mlextract (figure 17 (b)) and 0.125 mg / ml extract (figure 17 (c)).Figure 18 shows adherent head kidney cells from Salmo salar stained withGiemsa, captured using the EVOS cell imaging system.Figures 19 (a)-(b) show MTT results after 24 hours of exposure to varyingconcentrations of DMSO (figure 19 (a)) and salt controls (figure 19 (b)). Theabsorbance of treated cells was compared to that of control cells, with the control cells serving as the baseline (100%). Figure 20 shows MTT results after 24 hours of exposure to varying concentrations of EXT1, EXT2, EXT3, XAD, FERM, and FERM2. The absorbance of treated cells was compared to that of control cells, with the control cells serving as the baseline (100%). 82457PC01 10 Figure 21 shows evaluation of oxidative stress levels, assessed by detectingreactive oxygen species (ROS) generation using the CellROX reagent, in cells afterexposure to the extracts. Figure 22 shows assessment of phagocytic activity, using fluorescent bioparticles(pHrodoTM Red Escherichia coli Bioparticles), in cells after exposure to theextracts. Figure 23 shows MTT results after 24 hours of exposure to varying concentrationsof the extracts at five different concentrations. The absorbance of treated cellswas compared to that of control cells, with the control cells serving as the baseline (100%). Figure 24 shows bioelectrical characterization of discrete regions of the intestineof sea bass juveniles.Figures 25 (a)-(h) the three XAD compounds (figure 25 (a)-(c)) tested on gut Electrogenic amino acid transport (EAAT) assay behaved in a similar way in relation to the stimulation of the amino acid absorption current in the mid- intestine. The FERM compound (eluted in MqH2O) (figure 25 (d)) produces a clear inhibition on intestinal amino acid absorption, whereas a clear, linear and significant stimulation was observed after the addition of both XADs (figure 25 (e)) and FRAC3 compounds (both eluted in 30-40% EtOH). Both EXT2 and EXT3 compounds (figure 25 (g)-(h)) produces a clear, linear and significant increase of intestinal amino acid absorption, being more pronounced or evident after stimulation with compound EXT3. The present invention will now be described in more detail in the following. Detailed description of the invention Definitions Prior to discussing the present invention in further details, the following terms and conventions will first be defined: 82457PC01 11 Lignified saline biomass When used herein, lignified saline biomass refers to biomass grown in saline soilsor under saline conditions, brown or off-color compared to regular biomass colour,reaching the end of the life cycle for annual plants, and for perennial plants, in theend of the annual cycle just before, or after, dropping seeds, fruits, etc. Inaddition, as used herein, lignified saline biomass refers to biomass grown that can be used for the green fibre fraction left after green fractionation (juicing) of freshgreen halophyte biomass as described in Hulkko L. S., et al 2022(i) and Hulkko L.S., et al 2023. Fresh green saline biomassWhen used herein, fresh green saline biomass refers to biomass grown in salinesoils or under saline conditions, still remaining green, or potentially red, in color.The stage of maturity of the plant is before the plant sets seeds. Fresh greensaline biomasses are e.g. green leaves, green agro-residues, fresh halophytes.SWE (“sub-critical water extraction”)When used herein, SWE, sometimes also called “superheated water extraction” or“pressurized hot water extraction” (PHWE) in the art, is an extraction technique attemperatures between 100 and 374°C and pressure higher than steam pressure,high enough to maintain the liquid state. Two variants of SWE are described in theart, static SWE (SSCE) and dynamic SWE (DSWE). In SSCE, the extraction isperformed in a batch setting, and in DSWE, the extraction is performed in a percolation setting. This invention will work on-site in multiple industrial settings producing aquatic saline biomass high in bioactive compounds. Examples of target markets of theproducts are food, feed, nutraceuticals, pharmaceuticals, and cosmetics.Embodiments of the inventionOne embodiment of the invention relates to a multi-step method of extracting bio-active compounds from non-food fractions of saline biomass having a dry matter content (DMC) of 70-100%, wherein the method comprises the following steps: (Step 1a) pretreatment of the biomass by subjecting said biomass to shredding, sieving, and cold-water wash (10-20°C), followed by 82457PC01 12 (Step 2a) one or more extraction steps, followed by (Step 3a) separation of fibres and extracts of the slurry resulting from step 2a and 2b, followed by (Step 4a) concentration of the extracts by membrane filtration resulting from step 3a, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4a using permeate water as solvent or by using the extract obtained from 3a for extraction with possible auto-catalytic reactions.Another embodiment of the invention relates to a multi-step method of extractingpolyphenols (such as phenolic acids, hydroxycinnamic acids, flavonoids, flavanols,tannins, and stilbenes), terpenes, furanocoumarins, alkaloids, chitin, collagen, andother bioactive structural carbohydrates and proteins / peptides from non-foodfractions of saline biomass, such as halophytes, seaweed and algae having a drymatter content (DMC) of 70-100%, wherein the method comprises the following steps: (Step 1a) pretreatment of the biomass by subjecting said biomass to shredding, sieving, and cold-water wash (10-20°C), followed by (Step 2a) one or more extraction steps, followed by (Step 3a) separation of fibres and extracts of the slurry resulting from step 2a and 2b, followed by (Step 4a) concentration of the extracts by membrane filtration resulting from step 3a, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4a using permeate water as solvent or by using the extract obtained from 3a for extraction with possible auto-catalytic reactions. Still another embodiment of the invention relates to a multi-step method of extracting polyphenols from non-food fractions of halophytes selected from the group consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp. having a dry matter content (DMC) of 70-100%, wherein themethod comprises the following steps: (Step 1a) pretreatment of the biomass by subjecting said biomass to shredding, sieving, and cold-water wash (10-20°C), followed by (Step 2a) one or more extraction steps, followed by 82457PC01 13 (Step 3a) separation of fibres and extracts of the slurry resulting from step 2a and 2b, followed by (Step 4a) concentration of the extracts by membrane filtration resulting from step 3a, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4a using permeate water as solvent or by using the extract obtained from 3a for extraction with possible auto-catalytic reactions. Still another embodiment of the invention relates to a multi-step method of extracting polyphenols from non-food fractions of halophytes selected from the group consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp. having a dry matter content (DMC) of 70-100%, wherein themethod comprises the following steps: (Step 1a) pretreatment of the biomass by subjecting said biomass to shredding, sieving, and cold-water wash (10-20°C), followed by(Step 2a) one or more static sub-critical water extractions (SWE) of the slurryresulting from step (1a) carried out in a static sub-critical extractor (SSCE) at 100-140°C and 1-5 bar (absolute) pressure for 0.25-4 hours, followed by (Step 3a) separation of fibres and extracts of the slurry by membrane filtration with a filter mesh size of 150-100,000 Da resulting from step 2a and 2b, followed by(Step 4a) concentration of the extracts resulting from step 3a by in situmembrane filtration at 1-30 bar pressure for 0-50 hours, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4a using permeate water as solvent or by using the extract obtained from 3a for extraction with possible auto-catalytic reactions.Still another embodiment of the invention relates to a multi-step method ofextracting bio-active compounds from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugal juiced, saline biomass having a dry matter content (DMC) of 20-50%, wherein the method comprises the following steps: (Step 1b) drying of the biomass, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b usingwater as the sole extraction solvent, followed by 82457PC01 14 (Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b, followed by(Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds. Still another embodiment of the invention relates to a multi-step method of extracting bio-active compounds selected from the group consisting ofpolyphenols (such as phenolic acids, hydroxycinnamic acids, flavonoids, flavanols,tannins, and stilbenes), terpenes, furanocoumarins, alkaloids, chitin, collagen,and other bioactive structural carbohydrates and proteins / peptides from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugal juiced, saline biomass having a dry matter content (DMC) of 20-50%, wherein the method comprises the following steps: (Step 1b) drying of the biomass, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b usingwater as the sole extraction solvent, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b, followed by(Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds. Still another embodiment of the invention relates to a multi-step method of extracting bio-active compounds selected from the group consisting of polyphenols (such as phenolic acids, hydroxycinnamic acids, flavonoids, flavanols,tannins, and stilbenes), terpenes, furanocoumarins, alkaloids, chitin, collagen, andother bioactive structural carbohydrates and proteins / peptides from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugal juiced, halophytes, seaweed and algae having a dry matter content (DMC) of 20- 50%, wherein the method comprises the following steps: (Step 1b) drying of the biomass, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b usingwater as the sole extraction solvent, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b, followed by 82457PC01 15 (Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds.Still another embodiment of the invention relates to a multi-step method ofextracting polyphenols from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugal juiced, halophytes selected from thegroup consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp. having a dry matter content (DMC) of 20-50%, wherein themethod comprises the following steps: (Step 1b) drying of the biomass, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b usingwater as the sole extraction solvent, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b, followed by(Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. Still another embodiment of the invention relates to a multi-step method of extracting polyphenols from non-food fractions of screw-pressed, hydraulic- pressed, high-speed blended, centrifugal juiced, halophytes selected from the group consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp. having a dry matter content (DMC) of 20-50%, wherein themethod comprises the following steps:(Step 1b) drying of the biomass outside or at 40-105°C, for 1-48 hours resultingin DMC of the dried biomass of 8-16%, such as 12%, followed by (Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1b performed in a perforated basket with a mesh size of 0.5-5 mm using water asthe sole extraction solvent in a biomass:solvent ratio of 1:6, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severity hydrothermaltreatment of the extract produced in Step 2b carried out at 100-140°C and 1-5bar (absolute) pressure for 0.25-4 hours, followed by 82457PC01 16 (Step 4b) filtration of the extract obtained in Step 3b to obtain a final extract rich in bio-active compounds.Still another embodiment of the invention relates to non-therapeutic uses of anextract produced by a method according to the invention as a bactericidal orbacteriostatic agent. Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use as a medicament.Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating bacterial infections inaquatic animals, such as farmed fish. Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating diseases caused byTenacibaculum maritimum (such as Tenacibaculosis, Yellow Mouth Disease, SaltWater Columnaris) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout(Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchuslabrax))). Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating diseases caused byPhotobacterium damselae (such as Photobacteriosis (Pasteurellosis)) in farmedfish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))). Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating diseases caused by Vibrioanguillarum (such as vibriosis) in farmed fish (e.g. Salmon (Salmo salar),Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))). Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating diseases caused by Vibrio 82457PC01 17parahaemolyticus (such as Acute Hepatopancreatic Necrosis Disease (AHPND),Translucent Post Larvae Disease (TPD)) in farmed fish (e.g. Salmon (Salmo salar),Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))(. Still another embodiment of the invention relates to an an extract produced by amethod according to the invention for use in treating diseases caused byEdwardsiella tarda (such as Edwardsiellosis, Emphysematous PutrefactiveDisease) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchusmykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).Still another embodiment of the invention relates to an extract produced by amethod according to the invention for use in treating diseases caused by Yersiniaruckeri (such as Enteric Redmouth Disease (ERM), also known as Yersiniosis) infarmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))). Still another embodiment of the invention relates to an extract produced by a method according to any of claims 1-11 for use in treating diseases caused byAeromonas hydrophila (such as Hemorrhagic Septicemia, Ulcerative Disease,Motile Aeromonas Septicemia (MAS), Tail / Fin Rot) in farmed fish (e.g. Salmon(Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))). It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. 82457PC01 18 The invention will now be described in further details in the following non-limiting examples. ExamplesExample 1 – pretreatment of saline fresh green biomass1. Biomass is rinsed from sand, mud and other impuritiesThe washing process (unit 1 – figure 1) is designed to remove sand, small seacreatures e.g. snails, and surface salt from the biomass. The biomass is placed ina filter basket (see figure 2 for detailed drawing of the washing unit), placed in awashing drum. Tap water or demineralized water is spayed on the biomass as step one. Once the excess dirt is rinsed off the biomass is submerged in a bath oftap water. The drum reciprocator is turned on and the biomass is washed for agiven time to ensure all contaminants are removed. The washing water is drained of by opening the bottom valves.2. Screw pressA simple screw press (unit 2 – figure 1) is suitable for efficiently fractionatingsaline fresh green biomass, such as halophytes to juice and fibre fractions on a demonstration scale. The system fractionates the biomass, and the juice is separated through a coarse filter, allowing the presence of small suspendedparticles (juice dry matter approx. 4-15%). The juice is collected in a tank via ahose. Screw pressing also gives a preliminary mechanical distortion to thelignocellulosic fibres, and shredding reduces the size further. Screw pressing alsoallows for a good separation of solid and liquid fractions, leading up to relativelyhigh fibre dry matter content. Besides screw pressing, other juicing methods can be applied, such as a centrifugal juicer with rotating blades.3. Drying (optional)The drying step can optionally be performed after the washing step. This wouldapply if the biomass washing, juicing, and / or shredding is introduced at the site ofthe biomass production. This could be implemented to reduce the volume of the biomass and save on shipping cost involved in transporting the biomass from the cultivation site to the extraction plant. In that case the biomass would be washed, 82457PC01 19 sundried, oven-dried or subjected to other industrial drying processes such as fluid bed, tray, or rotary drying before shredding and shipping to the extraction facility.Example 2 - pretreatment of saline lignified or partly lignified biomassThe first step is washing of the saline biomasses to remove the salt and non-organic debris biomass as described in example 1.The full procedure for the pretreatment of the biomass prior to the extraction cascade is (unit numbers refers to figure 1):1. Biomass is rinsed with water in unit 1.a. The biomass is placed in the perforated basket.b. Water is added to unit 1 over the perforated basket.c. The biomass is shortly stirred in unit 1 to remove non-organic solid debris(sand, mud, etc.).d. The biomass is transferred to unit 3.2. The wet biomass is shredded in unit 3.3. The shredded biomass can be added back to unit 1.a. The biomass is placed in the perforated basket.b. Water is added.c. Water can be circulated using a pump connected to unit 3 (as shown infigure 1) over the biomass for several minutes to remove highly water soluble compounds and salt, and the salty water is then drained. The process is repeated multiple times until the salt concentration has been reduced to an acceptable level. Water is drained.d. The biomass is pressed by a hydraulic press (unit 5).e. The basket containing the pressed biomass is removed from unit 3.Example 3 – multi-step extractionExtraction is performed using the same technology as by conventional Soxhletapparatus and SSCE, using either Soxhlet extraction or SSCE extraction as theprimary extraction method and the other extraction method as the secondary.During SSCE, performed in unit 6 (see Figure 1), heating is applied, and theextraction is carried out under stirring and steam pressure. The temperatures(120 - 140°C) and residence times (15 - 240 min) are selected based on the 82457PC01 20properties of the biomass. SSCE with water is a non-toxic, easily scalable way toextract polyphenols from the biomass matrix and open up the lignocellulosestructure. In relation to saline lignified or partly lignified biomasses this method is applied as the primary extraction method.In relation to saline fresh green biomasses, such as green leaves, agro-residues,fresh halophytes this method is used as a secondary extraction method as due toprevious mechanical distortion, material removal in extraction, and naturally lowlignin content of e.g. green halophyte biomass, the conditions applied in SSCE canalso be used not only to remove the extractive compounds, but also as a low-severity hydrothermal treatment to open the lignocellulosic structure. Temperature and residence time are selected based on the properties of each biomass, and optimization of pretreatment conditions for each type of biomass is required to prevent the production of toxic furans.In the Soxhlet extraction (unit 8), the biomass can be placed in a perforatedbasket which allows the extraction solvent, water, to pass through but has enough biomass particle retention. This extraction step removes most of the total waterextractive material when used for the treatment of saline fresh green biomassunder normal extraction conditions. For the treatment of saline lignified or partly lignified biomass, Soxhlet can be used as either a secondary or primary extraction method.The extracts are passed through a nanofiltration unit (unit 10), separating theretentate and permeate. The permeate is then heated to boiling point temperatureand siphoned into the Soxhlet extractor (unit 8) or SSCE (unit 6) to fill theextraction chamber until the drainage level is reached, and the extract is drainedinto another vessel (unit 7), for solvent recovery through nanofiltration. Theobtained extract is drained, and the fibres can be pressed to remove the extract from the fibres. 82457PC01 21 The system allows flexible use of obtained plant extract, which can be used as is or combined with the concentration and purification processes. The extracts from partly lignified halophyte fibres are rich in bioactive compounds and can be used in various applications.The extraction system consists of the following sequences (see Figure 1 fordetails):1. Extraction of fibres in unit 6.a. The biomass is placed in the tank.b. Water is added to yield approximately 5% dry matter loading.c. In the first process step, the biomass can be recirculated using a particlepump in a loop to unit 7 and subjected to ultrasound in a pipe-chamber.The settings for using ultrasound are determined by the type of biomass used.d. Heating is applied under stirring and steam pressure. SSCE is carried out at120 - 140°C for 15 - 240 min (should be optimized for each biomass).e. Cooling.f. Extracted fibres and extract are transferred into the perforated basket inunit 1 – figure 2 through a flexible hose connection.2. Efficient separation of extract and fibres can be done in unit 1 applying thehydraulic press.a. The extract filtered through the perforated basket is transferred to unit 9and retained.b. The biomass in the perforated basket is pressed by a hydraulic press torelease all extract.c. The perforated basket, containing the pressed biomass, is moved into unit8 to set-up for a Soxhlet percolation.3. Nanomembrane filtration using unit 10.a. The extract stored in unit 8 can be pH adjusted.b. The extract in unit 8 is filtered through a cartridge filter and pumped to unit9 for storage.c. From unit 9, the extract is pumped using a feeding pump and high-pressure pump to a sequential membrane filter (pressure up to 35 bars – 82457PC01 22 concentrates molecules down to 150 g / mol by sequential membrane filtrations).d. During start-up, both retentate and permeate are led back to unit 9.e. Cooling on Tank unit 9.f. After start-up, the permeate is sent to unit 6 and the retentate back totank unit 9.g. The permeate can be used for extraction again in unit 8 – e.g. togetherwith once extracted biomass or new fresh fibres, or heated by unit 6, and transferred to unit 8 to perform a Soxhlet percolation.h. CIP (cleaning in place) of membrane and nanofiltration unit.This system is designed to efficiently extract bioactive compounds, such aspolyphenols and alkaloids, from most circular (agricultural residue) and aquatic(algae) biomasses, but for the time being, with most documentation on salinehalophyte biomasses, such as Salicornia spp., Sarcocornia spp., Tripolium spp.,and Crithmum spp.This system is designed to avoid the limitations of traditional single-stepextraction systems. When SSCE is used alone, mass transfer limitation can hinderfurther extraction of the desired compounds. This is avoided by introducingmembrane filtration, where the extract can be concentrated, and the permeatecan be re-used as a solvent in the second extraction cycle. Not only will thisovercome the mass transfer limitations, but it can also have a catalytic effect inthe second extraction cycle due to the permeation of small organic molecules. This cascade step can be repeated several times, and the permeate from themembrane filtration can be recycled back into unit 6 for the complete extraction ofvaluable compounds. In evaporation extraction systems, such as traditional Soxhlet extraction, extraction may be ineffective due to low contact between biomass and solvent and be very energy consuming and require an extended residence time to carry out the needed multiple evaporation cycles. By combining the systems as suggestedhere, the opening of the biomass structure can be achieved in the first steps inunit 6 and efficient mass transfer and washing of the extractives from the fibres isachieved in unit 8 or unit 1 with the permeate from the membrane filtration 82457PC01 23 without introducing fresh water into the system (and thereby diluting the produced extracts). An optional pressing using unit 5 or centrifugation step (in scaled up systems) can be carried out between the cascade steps ensuring very efficient removal of extracted compounds from the biomass and complete removal of macroscopic particles.The described cascade extractor has the following advances in extractiontechnology:• Cascade / multi-step extraction with centrifugation and membrane filtrationfor optimal removal of extractive compounds from the fibres and concentration of extractives.• SSCE combined with recirculating membrane filtration and Soxhletpercolation.• Possibility of cascade steps using permeate from the membrane filtration toovercome mass transfer limitations and possible auto-catalytic effect.• Very flexible processing – and cascades can be optimized for variousagricultural and aquatic biomasses and target compounds.• Possibility to utilize and valorize the fibre fraction of saline fresh greenbiomass from screw-pressed biomass often used directly to low-value high- volume products (e.g. bioenergy).Example 4 - Cascade extraction processThis example describes the proof-of-concept trials that were performed on themulti-step cascade extraction process for extracting saline, fresh green, or lignified biomasses as it was developed and scaled up as part of the “Horizon2020Innovation action project” called Aqacombine.The processThe cascade extraction process consists of the following units: Shredder (unit 3),Wash (unit 1), Soaking (unit 4), Press (unit 5), subcritical reactor (unit 6) withloop to ultrasound cell (unit 7), percolation reactor (unit 8), and liquid storagetank (unit 9) with nanofiltration unit (unit 10) for concentration of compounds andrecycling of water in the process. 82457PC01 24 After the shredding the biomass is placed into caskets designed to carry the biomass through the process and to fit with the press, which enables increased separation of the extraction liquid from the residual fibres after the individual process steps. The capacity of the demonstration scale plant (at the time of proof-of-concept) is 100-liter solvent with a maximum biomass packing capacity of 5 kg of biomass per batch. The biomass Fully lignified Salicornia ramosissima was harvested in Murosa, Portugal (RIASEARCH) and shipped to AAU. The biomass was shredded using the 2- EM01 / Biomass Cutting Mill CM2500 from NordicEngineering to a particle size of 2– 5 mm and then washed three times in tap water using the washing process andthen drained for excess water using the press. The biomass was dried in a forcedconvection heating cabinet in 48h at 60 °C prior to being placed in the extractionunits. Reference and cascade extraction trials To show the effect of the cascade process several extractions were run on thedemonstration pilot. Table 1 shows biomass loading, total volume, processtemperature, and residence time for the different units. Table 2 gives an overviewand explanation of the trials that were run to document the effect of the cascade process.Table 1. Biomass loading and process parameters for the different extraction unitoperations. Extraction BiomassTemperature Residence time loading (°C) (hour) (g / l) Soxhlet (4 runs) 13 100 0.5 (per cycle)Subcritical 17 130 0.5Ultrasound (Two20 1separate containers) 82457PC01 25Table 2. Explanation for the different trails, which were run as part of thereference and cascade extraction trials. Extraction method ID SWE1 Subcritical Water Extraction (SWE) 1 (for reference and replicabilityof the plant) SWE2 Subcritical Water Extraction (SWE) 2 (for reference and replicabilityof the plant) SSoxhlet (S) (for reference)USWE Combined Ultrasound (U) and Subcritical Water Extraction (SWE)US Combined ultrasound (U) and SoxhletSWE2+S Combined Subcritical Water Extraction (first) and Soxhlet (second)S+SWE Combined Soxhlet (first) and Subcritical Water Extraction (second)USWE+S Ultrasound (first) Subcritical Water extraction (second) and Soxhlet(third) US+SWE Ultrasound (first), Soxhlet (second), and Subcritical Water extraction(third) Soxhlet cycle optimization trials Besides the refence and cascade extractions, trials were also performed to optimize the amount of Soxhlet cycles to perform in the cascade. One to four Soxhlet cycles were run for Soxhlet alone and for Soxhlet as part of a cascade. An overview of the trials carried out in this study is shown in Table 3.Table 3. Explanation for the different trials carried out in order to optimize thenumber of Soxhlet cycles in the cascade. Extraction method ID S1-S4 Four Soxhlet cycles run on raw biomassSWE+S1- Four Soxhlet cycles run on the fiber fraction after Subcritical Water S4 Extraction US1-S4 Four Soxhlet cycles run on Ultrasound pretreated fibres 82457PC01 26 USWE+S1- Four Soxhlet cycles run on the fibres after extraction with both S4 Ultrasound and SWE Pre-soaking cascade step As part of the cascade approach pre-soaking in in-situ produced pre-treatment liquid was investigated. Phenolic compounds, organic acids, and minerals produced during the extraction of saline biomasses such as Salicornia ramosissimahave the ability to act as natural, safe, and non-flammable co-solvents. Hence theeffect of pre-soaking in different Salicornia ramosissima extracts prior to Soxhletextraction was tested. Table 4 gives an overview of the different pre-soaking liquids tested.Table 4. Overview of the trials carried out in order to investigate the effect ofpre-soaking as part of the extraction cascade. Extraction method ID Extract1 Salicornia ramosissima extracted at 210°C for 10 minutesExtract2 Salicornia ramosissima extracted at 130°C for 1 hourExtract1D Drained liquid after soaking raw Salicornia ramosissima biomass inEXT1 for ½ hour at 20°C Extract2D Drained liquid after soaking raw Salicornia ramosissima biomass inEXT1 for ½ hour at 20°C WaterD Drained liquid after soaking raw Salicornia ramosissima biomass inWater for ½ hour at 20°C Extract1+SWE Subcritical water extraction of Salicornia ramosissima biomasssoaked in EXT1 for ½ hour at 20°C Extract2+SWE Subcritical water extraction of Salicornia ramosissima biomasssoaked in EXT1 for ½ hour at 20°C Water+SWE Subcritical water extraction of Salicornia ramosissima biomasssoaked in EXT1 for ½ hour at 20°C SWE Subcritical water extraction with no soaking (reference)Temperature optimization for the Subcritical Water Extraction step Even though the Subcritical Water Extraction step had previously been optimized in lab scale (1-5 liter) a set of trials were performed to examine the optimal 82457PC01 27 temperature for the SWE in this new demonstration scale plant. Temperatures of 100, 110, 120, 130, 140, and 150°C were tested with a residence time of one hour. Analytical methods Total extractives and Dry matter determination To find the conversion factor between biomass and dry matter, dry matter was determined at the washed and shredded biomass in crucibles in a heating cabinetin 24 h at 105 °C according to Sluiter A. et al.To find total extractives, dry matter was determined at extracts in the same manner as above.Antioxidant capacity - DPPHThe method developed by Brand-Williams et al. was used which was adapted to 96-well microplates by Moreno et al. with modifications.Gallic acid (0.01 mg / ml) in methanol (MeOH) was used as a reference. DPPHsolution (0.5 mg / ml) was prepared with DPPH and MeOH. Serial dilution of thereference solution was performed in Eppendorf tubes and transferred in each 96-well plate microwell plate to construct a calibration curve (R2>0.99). Samples were transferred in a dilution series, and total antioxidant capacity was found using an intern calculation sheet and expressed in gallic acid equivalents of g pr.kg dry matter (DM) Biomass.Total phenolic and flavonoid content The following methods are used to measure the approximated concentrations oftotal phenolic compounds (TPC), total flavonoids (TFC) (cf. Velioglu Y. S. et al.and Pirbalouti A. G. et al.).Total phenolic content Gallic acid in Milli Q water was used as a reference. 20 % [w / v] Sodium carbonate solution was prepared with Anhydrous Sodium carbonate and Milli Q water. 82457PC01 28 Both samples and standard solutions were prepared as follows: 1.5 mL of Milli Q water, 0.1 mL of sample, 400 μL of Folin-Ciocalteu phenol reagent, 600 μL Sodium Carbonate 20 % [w / v] solution and 2.4 mL Milli Q water. All solutions were vortexed briefly and left for two hours at room temperature in total darkness. The prepared samples were transferred to micro cuvettes and then measured spectrophotometrically at 760 nm. Total phenolic content was expressed in mg of gallic acid equivalents (GAE). Afterwards converted to the unit of: Total flavonoid content Quercetin (2 mg / ml) in Milli Q water was used as a reference. 2 % [w / v] Aluminum chloride solution was prepared with Aluminum chloride and Milli Q water. Serial dilutions of the reference solution were performed in a 96-well plate microwell plate to construct a calibration curve (R2>0.99).Afterward, samples were transferred into 6 replicates with two color correctionwells, and total flavonoid content was calculated and expressed as follows in the concentration of flavonoids in Quercetin equivalents: ^^^@420 ^^^^^=^^^^^ ^^ ^^^^^^^^^^^ ^^^^^Afterwards converted to the unit of: 82457PC01 29Results - Cascade extractionsFigure 3 shows the results of the reference extractions and the cascade extractiontrials. The extractions have been compared concerning the total extractivesfraction obtained (a), total polyphenolic content (b), total flavonoid content (c),and antioxidant capacity in the produced extracts (d).Two subcritical water extractions were performed in order to investigate the replicability of this process, as it is a high-temperature pressurized process and one of the more complex steps in the cascade. As it can be seen from Figure 5, slightly more total amount of extract was produced in SWE1 than SWE2, however the content of total phenolics and antioxidant capacity was very similar in the two experiments. In general, good replicability was found in these two trials. In the future more runs will be performed. For the reference trials SWE and Soxhlet performed similar, but with a higheramount of total phenolics and flavonoids being extracted in SWE, as the inventorshave also documented before in lab trials. However, the antioxidant capacity of the two types of extracts were very similar. The effect of ultrasound as a pre-treatment to the reference extraction (withSalicornia ramosissima) were minimal, as the inventors have also seen before inlab scale trials. In all trials presented in Figure 3 ultrasound was performed as apretreatment on the raw biomass as the first step in the cascade. However, when looking at applying ultrasound as part of the cascade there seem to be an effect on the flavonoid content. In Figure 3, it can be seen that both the SWE+S and S+SWE cascade yields a higher total content of flavonoids when ultrasound is applied as a pretreatment. Especially, the cascade where ultrasound is applied prior to SWA followed by Soxhlet. In this trial the flavonoid content achieved were almost five times higher than in SWE alone. In the future, it should also be investigated if ultrasound can have an effect on recalcitrant fiber residues later in the cascade. Also, detailed analysis of individual compounds might reveal specific compounds that will react to ultrasounds. 82457PC01 30 Furthermore, the ability to apply ultrasound could be an important cascade step when looking at other biomasses in the future. Looking at the cascades that have been run in these proof-of-concept trials the positive effect of the cascade extractions is clear. In all combinations of ultrasound, SWE, and Soxhlet significantly higher amount of total extractives is achieved, with higher total phenolics, flavonoids, and antioxidant capacity as a results. The best cascade combination (of the ones investigated in this initial study) is the subcritical water extraction followed by a Soxhlet cycle, where the amount of total phenolics is doubled compared to the traditional Soxhlet extraction method, and the flavonoid content in the extract is increased five times (compared to both Soxhlet and SWE alone) when ultrasound is also applied. In-situ concentration of extract and recycling of water in the processThe last two columns in the antioxidant figure in Figure 3d shows the permeateand retentate after the nanofiltration concentration step carried out on the SWE2extract in the cascade. It is clear from Figure 3d that no antioxidant capacity islost to the permeate, but is maintained in the retentate, showing that the nanofiltration is very efficient in retaining the polyphenolic compounds, and can be applied as a successful method to concentrate the produced extracts and re-cycle water in the process, as illustrated in figure 1. Comparison to traditional, pilot scale, solvent based extraction carried out by commercial partner For comparison with a more traditional solvent based process, the inventors madea comparison with pilot scale trials run by the inventors AQUACOMBINE partner,Celabor. They have run pilot extractions using Salicornia ramosissima. Theprotocol for the Celabor pilot run was as follows: After drying and crushing oflignified Salicornia ramosissima biomass, 500 g were poured into the 6 L reactorof the subcritical water pilot plant at CEL facilities. A 30 min-cycle water bath at room temperature was first done to remove salt from the biomass. The washwater was discarded. Two 30 min-cycles were then performed with 140 °Csubcritical water. Both extracts were pooled, and after cooling, 200 g of theresidual material was placed in a Teflon covered 5 L reactor, and 2 L 630 mMNaOH was added, and the hydrolysis was done at 80 °C for 1 hour. After 82457PC01 31acidification to pH 2.5 with 6 M HCl, the supernatant was pooled with thesubcritical water extract. Table 5 shows the results of the solvent based subcritical extraction performed at Celabor.From these results it can be seen that Celabor achieved a total phenolics contentof 78 g / kg dry extract in their three-step solvent-based process. With the extraction yields also reported in Table 5, this corresponds to approximately 24 g total phenolics per kg raw biomass in comparison to the 120 g / kg achieved in the SWE+S solvent free cascade process, as seen in Figure 3. Parameters 1st cycle2ndcycle 3rdcycle 140 °C 140 °C 140 °C Mass yield (% dry extract vs raw18.3 % 10.2 % 3.1 %material) Total phenolic content (mg GAE / g42.9 58.0 78.0dry extract) Protocatechuic acid* <QL 59.6 44.8Coumaric acid* 64.8 N.D. N.D.Caffeic acid* <QL <QL 30.9Ferulic acid* 238.5 323.9 376.2Quercetin* <QL <QL 48.1Neochlorogenic acid* 240.6 255.5 175.7Cryptochlorogenic acid* 201.2 183.3 126.7Chlorogenic acid* 241.4 232.1 163.1Hyperoside* 68.1 87.0 60.0Isoquercitrin* 638.3 746.7 533.8Table 5. Results of pilot scale solvent based (NaOH+HCL) subcritical extraction carried out at Celabor in the Aqaucombine project. Analysis of multistep Soxhlet cycle Based on the results that SWE followed by Soxhlet is a very promising extraction cascade for the extraction of polyphenols and flavonoids from Salicorniaramosissima, it was investigated how many Soxhlet cycles would be needed toachieve the optimal result. Figure 4 shows the detailed results of the increase inextractives (a), total phenolics (b), flavonoids (c), and antioxidant capacity (d)with up to four Soxhlet cycles on it own (S) or used in cascade with SubcriticalWater Extraction (SWE), Ultrasound (U), and Ultrasound flowed by Subcritical Water Extraction (USWE). 82457PC01 32From Figure 4 it is clear that increased extraction is achieved by multiple Soxhletruns as part of a cascade extraction after subcritical extraction in terms of totalamount of extractives. In terms of flavonoids and phenolics, it seems that theSoxhlet cycle can be limited to a single or two runs, which is a promising results as this will save energy in the process. Antioxidant capacity seems to follow the same trend as the total extractives and is increased by multiple Soxhlet steps. Pre-soaking trials The effect of pre-soaking the biomass prior to extraction was investigated as a cascade step for the multiple step cascade extraction process. Two different pre- soaking liquids were investigated (EXT1 and EXT2) and the pre-soaking wascarried out for half an hour at room temperature. Figure 5 shows the results oftotal phenolics (a) and flavonoid content (b) of the drained liquids after the pre-soaking (cold extraction) and the liquids (extracts) after subcritical waterextraction. As a reference, Subcritical Water Extraction is added to the charts.From Figure 5 it is clear that, especially soaking in EXT1 is very efficient inincreasing the extraction yield in the subcritical water extraction. By soaking in EXT1 the amount of total phenolics extracted is doubled and the obtained contentof flavonoids in the extracts is increased with approx. four times compared tosubcritical water extraction of the raw Salicornia ramosissima fibres. Even, justthe cold extraction for half an hour with EXT1 as extraction medium gives the same flavonoid yield as subcritical water extraction alone. The pre-soaking liquid EXT1 is produced by pre-treating the extractive free fibres(after Soxhlet or Subcritical water extraction) of Salicornia ramosissima in anautoclave reactor at 210°C for 10 minutes. This process produces a liquid rich inorganic acids, furanic compounds and phenolic acids, and monomeric sugars(Figure 6). The inventors assume that the organic compounds extracted incombination with minerals in Salicornia ramosissima form an ionic liquid orNatural Deep Eutectic Solvents (NADES), which are natural functional liquid media which can dissolve natural or synthetic chemicals of low water solubility or increase the water solubility in e.g., extraction processes. 82457PC01 33Under certain stressed cultivation conditions Salicornia sp. contains high amountsof proline and choline (Ozturk, M. et al.) which are a well know NADES (Wu, S. etal.), especially in combination with biomass sugars (Putu, N. et al.). Furthermore, carboxylic acids have been reported as NADES and the inventors know that EXT1 contains high amounts of 2-furoic acid which is a carboxylic acidconsisting of a furan ring and a carboxylic acid side-group. The inventors believethat the content of 2-furic acid in EXT1 contributes significantly to its function as a natural solvent. This finding is of outmost importance to the cascade, as more research into the pre-soaking effects, can allow for more efficient extraction at lower temperatures, and hence energy saving in the cascade process. Also, more research is needed to optimize the pre-soaking time and to examine if the soaking liquid can be used multiple times (as it is produced at high reaction temperature). Temperature optimization For the subcritical water extraction (without soaking), a temperature study was performed to investigate the influence between total phenolics extraction flavonoid content. From Figure 7, it can be seen that the content of phenolic compounds (a) is increasing with increasing temperatures whereas the flavonoidcontent (b) has an optimum around 140°C. This can be due to the extraction ofnon-flavonoid phenolics at temperatures of 150°C and above, probably stemming from the degradation of the biomass lignin at these temperatures. More research is needed to analyze the extraction kinetics around this critical temperature of 140-150°C and to further optimize the process parameters for each step in the cascade, especially also including the pre-soaking step.Example 4 – main conclusionsIn this section the main conclusions from the proof-of-concept trials on the novel multi-step cascade extraction process is described. 82457PC01 34 Six different cascades were tested and compared to traditional Soxhlet and subcritical water extraction (SWE). In all combinations of ultrasound, SWE, andSoxhlet significantly higher amounts of total extractives were achieved, withhigher total phenolics, flavonoids, and antioxidant capacity as result. The best cascade combination (of the ones investigated in this initial study) was the subcritical water extraction followed by a Soxhlet cycle, where the amount of total phenolics was doubled compared to the traditional Soxhlet extraction method, and the flavonoid content in the extract was increased five times (compared to bothSoxhlet and SWE alone) when ultrasound was also applied. Adding ultrasound tothe cascade especially showed a positive effect on the flavonoid content. Increased extraction was achieved by multiple Soxhlet runs (4) as part of a cascade extraction after SWE in terms of total amount of extractives. In terms offlavonoids and phenolics it seems that the Soxhlet cycle can be limited to a singleor two runs, which is a promising result as this will save energy in the process. Antioxidant capacity seems to follow the same trend as the total extractives and is increased by multiple Soxhlet steps. More research is needed on the optimizationof the Soxhlet cycles for Salicornia ramosissima and other biomasses.A comparison of our best extract was made to a Salicornia ramosissima extractproduced by traditional solvent based extraction in pilot scale trials run by ourAQUACOMBINE partner, Celabor. The Celabor process yielded a total phenolicscontent of approximately 24 g total phenolics per kg raw biomass in comparison to the 120 g / kg achieved in the SWE+S solvent free cascade process. Hence, the multistep cascade extraction yielded five times higher phenolic content compared to traditional solvent extraction. Pre-soaking in “in-situ” produced extracts was investigated as a step in the cascade. EXT1 is an extract produced from extractives-free Salicornia ramosissima fibres pretreated at 201°C for 10 minutes. Pre-soaking in EXT1 showed to be very efficient in increasing the extraction yield in the subcritical water extraction (as part of the cascade). By soaking in EXT1 the amount of total phenolics extracted was doubled and the obtained content of flavonoids in the extracts was increased with approx. four times compared to subcritical water extraction of the rawSalicornia ramosissima fibres. 82457PC01 35 This finding is of outmost importance to the cascade, as more research into the pre-soaking effects, can allow for more efficient extraction at lower temperatures, and hence energy saving in the cascade process. Finally, it was shown that the nanofiltration cascade step was very efficient in retaining the polyphenolic compounds and could be applied as a successful method to concentrate the produced extracts and re-cycle water in the process, which is a cost saving and water saving feature of the novel multiple step cascade extraction process.As a main conclusion the inventors can state that based on these proof-of-concepttrials the amount of extract and content of bioactive polyphenolic compound inextracts of Salicornia ramosissima can be increased by up to five times using thisnovel multi-step cascade extraction process – even when comparing to moreexpensive solvent-based systems. Furthermore, process innovations to increase yield and save energy includes:• Pre-soaking step with “in-situ” produced NADES increases yield and savesenergy and process cost.• Biomass transfer using caskets which can be pressed between processsteps for increased yield and retrieval of products.• Nanofiltration unit as part of the Soxhlet cycle which allows forconcentration of products and water re-cycling in the process.Example 5 - Integrated biorefinery set-upThe extraction system described above has been designed to fit into a fully integrated circular aquaculture and biorefinery system, such as theAQUACOMBINE system. The AQUACOMBINE project demonstrated combinedaquaculture and halophyte farming using the principles of circular economy, where waste and residues are utilized within the system to create both internal value and new products. Excess nutrients from the fish production was used as fertilizer for halophyte plants and filtered through a microbial water treatment system to enable recirculation of the water back into the aquaculture tanks 82457PC01 36 (Recirculating aquaculture system). This aquaponics system is very flexible and can be adapted to various locations, including rural and remote areas. The valorization of halophyte biomass can range from simple "cheap" conversion -yielding medium-value products - to more sophisticated conversion - yielding veryhigh-value products. The AQUACOMBINE project demonstrated the combined,synergistic production of fish, food, botanical extracts, feed protein, and biogas.Example 6 – Production and application of natural, biomass derivedsolvent in the cascade extraction process This example relates to the surprisingly beneficial effects of pre-soaking of shredded and washed biomass prior to (or as part of the) cascade extraction process in a natural, biomass derived solvent. Production of the natural biomass derived solvent (NS)The Salicornia based natural solvent is produced by physio-chemical pretreatmentof Salicornia / Sarcocornia / Arthrocnemum or any other species in theSalicornioideae subfamily of the Amaranthaceae family. The shredded and washed biomass is subjected to a high severity aqueous pre-treatment at elevated temperatures of 190-220^C for 2-60 minutes. The produced slurry is filtrated, and the fibrous residue is removed. The produced liquid can be used as a natural biomass derived solvent / catalyst (NS) in the cascade extraction process. The solvent can be recycled and re-used several times. Pre-soaking process The raw biomass to be extracted upon is pre-soaked in the produced NS for 15 min to several hours at temperatures varying from room temperature to 80^C.Figures 8a and 8b show the comparison of total flavonoid and total phenolicsextracted from raw biomass of Salicornia ramosissima at 40^C (NS1), 60^C (NS2),and 80^C (NS3). The pre-soaking of the raw biomass prior to extraction isincreasing the flavonoid yield with up to four times and the phenolics content upto three times.Decreased recalcitrance of residual fibres 82457PC01 37An additional advantage of the pre-soaking is the increased enzymaticconvertibility of the residual fibres (left after the phytochemicals extraction) asshown in Figure 9. This means that the residual fibres can easily be hydrolysed toyield sugars to be used in fermentation processes. This is an advantage compared to current state of the art processes where fibres need to be pre-treated (physio- chemical pretreatment) prior to enzymatic hydrolysis in order to overcome biomass fibres natural recalcitrance (e.g. cellulose crystallinity).Example 7 – Bactericidal activity of the extracts produced according tothe methods of the inventionThis example relates to the bactericidal activity of the extracts produced accordingto the methods of the present invention. Bactericidal activity displays the extractability to kill a bacterium after a determined period of time. In the presentexample, the bactericidal activity was quantified after 2.5 h incubation, utilizing the reduction of a tetrazolium dye, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, to detect the number of surviving bacteria after incubation with the extract.Details of the abbreviations of extracts referred to in Examples 7-11 are as follows(see also Tables 2-3 for further details).EXT1 (SWE+S) 1.7 kg biomass was rinsed with water in unit 1, and placed in the perforated baskets. Water was added to unit 1 over the perforated baskets. The biomass was shortly stirred in unit 1 to remove non-organic solid debris (sand, mud, etc.). The biomass was transferred to unit 3. The wet biomass was shredded in unit 3. The shredded biomass was placed in perforated baskets in unit 6, and 100 L water was added, and extracted at 130 °C for 30 min. After extraction, the extractor and extract were cooled to 50 °C . The produced extract was transferred to unit 9. The baskets containing the wet biomass were transferred to unit 8. The liquid in unit 9 was subject to cross-flow filtration in unit 10, with the permeate being transferred to unit 6 and the retentate, transferred to unit 9. The watertransferred to unit 6 was heated to 100 °C, and then percolated over the biomass.The produced percolation extract was pumped to unit 9, and the cycle was 82457PC01 38 repeated. The percolation extractions were done in three cycles. The biomass in unit 8 was moved to unit 5 and pressed by a hydraulic press. The concentrated extract in unit 9 was collected. The pooled extract was frozen in liquid nitrogenand freeze-dried to obtain a powder. The powder was shipped to partners for fishtrials. EXT2 (USWE+S) 1.7 kg biomass was rinsed with water in unit 1, and placed in the perforated baskets. Water was added to unit 1 over the perforated baskets. The biomass was shortly stirred in unit 1 to remove non-organic solid debris (sand, mud, etc.). The biomass was transferred to unit 3. The wet biomass was shredded in unit 3. The shredded biomass was placed in perforated baskets in unit 7 and 20 L water was added, and the biomass was subjected to an ultrasound pretreatment at 20 °C for 60 min. The shredded biomass and the 20 L extract were transferred to unit 6 and volume adjusted to 100 L, and extracted at 130 °C for 30 min. After extraction, the extractor and extract were cooled to 50 °C . The produced extract was transferred to unit 9. The baskets containing the wet biomass were transferred to unit 8. The liquid in unit 9 was subject to cross-flow filtration in unit 10, with the permeate being transferred to unit 6 and the retentate, transferred to unit 9. The water transferred to unit 6 was heated to 100 °C, and then percolated over the biomass. The produced percolation extract was pumped to unit 9, and the cycle was repeated. The percolation extractions were done in three cycles. The biomass in unit 8 was moved to unit 5 and pressed by a hydraulic press. The released liquid was added to the pool of extract. The concentrated extract in unit 9 was collected. The pooled extract was frozen in liquid nitrogen and freeze-dried to obtain a powder. The powder was shipped to partners for fish trials. EXT3 (S+SWE) 1.7 kg biomass was rinsed with water in unit 1, and placed in the perforated baskets. Water was added to unit 1 over the perforated baskets. The biomass was shortly stirred in unit 1 to remove non-organic solid debris (sand, mud, etc.). The biomass was transferred to unit 3. The wet biomass was shredded in unit 3. The shredded biomass was placed in perforated baskets in unit 8. 100 L water was added to unit 6 and heated to 100 °C, and then percolated over the biomass in unit 8. The produced extract was transferred to unit 9. The liquid in unit 9 was 82457PC01 39 subject to cross-flow filtration in unit 10, with the permeate being transferred to unit 6 and the retentate, transferred to unit 9. The water transferred to unit 6 was heated to 100 °C, and the cycle was repeated. The percolation extractions were done in three cycles. The biomass in unit 8 was transferred to unit 6. The water from filtration recycling was added, and the volume was adjusted to 100 L. The biomass was extracted at 130 °C for 30 min. After extraction, the extractor and extract was cooled to 50 °C . The produced extract was transferred to unit 9. The biomass was transferred to unit 5 and pressed by a hydraulic press. The released liquid was added to the pool of extract. The concentrated extract in unit 9 was collected. The pooled extract was frozen in liquid nitrogen and freeze-dried to obtain a powder. The powder was shipped to partners for fish trials. XAD3 0.075 kg biomass was rinsed with water in unit 1. The biomass was shortly stirred in unit 1 to remove non-organic solid debris (sand, mud, etc.). The biomass was transferred to unit 3. The wet biomass was shredded in unit 3. The shredded biomass was placed in a subcritical reactor and 1.5 L water was added. The biomass was extracted at 130 °C for 30 min. The extract was removed from thereactor and filtered using a 100 µm and 50 µm sintered glass filter. The solids-free extract was then subjected to a dynamic column adsorbtion using 300 ml (1 bed volume) Amberlite XAD-4 resin, hence 5 BV being adsorbed. The resin was cleaned with 2 BV of water, and desorbed with 2 BV EtOH. The desorbate was collected and subjected to rotary evaporation. The remaining fraction containing the desorbed dry matter was frozen in liquid nitrogen and freeze-dried to obtain a powder. The fraction was dissolved in 40 % EtOH and shipped to partners for fish trials. XAD 0.075 kg biomass was rinsed with water in unit 1. The biomass was shortly stirred in unit 1 to remove non-organic solid debris (sand, mud, etc.). The biomass was transferred to unit 3. The wet biomass was shredded in unit 3. The shredded biomass was placed in a subcritical reactor and 1.5 L water was added. The biomass was extracted at 130 °C for 30 min. The extract was removed from thereactor and filtered using a 100 µm and 50 µm sintered glass filter. The solids-free extract was then subjected to a dynamic column adsorbtion using 300 ml (1 82457PC01 40 bed volume) Amberlite XAD-4 resin, hence 5 BV being adsorbed. The resin was cleaned with 2 BV of water, and desorbed with 2 BV EtOH. The desorbate was collected and subjected to rotary evaporation. The remaining fraction containing the desorbed dry matter was frozen in liquid nitrogen and freeze-dried to obtain a powder. The powder was shipped to partners for fish trials. FRAC 1 ml XAD3, as sent to the partners, was loaded to a prepared 50 ml Sephadex LH- 20 flash chromatography column. Chromatography was performed using gravitational pull with 100 % methanol to obtain 55 subfractions. The subfractions were analyzed using a full-spectra (UV-vis) scan for their absorption at 320, 350 and 370 nm (specific wavelengths for polyphenols). The subfractions were then, based on their absorption characteristics, pooled in 5 fractions. Fraction 3, determined to have the largest amount of DM and largest absorption in the specified wavelengths, was subjected to evaporation under nitrogen, and sent to partners for fish trials. Now, NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT (yellow colour) to its insoluble formazan, which has a purple colour. Dead bacteria do not react with MTT. Bacterially reduced formazan is solubilized by adding DMSO and formazan production is quantitated by measuring absorption at 560 nm. Absorption of formazan is directly related to viable bacteria cell number and is used to determine the number of bacteria not killed by the product to be tested. Figures 10 (a)-(g) show the bactericidal activity of extracts at differentconcentration, against Tenacibaculum maritimum (A), Photobacterium damselaesubsp. piscicida (B), Vibrio anguillarum (C), Vibrio parahaemolyticus, (D),Edwardsiella tarda (E), Yersinia ruckeri (F) and Aeromonas hydrophila (G). 82457PC01 41 Extra Conce Photobac Tenaciba Aeromo ct ntratio culum terium Vibrio Vibrio Edward Yersinia nas n maritmiu damselae anguill parahaem siella ruckeri hydroph (mg / m subsp. arum olyticus tarda ila ml) piscicida % live bacteria (compared to control)EXT1 0.5 81.14 81.05 93.45 72.53 108.35 67.85 90.400.25 77.52 92.06 78.24 111.02 144.34 114.15 96.290.125 85.35 78.87 90.33 115.73 124.42 114.59 92.17EXT2 0.5 82.90 96.28 90.64 78.57 98.08 73.49 85.290.25 80.44 73.03 90.68 114.47 107.20 111.17 88.790.125 80.33 78.40 82.67 104.98 112.65 94.23 90.52EXT3 0.5 83.54 85.92 94.88 80.62 98.43 75.42 89.130.25 80.79 83.60 94.61 102.79 108.18 105.59 91.940.125 80.74 81.44 91.26 95.62 92.51 97.45 91.90XAD3 0.5 74.37 85.83 93.68 103.72 113.76 97.02 81.600.25 78.98 85.58 89.33 124.42 116.72 105.09 87.180.125 80.33 85.29 99.61 120.57 111.55 96.90 81.63XAD 0.5 74.55 89.29 95.45 103.25 103.25 96.59 98.990.25 76.88 81.55 81.59 116.12 128.50 100.93 94.980.125 79.92 81.55 70.45 115.73 99.25 98.88 90.80FREM 0.5 75.31 82.20 96.69 105.11 99.36 98.32 87.970.25 79.22 84.53 89.60 112.81 110.10 97.14 87.590.125 76.01 81.26 80.20 111.68 111.90 98.14 82.98FRAC0.5 81.03 85.05 96.65 105.31 101.10 98.51 92.7330.25 80.85 84.53 99.96 109.36 109.92 94.69 85.270.125 77.93 81.32 74.92 110.29 110.39 86.59 81.24Table 6(a). Bactericidal activity (% live bacteria compared to control) of the extracts at different concentration against bacteria after a 2.5h incubation period.

[0002] 82457PC01 42 Extra Photobac Concent Tenaciba Vibrio Aeromo ct ration culum terium Vibrio paraha Edward Yersinia nas (mg / ml maritmiu damselae anguilla emolyti siella ruckeri hydroph ) m subsp. rum cus tarda ila piscicida % dead bacteria (compared to control)EXT1 0.5 18.86 18.95 6.55 27.47 0.00 32.15 9.600.25 22.48 7.94 21.76 0.00 0.00 0.00 3.710.125 14.65 21.13 9.67 0.00 0.00 0.00 7.83EXT2 0.5 17.10 3.72 9.36 21.43 1.92 26.51 14.710.25 19.56 26.97 9.32 0.00 0.00 0.00 11.210.125 19.67 21.60 17.33 0.00 0.00 5.77 9.48EXT3 0.5 16.46 14.08 5.12 19.38 1.57 24.58 10.870.25 19.21 16.40 5.39 0.00 0.00 0.00 8.060.125 19.26 18.56 8.74 4.38 7.49 2.55 8.10XAD3 0.5 25.63 14.17 6.32 0.00 0.00 2.98 18.400.25 21.02 14.42 10.67 0.00 0.00 0.00 12.820.125 19.67 14.71 0.39 0.00 0.00 3.10 18.37XAD 0.5 25.45 10.71 4.55 0.00 0.00 3.41 1.010.25 23.12 18.45 18.41 0.00 0.00 0.00 5.020.125 20.08 18.45 29.55 0.00 0.75 1.12 9.20FREM 0.5 24.69 17.80 3.31 0.00 0.64 1.68 12.030.25 20.78 15.47 10.40 0.00 0.00 2.86 12.410.125 23.99 18.74 19.80 0.00 0.00 1.86 17.02FRAC0.5 18.97 14.95 3.35 0.00 0.00 1.49 7.2730.25 19.15 15.47 0.04 0.00 0.00 5.31 14.730.125 22.07 18.68 25.08 0.00 0.00 13.41 18.76Table 6(b). Bactericidal activity (% dead bacteria compared to control) of theextracts at different concentration against bacteria after a 2.5h incubation period.

[0003] 82457PC01 43 Bactericidal activity against: (all extracts at all concentrations) Tenacibaculum maritimum Photobacterium damselae subsp. piscisidaVibrio anguillarum Aeromonas hydrophila Bactericidal activity against: Vibrio parahaemolyticus (EXT1, 2 and 3 at 0.5 mg / ml)Yersinia ruckeri (EXT1, 2 and 3 at 0.5 mg / ml)Table 7. Summary of the bactericidal activity of the extracts after 2.5h.Example 8 - Bacteriostatic activity of the extracts produced according tothe methods of the invention The bacteriostatic activity of the extracts produced according to the methods ofthe invention was tested against bacterial pathogens. The extracts were able toinhibit pathogens growth without necessarily inducing bacterial death. The present protocol enables the evaluation of the extracts ability to inhibit bacterial growth during a 24h period. The extracts at the desired concentration were incubated during 24h at 25°C and continuous shaking (100 rpm). The absorbance was read every hour for the first 4 hours and every 4 hours after that. Bacteriostatic activity was then calculated as the difference between continuous growth of bacteria (positive control, bacteria alone, 100%) and the test wells (bacteria and extracts).Figures 11 (a)-(c) show the bacteriostatic activity of the extracts againstTenacabaculum maritimum at extract concentrations of 0.5 mg / ml extract (Figure11 (a)), 0.25 mg / ml extract (Figure 11 (b)) and 0.125 mg / ml extract (Figure 11(c)). Figures 12 (a)-(c) show the bacteriostatic activity of the extracts againstVibrio anguillarum at extract concentrations of 0.5 mg / ml extract (Figure 12 (a)),0.25 mg / ml extract (Figure 12 (b)) and 0.125 mg / ml extract (Figure 12 (c)).Figures 13 (a)-(c) show the bacteriostatic activity of the extracts against Vibrioparahaemolyticus at extract concentrations of 0.5 mg / ml extract (figure 13 (a)), 0.25 mg / ml extract (Figure 13 (b)) and 0.125 mg / ml extract (Figure 13 (c)).Figures 14 (a)-(c) show the bacteriostatic activity of the extracts against Vibrioharveyi at extract concentrations of 0.5 mg / ml extract (Figure 14 (a)), 0.25mg / ml extract (Figure 14 (b)) and 0.125 mg / ml extract (Figure 14 (c)). Figures15 (a)-(c) show the bacteriostatic activity of the extracts against Edwardsiellatarda at extract concentrations of 0.5 mg / ml extract (Figure 15 (a)), 0.25 mg / ml 82457PC01 44extract (Figure 15 (b)) and 0.125 mg / ml extract (Figure 15 (c)). Figures 16 (a)-(c) show the bacteriostatic activity of the extracts against Photobacteriumdamselae subsp. piscicida at extract concentrations of 0.5 mg / ml extract (Figure16 (a)), 0.25 mg / ml extract (Figure 16 (b)) and 0.125 mg / ml extract (Figure 16(c)). Figures 17 (a)-(c) show the bacteriostatic activity of the extracts againstYersinia ruckeri at extract concentrations of 0.5 mg / ml extract (Figure 17 (a)),0.25 mg / ml extract (Figure 17 (b)) and 0.125 mg / ml extract (Figure 17 (c)). Vibrio Vibrio T. maritimum anguillarum parahaemolytic Vibrio harveyi us 0.5 0.25 0.125 0.5 0.25 0.125 0.5 0.25 0.125 0.5 0.25 0.125 mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / ml ml ml ml ml ml ml ml ml ml ml ml Contr 100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0 ol 0 0 0 0 0 0 0 0 0 0 0 0 EXT1 99.3899.0699.8191.4992.1491.91115.0109.6114.2109.3106.7113.2 7 8 2 7 2 2 EXT2 98.2996.0499.9193.0994.6193.85113.1100.8109.788.9384.8592.628 8 4 EXT3 99.3198.5099.8194.2792.9593.12109.3103.4108.190.0384.2493.886 7 0XAD3 97.0497.6998.4475.4891.6092.95107.2102.2104.688.1087.2789.375 4 7 XAD 97.10100.899.7892.5379.0887.14125.4106.8104.088.3281.9891.744 7 1 4FREM 98.4798.6699.6395.8295.0993.4899.97100.1105.491.1384.9088.433 2 FREC 98.2598.3598.8594.8098.7695.1199.43101.6105.093.6685.0792.181 1 Photobacterium Edwardsiella damselae Aeromonas Yersinia ruckeri tarda hydrophila subsp. piscicida 0.5 0.25 0.125 0.5 0.25 0.125 0.5 0.25 0.125 0.5 0.25 0.125 mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / ml ml ml ml ml ml ml ml ml ml ml ml Contr 100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0100.0 ol 0 0 0 0 0 0 0 0 0 0 0 0 EXT1 108.2110.5102.773.4473.2381.88102.9102.298.59101.4103.196.03 4 5 5 8 9 6 2 EXT2 115.5115.7108.293.1490.9099.76103.5105.0101.6106.1105.5104.3 6 2 4 4 5 8 4 3 3 EXT3 110.4111.3100.595.6991.9694.23104.6103.9101.8133.3106.5104.5 2 1 3 6 5 0 5 4 3XAD3 111.3111.3105.992.4092.8594.80100.1102.998.8099.6596.7394.729 1 4 0 0 XAD 111.7114.1106.793.1891.3193.26116.7111.1104.1102.0100.0100.1 2 0 5 6 0 2 6 0 5 82457PC01 45 FREM 106.7112.7101.691.9290.7894.1593.66101.495.9596.2392.6192.001 7 2 1 FREC 108.5108.2103.792.1691.7196.3994.5694.9094.80103.294.5796.083 8 6 7 Table 8. Bacteriostatic activity of the extracts at 24h against bacterial pathogens. Photobacterium Vibrio Aeromonas Yersinia anguillarum Vibrio harveyi damselae hydrophila ruckeri subsp. piscicida 0.50.250.1250.50.250.125 0.5 0.250.125 0.5 0.250.1250.5 0.250.125 mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Inhibitory Cumulative Inhibitory conditions growth conditions > 10% inhibition Table 9: Percentage of inhibition of bacterial growth at 24h. Example 9 – In vitro determination of effects of the extracts producedaccording to the methods of the invention on cytotoxicity and immunomodulatory responses in head kidney primary adherent cells from Salmo salar Head kidney primary adherent cells enriched in macrophages (Figure 18), as an in vitro model to evaluate the effects of various extracts derived from halophytes. The primary goal is to assess the cytotoxicity and immunomodulatory potential of these extracts and select optimal concentrations for further functional studies. Pre-Screening Analysis 82457PC01 46 The pre-screening phase involved testing five different halophyte extracts: EXT1, EXT2, EXT3, XAD, and FERM. Two forms of the FERM extract were included, one with salt removed (FERM) and the other without salt removed (FERM2). The extracts were dissolved in water, except for XAD, which was dissolved in 80% DMSO. The tested concentrations ranged from 4 mg / mL to 0.25 mg / mL. Corresponding controls, including DMSO and NaCl, were added to account for any effects of the solvents and salts used in the experiment. The MTT assay was employed to assess the impact of the treatments on cell function compared to control cells that were not exposed to the extracts. Results from this phase indicated that neither the DMSO nor the salt controls had asignificant effect on the metabolic activity of the cells (Figures 19 (a)-(b)). Thisfinding suggested that the cellular effects observed were likely due to the bioactive compounds present in the halophyte extracts rather than the solvents or salts themselves. As shown in Figure 20, for EXT1, EXT2, and EXT3, higher concentrations (4, 2, and 1 mg / mL) significantly reduced cell metabolic activity, resulting in a decrease of 15-90%. In contrast, the lower concentrations (0.5 and 0.25 mg / mL) had a minimal impact on cell metabolic activity, making them more suitable for further analysis. For the XAD extract, cell metabolic activity decreased across all tested concentrations, with reductions ranging from 20% to 50%. Nevertheless, the lower concentrations (0.5 and 0.25 mg / mL) were the least disruptive to cell metabolism and were therefore selected for further study. Regarding FERM and FERM2, no significant differences in cell metabolic activity were observed between the two extracts, regardless of the concentration tested. Both were well tolerated at all concentrations. FERM2 was considered more suitable for further analysis due to its cost-effectiveness, as it does not require the additional step of salt removal.For the next stage, concentrations of 2 mg / mL and 0.25 mg / mL were selected forfurther testing. Screening Analysis Following the pre-screening phase, two concentrations of each extract were selected for more detailed screening. The cells were exposed to the extracts for 24 hours, and subsequent analyses measured oxidative stress through reactive oxygen species (ROS) production using CellROX staining and phagocytosis using fluorescent bioparticles, both assessed via Imaging Flow Cytometry (IFC). The XAD extract at both 0.5 and 0.25 mg / mL significantly reduced oxidative stress compared to control cells (CTRL). In contrast, the other extracts (EXT1, EXT2, 82457PC01 47 EXT3, FERM, and FERM2) did not show a notable impact on oxidative stress levels (Figure 21). Additionally, XAD at 0.5 mg / mL enhanced phagocytic activity compared to the CTRL. While the other extracts did not significantly alter phagocytic activity, there was a tendency toward increased phagocytosis across all groups and compared to CTRL (Figure 22). Based on these findings, one concentration per extract was selected for further experiments: 0.25 mg / mL for all extracts except for XAD, for which 0.5 mg / mL was chosen. All results are based on three independent repetitions, each performed in triplicate. The pre-screening and screening phases have successfully identified concentrations for each extract that minimize cytotoxicity while modulating immune functions.Example 10 - RTS11 cell line. Rainbow trout spleen macrophages.(CIIMAR) Pre-Screening Analysis The pre-screening phase involved testing five different halophyte extracts: EXT1, EXT2, EXT3, XAD, XAD 3, FERM and FRAC3. The tested concentrations ranged from 2 mg / mL to 0.125 mg / mL that were tested at least in six independent days(cells resulted from six different passages of the same stock cells). The MTT assaywas employed as previously described in order to assess the impact of the treatments on cell function compared to control cells that were not exposed to theextracts (Figure 23).Example 11 - Extracts effect on gut Electrogenic amino acid transport(EAAT) – UCAEAAT assays were performed in a set of preliminary assays to establish putative region-dependent effects in electrogenic amino acid transport using the anterior, mid, and posterior intestinal regions. The principle of the test is that the presenceof amino acids stimulates the epithelium, thus generating a change in the currentdue to the cotransport of amino acids with ions. Discrete portions of the intestineof European sea bass juveniles (50-60 g) were isolated and mounted in a tissueholder, and positioned between two half-Ussing chambers containing 2 mL of physiological saline prepared according to the previous characterization of sea bass plasma ion composition. In this type of assay, the apical side of the preparation is stimulated with an amino acid mixture. 82457PC01 48 Basal bioelectrical characterization of discrete regions of the intestine of seabassjuveniles showed that all intestinal regions are in net anion absorption (Figure24). Moreover, the sequential apical addition of 4, 8, and 16 mM essential amino acid pools evoked changes in short circuit current (Delta Isc, µA.cm-2). Regardless of the essential amino acid pool concentration tested, the Delta Isc observed was always significantly higher in the mid-intestine, and for this reason was chosen to perform the following experiments with those extracts provided. Seven extracts were then tested at the concentration of 1 mg / ml in the mid- intestine as previously described. In general, the three XAD compounds (Figure 25 (a)-(c)) tested behave in a similar way in relation to the stimulation of the amino acid absorption current in the mid-intestine. FERM compound (eluted in MqH2O) (Figure 25 (d)) produces a clear inhibition on intestinal amino acid absorption, whereas a clear, linear and significant stimulation was observed after the addition of both XADs (Figure 25 (e)) and FRAC3 compounds (both eluted in 30-40% EtOH). Finally, both EXT22 and EXT3 compounds (Figure 25 (g)-(h)) produces a clear, linear and significant increase of intestinal amino acid absorption, being more pronounced or evident after stimulation with compound EXT3.

[0004] 82457PC01 49 ReferencesAltemimi A. et al., "Phytochemicals: Extraction, Isolation, and Identification ofBioactive Compounds from Plant Extracts," Plants, vol. 6, no. 4, Sep. 2017, doi: 10.3390 / plants6040042.Brand-Williams W. et al., “Use of a Free Radical Method to Evaluate AntioxidantActivity,” LWT - Food Sci. Technol., vol. 28, no. 1, pp. 25–30, 1995, doi:10.1016 / S0023-6438(95)80008-5. Carreira Casais et al., Int. J. Environ. Res. Public Health (2021), 18, 9153“Benefits and Drawbacks of Ultrasound-Assisted Extraction for the Recovery ofBioactive Compounds from Marine Algae”. Correia et al., Antioxidants (2022), 11, 2449 “Salicornia ramosissima: A NewGreen Cosmetic Ingredient with Promising Skin Effects”.Hulkko L. S. S. et al., Nature Scientific Reports (2022) 12:20507 “Cultivation andcharacterization of Salicornia europaea, Tripolium pannonicum and Crithmummaritimum biomass for green biorefinery applications”.Hulkko, L. S. S., Turcios, A. E., Kohnen, S., Chaturvedi, T., Papenbrock, J. &Thomsen, M. H. (2022 (i)). Cultivation and characterisation of Salicorniaeuropaea, Tripolium pannonicum and Crithmum maritimum biomass for green biorefinery applications. Scientific Reports. 12(1). 2045-2322. Hulkko, L. S. S. Chaturvedi, T., Custódio, L. & Thomsen, M. H. (2023). Harnessing the Value of Tripolium pannonicum and Crithmum maritimum Halophyte Biomass through Integrated Green Biorefinery. Marine Drugs 21(7), 1660-3397Ko et al., Natureresearch Scientific Reports (2020) “Subcritical water extraction ofbioactive compounds from Orostachys japonicus A. Berger (Crassulaceae)”. 82457PC01 50Moreno S. et al., “Antioxidant and antimicrobial activities of rosemary extractslinked to their polyphenol composition,” Free Radic. Res., vol. 40, no. 2, pp. 223– 231, 2006, doi: 10.1080 / 10715760500473834. Ozturk, M. et al. (2018) A Little-Known and a Little-Consumed Natural Resource: Salicornia, p. 83-108. In: Ozturk, M. et al. Global Perspectives on Underutilized Crops. Springer.Pirbalouti A. G. et al., “Antioxidant activity, total phenolic and flavonoid contentsof some medicinal and aromatic plants used as herbal teas and condiments in Iran,” J. Med. Food, vol. 17, no. 10, pp. 1151–1157, 2014, doi: 10.1089 / jmf.2013.0057. Pujol D. et al., “The chemical composition of exhausted coffee waste,” Ind. Crops Prod., vol. 50, pp. 423–429, Oct. 2013, doi: 10.1016 / j.indcrop.2013.07.056. Putu, N. et al. (2021) Natural Deep Eutectic Solvents (NADES): Phytochemical Extraction Performance Enhancer for Pharmaceutical and Nutraceutical Product Development. Plants (Basel), 10(10): 2091.Radovanovic K., et al., Processes (2023), 11, 86 “Subcritical Water Extraction asan Effective Technique for the Isolation of Polyphenols of Achillea Species”.Selvamuthukumaran M. and Shi J., “Recent advances in extraction of antioxidants from plant by-products processing industries,” Food Qual. Saf., vol. 1, no. 1, pp. 61–81, Mar. 2017, doi: 10.1093 / fqsafe / fyx004. Sluiter A. et al., “Determination of total solids in biomass and total dissolved solids in liquid process samples,” National Renewable Energy Laboratory (NREL), no. March. pp. 3–5, 2008.Topuz et al., Journal of Aquatic Food Product Technology (2015) “Optimisation ofantioxidant activity and polyphenol extraction conditions from red seaweed(Laurencia obtuse)”. 82457PC01 51Velioglu Y. S. et al., “Antioxidant Activity and Total Phenolics in Selected Fruits,Vegetables, and Grain Products,” J. Agric. Food Chem., vol. 46, no. 10, pp. 4113– 4117, 1998, doi: 10.1021 / jf9801973.Wu, S. et al., (2021) Isolation of choline monooxygenase (CMO) gene fromSalicornia europaea and enhanced salt tolerance of transgenic tobacco with CMO genes. Indian J Biochem Biophys, 47(5):298-305.

Claims

82457PC01 52 Claims 1. A multi-step method of extracting bio-active compounds from non-foodfractions of saline biomass having a dry matter content (DMC) of 70-100%,wherein the method comprises the following steps:(Step 1a) pretreatment of the biomass by subjecting said biomass toshredding, sieving, and cold-water wash (10-20°C), followed by(Step 2a) one or more extraction steps, followed by(Step 3a) separation of fibres and extracts of the slurry resulting from step2a and 2b, followed by(Step 4a) concentration of the extracts by membrane filtration resultingfrom step 3a, followed by (Step 5a) Soxhlet percolation extraction of fibres resulting from step 4ausing permeate water as solvent or by using the extract obtained from 3a forextraction with possible auto-catalytic reactions.

2. The method according to claim 1, wherein the bio-active compounds areselected from the group consisting of polyphenols, alkaloids, terpenes,furanocoumarins, chitin, collagen, and other bioactive structural carbohydratesand proteins / peptides.

3. The method according to claim 2, wherein the polyphenols are selected from the group consisting of phenolic acids, hydroxycinnamic acids, flavonoids, flavanols, tannins, and stilbenes.

4. The method according to any of claims 1-3, wherein the saline biomass isselected from the group consisting of halophytes, seaweed and algae.

5. The method according to claim 4, wherein the halophytes are selected from thegroup consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp.

6. The method according to any of claims 1-5, wherein the shredded biomass ofstep (1a) has particle sizes between 0.1 and 5 cm.82457PC01 53 7. The method according to any of claims 1-6, wherein the one or more extractionsteps in step (2a) is static sub-critical water extraction (SWE) of the slurryresulting from step (1a) carried out in a static sub-critical extractor (SSCE) at100-140°C and 1-5 bar (absolute) pressure for 0.25-4 hours.

8. The method according to any of claims 1-7, wherein the separation step (3a) is filtration by membrane filtration with a filter mesh size of 150-100,000 Da.

9. The method according to any of claims 1-8, wherein the concentration step(4a) is carried out by in situ membrane filtration at 1-30 bar pressure for 0-50hours.

10. The method according to any of claims 1-9, wherein the Soxhlet percolationextraction step (5a) is running for 1-12 cycles.

11. The method according to any of claims 1-10, wherein the one or moreextraction steps (Step 2a) is accompanied by ultrasound treatment for 0-5 hours,12. A multi-step method of extracting bio-active compounds from non-food fractions of screw-pressed, hydraulic-pressed, high-speed blended, centrifugaljuiced, saline biomass having a dry matter content (DMC) of 20-50%, wherein themethod comprises the following steps: (Step 1b) drying of the biomass, followed by(Step 2b) Soxhlet percolation extraction of the dried biomass from Step 1busing water as the sole extraction solvent, followed by(Step 3b) static sub-critical extraction (SSCE) as a low-severityhydrothermal treatment of the extract produced in Step 2b, followed by(Step 4b) filtration of the extract obtained in Step 3b to obtain a finalextract rich in bio-active compounds.

13. The method according to claim 12, wherein the bio-active compounds areselected from the group consisting of polyphenols, alkaloids, terpenes,furanocoumarins, chitin, collagen, and other bioactive structural carbohydratesand proteins / peptides.82457PC01 54 14. The method according to claim 13, wherein the polyphenols are selected from the group consisting of phenolic acids, hydroxycinnamic acids, flavonoids, flavanols, tannins, and stilbenes.

15. The method according to any of claims 10-14, wherein the saline biomass isselected from the group consisting of halophytes, seaweed and algae.

16. The method according to claim 15, wherein the halophytes are selected fromthe group consisting of Salicornia spp., Sarcocornia spp., Tripolium spp., andCrithmum spp.

17. The method according to any of claims 10-16, wherein the drying of thebiomass in Step 1b is performed outside or at 40-105°C, for 1-48 hours resultingin DMC of the dried biomass of 8-16%, such as 12%.

18. The method according to any of claims 10-17, wherein extraction solvent(water) is added in Step 2a in a biomass:solvent ratio of 1:6.

19. The method according to any of claims 10-18, wherein the Soxhlet extraction in Step 2b is performed in a perforated basket with a mesh size of 0.5-5 mm.

20. The method according to any of claims 10-19, wherein the hydrothermaltreatment in Step 3b is carried out at 100-140°C and 1-5 bar (absolute) pressurefor 0.25-4 hours.

21. The method according to any of claims 10-20, wherein the filtration in Step 4bis carried out with a filter mesh of 0.1-1 mm.

23. Non-therapeutic use of an extract produced by a method according to any ofclaims 1-11 as a bactericidal or bacteriostatic agent.

24. An extract produced by a method according to any of claims 1-11 for use as amedicament.82457PC01 5525. An extract produced by a method according to any of claims 1-11 for use intreating bacterial infections in aquatic animals, such as farmed fish.

26. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Tenacibaculum maritimum (such as Tenacibaculosis,Yellow Mouth Disease, Salt Water Columnaris) in farmed fish (e.g. Salmon (Salmosalar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass, Dicentrarchus labrax)).

27. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Photobacterium damselae (such as Photobacteriosis(Pasteurellosis)) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

28. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Vibrio anguillarum (such as vibriosis) in farmed fish(e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

29. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Vibrio parahaemolyticus (such as AcuteHepatopancreatic Necrosis Disease (AHPND), Translucent Post Larvae Disease(TPD)) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchusmykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

30. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Edwardsiella tarda (such as Edwardsiellosis,Emphysematous Putrefactive Disease) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

31. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Yersinia ruckeri (such as Enteric Redmouth Disease(ERM), also known as Yersiniosis) in farmed fish (e.g. Salmon (Salmo salar),82457PC01 56 Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

32. An extract produced by a method according to any of claims 1-11 for use intreating diseases caused by Aeromonas hydrophila (such as HemorrhagicSepticemia, Ulcerative Disease, Motile Aeromonas Septicemia (MAS), Tail / Fin Rot) in farmed fish (e.g. Salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Sea bass (such as European sea bass (Dicentrarchus labrax))).

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