Process for the preparation of a hydrolyzed extract from algae

Abstract

The present invention relates to a process to obtain a hydrolysate from an algal biomass. The invention further relates to the hydrolysate from an algal biomass obtained by such process, and its use as plant bio-stimulant.

Description

[0001] PROCESS FOR THE PREPARATION OF A HYDROLYZED EXTRACT FROM ALGAE

[0002] Technical field of the invention

[0003] The present invention relates to a process to obtain a hydrolysate from a biomass of nonanimal origin, in particular from an algal biomass, the hydrolysate of algal origin obtained by such process, and its use as plant bio-stimulant.

[0004] State of the art

[0005] In the agricultural field, the interest in bio-stimulants is steadily growing. A plant biostimulant, such as defined by the Community Regulation 2019 / 1009, means any product stimulating the nutritional processes of plants independently from its nutrient content, with the purpose to improve one or more of the following characteristics of the plant or rhizosphere: a) efficiency of the nutrient use; b) tolerance to abiotic stress; c) qualitative characteristics; d) availability of the nutrients confined in the soil or rhizosphere.

[0006] In agriculture, the use of bio-stimulants reduces the use of fertilizers and plant protection products, optimizing their use, with a lower overall impact on the environment.

[0007] Hydrolysates are an important group of bio-stimulants, in particular those of plant origin, mainly containing a mixture of oligosaccharides, polyphenols, peptides and amino acids, phytohormones, etc. showing a notable potential in improving the productivity, nutritional quality, and tolerance to the factors of abiotic stress of plants in different agricultural crops. The use of plant-origin hydrolysates as bio-stimulants is an efficient complement to the use of mineral fertilizers, whose excessive use has caused over time negative effects connected to the degradation of soils and pollution of waters (both superficial and underground) and whose productive processes have an excessive environmental impact both in terms of emissions of greenhouse gases and the consumption of not-renewable resources.

[0008] In particular, algal extracts occupy an important position in the market of bio-stimulants and represent the fastest-growing product category. In literature, there are numerous and different studies relating to the possible action mechanism of such extracts on model plants and crops and, among their effects, improvement of the plant vigor, root development, increase of the chlorophyll synthesis, positive effects on blooming and fructification, senescence delay, tolerance to abiotic stress are counted. Bio-stimulants originating from macro-algae are a very heterogenous category since their own composition depends on: family and species of the used algae (for example, brown algae, green algae, red algae), place and harvest period thereof (for example, geography, season, temperature, and water salinity), the used process to obtain them (for example, mechanical processes, extraction by acidic or basic agents, enzymatic hydrolysis, or fermentation).

[0009] Macro-algae, especially brown algae, are known as a rich source of high-biodiversity carbohydrates able to act in numerous biologic applications. For this reason, among others, they are the most used algae for the preparation of bio-stimulants.

[0010] Among these carbohydrates, the alginate plays an important role in the food, cosmetic, and pharmaceutical industry, for use as a gelling agent, thickener, stabilizer, and emulsifier. Further, the alginate showed to have an interesting potential even for the agricultural field.

[0011] The alginate is a linear anionic polysaccharide, consisting of the P-D-mannuronic acid (unit M) and a-L-guluronic acid (unit G) monomers, bound through P-(l — >4) or a-( l ^4) covalent bonds.

[0012] The alginate content, as well as its composition (M / G ratio and sequences), is variable in the alga depending on the species, harvest period, geography, alga age, and alga portion.

[0013] In the case of brown algae, it can constitute up to 40% of the biomass and it is the main component of their cell wall.

[0014] In the brown algae, it is present as a calcium, magnesium, potassium, and sodium salt, with calcium alginate as the most represented form. The alginic acid is insoluble in itself, but its alkali metal salts are water soluble. Differently, alkaline earth metal salts jellify instantaneously since junction zones are formed involving the G-G-G-G blocks, whereby, for example, Ca++ions complex with four unities of L-guluronate, in a structure named “egg-box,” which, in the plurality of the polymer, generate a net able to withhold water, i.e. to form a gel.

[0015] In the state of the art, the extraction of alginate from brown algae occurs employing a basic solution (extraction or basic hydrolysis), so to break down the structures and solubilize the alginate.

[0016] Anyway, the depolymerization reaction carried out by a basic route leads only in part to the production of alginic acid oligomers, forming monocarboxylic and dicarboxylic acids as well, in a high or low amount depending on the amount of used base, reaction temperature, and process time. Meanwhile, the base added for the extraction contributes to the composition of the dried extract resulting from the basic hydrolysis; therefore, the so produced extracts have a dry matter characterized by an alga-extracted lower carbon content.

[0017] In the agronomic field, the alginate structure, specifically its polymerization grade, is relevant to the bio-stimulant properties on plants. In this sense, the oligosaccharide fractions are more bioactive with respect to the alginate as a polymer. Among the reported effects are increase of the tolerance to abiotic stresses such as drought, salinity, and heavy metals. For these reasons, the production of extracts for use in agriculture as bio-stimulants provides that the extraction leads to a depolymerization of the alginate, solubilizing it.

[0018] There is still the need to find a process to obtain a preferably improved hydrolysate from an algal biomass, to be used as plant bio-stimulant.

[0019] Brief Description of Figures

[0020] Figure 1 schematically shows a process according to the invention.

[0021] Figure 2 shows a process according to the invention starting from the brown alga of the species Macrocystis pyrifera.

[0022] Figure 3 shows a process according to the invention starting from the brown alga of the species Durvillaea antarctica.

[0023] Summary of the Invention

[0024] Object of the present invention is a process for the preparation of hydrolysates from algae comprising the steps of: i) starting from an algal biomass; ii) optionally rehydrating said algal biomass; iii) subjecting the algal biomass to basic extraction; iv) subjecting to one or more cycles of enzymatic hydrolysis wherein at least one polysaccharide-degrading enzyme is used in each cycle; v) separating by centrifugation and / or filtration to obtain: a. a liquid phase (hydrolyzed extract); and b. a solid phase (sludge); vi) recovering the hydrolyzed extract; and vii) concentrating the hydrolyzed extract.

[0025] Such sludge can be used as such or dried, as a soil improver, or it can be used for the formulation of organo-mineral fertilizers.

[0026] A further object of the present invention are the hydrolysates of algal origin obtainable by the process of the invention.

[0027] A further object of the present invention are the hydrolysates of algal origin according to the invention as plant bio-stimulants.

[0028] Detailed Description of the Invention

[0029] It has been now surprisingly found that a process providing the use of an enzymatic hydrolysis after an extraction step by basic conditioning (basic extraction) allows to obtain biostimulants with a high level of algal substance final concentration, comprising carbon and oligomers of the hydrolyzed alginic acid. Further, surprisingly, the process allows to obtain an extraction yield of the total carbon (Got) from the alga > 70% and with a high degree of hydrolysis. The hydrolyzed extract is characterized by a lower content of metallic salts, thus resulting in a higher purity of components extracted from the alga which are active in the stimulation of the plant development. Such active compounds play their function on adult or developing plants when applied both by foliar route, and by root route. A further administration route is the one to the seed.

[0030] Object of the present invention is a process for the preparation of hydrolysates from algae comprising the steps of: i) starting from an algal biomass; ii) optionally rehydrating said algal biomass; iii) subjecting the algal biomass to basic extraction; iv) subjecting to one or more cycles of enzymatic hydrolysis wherein at least one polysaccharide-degrading enzyme is used in each cycle; v) separating by centrifugation and / or filtration to obtain: a. a liquid phase (hydrolyzed extract); and b. a solid phase (sludge); vi) recovering the hydrolyzed extract; and vii) concentrating the hydrolyzed extract.

[0031] The sludge can be subjected to a further step of squeezing or washing viii) to recover a higher amount of liquid phase, i.e. to increase the extraction yield.

[0032] Such sludge can be used as such or dried, as a soil improver, or it can be used for the formulation of organo-mineral fertilizers.

[0033] The biomass used in the process of the present invention is of algal origin, preferably originating from, comprising, or consisting of, brown algae, more preferably brown algae of species Macrocystis pyrifera or Durvillaea antarctica, and combinations thereof.

[0034] The algal biomass can be in a fresh or dried form.

[0035] The biomass in a dried form is preferably grounded or powdered.

[0036] When the biomass is in a dried form, it is rehydrated in water for a time ranging from 2 hours to 12 hours according to the type of the used alga and its physical state (powder, pieces, etc.).

[0037] The fresh biomass can be crushed before being subjected to the subsequent steps of the process. The crushed biomass has a particle size ranging preferably from 5 to 10 cm.

[0038] The algal biomass, dried or fresh, can be diluted in a dry matter (DM): water ratio from 1 : 15 to 1 :35, preferably equal to 1 :20 weight / weight (w / w). Basic extraction iii)

[0039] The basic extraction process of step iii) comprises the following steps: a) addition of a basifying agent; and b) addition of dilution water.

[0040] The basifying agent of step a) is preferably selected from Na2COa, K2CO3, NaOH, NH3 and KOH, preferably it is KOH.

[0041] Preferably, the concentration of the basifying agent ranges from 0.2% w / w to 2.0% w / w with respect to the total weight of the reaction mixture, more preferably it is 0.5% w / w.

[0042] Further preferably the pH reached in the basic solution used during the basic extraction step is higher than 9, more preferably ranges from 10 to 12, even more preferably it is equal to 10.5.

[0043] The basic extraction preferably occurs at a temperature ranging from 60 to 90 °C, preferably at 80 °C.

[0044] Preferably the basic extraction occurs for a time ranging from 2 to 10 hours, preferably 4 hours.

[0045] During this step the insoluble alginic acid salts, constituting the cell wall of the alga cells, are turned into soluble salts such as sodium alginate or potassium alginate.

[0046] Such basic extraction conditions allow to solubilize and therefore to extract the alginate without causing its depolymerization.

[0047] An increase in viscosity is therefore observed at this stage, indicating the solubilization of alginate.

[0048] Enzymatic hydrolysis iv)

[0049] The enzymatic hydrolysis process of step iv) comprises the following steps: c) optional pre-conditioning at basic pH (for example at pH 8.7 for 2 hours); d) pH adjustment by a basicity corrector and temperature adjustment; e) addition / inoculation of at least one polysaccharide-degrading enzyme; f) optional enzymatic inactivation; g) centrifugation and / or filtration for separation of the liquid phase (broth) from the solid phase (sludge) and subsequent optional squeezing of the sludge; and h) broth concentration.

[0050] The sludge squeezing of step g) preferably occurs by a filter-press.

[0051] The broth obtained in step h) can be stabilized by adding at least one preservative or by dehydration.

[0052] The sludge can be used as such or dried, as a soil improver, or it can be used for the formulation of organo-mineral fertilizers.

[0053] During the enzymatic hydrolysis step, the depolymerization of the alginate occurs and therefore the formation of the oligomers thereof, acting as bio-stimulant molecules.

[0054] The enzymatic hydrolysis occurs employing enzymes endowed with hydrolytic activity on the alginate and for a period of time ranging from 1 to 4 hours, preferably 2 hours.

[0055] Optionally, a pre-conditioning step c) is carried out at a temperature ranging from 45 to 70 °C, preferably equal to 60 °C and at a pH from 6.5 to 9.0, preferably equal to 8.7, more preferably for 2 hours.

[0056] In step d) the pH can be set to a range from 6.5 to 8.0, preferably at a pH equal to 6.7 and preferably at a temperature comprised from 40 to 55 °C, preferably at 45 °C.

[0057] In step e) the at least one polysaccharide-degrading enzyme is inoculated.

[0058] The used polysaccharide-degrading enzymes hydrolyze the polysaccharide components of the alga cell wall (for example cellulose / s, hemicellulose / s, alginates). The category of the polysaccharide-degrading enzymes includes cellulases, hemicellulases, and alginate-degrading enzymes, including alginate lyase and alginate hydrolase.

[0059] The cellulase is an enzyme able to degrade cellulose.

[0060] The hemicellulase belongs to the enzymatic family of glycosidic hydrolases, and the enzymatic reaction leads to the hydrolysis of hemicellulose.

[0061] Depending on the composition of the polysaccharides, one or more enzymes more suitable for the hydrolytic action can be used, such as specific alginate-degrading enzymes such as, preferably, alginate lyase, alginate hydrolase, and mixtures thereof, such as Kelp Hydrolase (produced by Creative Enzymes), more preferably alginate hydrolase, even more preferably a mixture comprising alginate hydrolase, as in Kelp Hydrolase, and optionally alginate lyase. In a further preferred aspect of the invention, the mixture comprises, or consists of, alginate hydrolase and alginate lyase.

[0062] The enzymes alginate hydrolase and alginate lyase, constitute the enzymatic complex appointed to alginate degradation, as described and represented in Figure 9 by Schumann K. and Weide G., Enzymatic degradation of alginate by marine fungi, Hydrobiologia 204 / 205: 589-596, 1990.

[0063] A mixture of enzymes used in the present invention is a mixture comprising alginate hydrolase, as in Kelp Hydrolase, i.e. the commercial name of the product of Creative Enzymes, comprising alginate hydrolase.

[0064] Further, by the use of the aforesaid enzymes, and in particular by the use of alginate hydrolase, for example a mixture comprising alginate hydrolase (as Kelp Hydrolase), a fluid and less viscous extract is obtained, indicative result of the efficacy of the aforesaid enzymes in hydrolyzing the alga polysaccharides.

[0065] Preferably, in step d) the basicity corrector is selected from citric acid, acetic acid, glycolic acid, and phosphoric acid (H3PO4), more preferably is phosphoric acid or citric acid, even more preferably is citric acid.

[0066] Said basicity corrector is preferably added to reach a pH ranging from 6.5 to 8.5, preferably from 6.5 to 7.0, more preferably of 6.7.

[0067] Preferably said basicity corrector is added in a concentration from 0.2% w / w to 2.0% w / w with respect to the total weight of the reaction mixture, more preferably of 0.5% w / w.

[0068] The optional enzymatic inactivation of step f) can occur by thermal inactivation, at a temperature ranging from 60 °C to 90 °C, preferably of 85 °C and for a time ranging from 30 to 10 minutes, preferably of 15 minutes, and / or subjecting the mixture to a pressure from 20 mbar (2000 Pa) to 200 mbar (20000 Pa) for a period of time from 15 to 30 minutes.

[0069] The process of the invention, by the specific combination of basic extraction and enzymatic hydrolysis, allows to maximize the extraction of the organic carbon from the alga with respect to the basic hydrolysis alone or enzymatic hydrolysis alone.

[0070] The chemical-enzymatic process according to the invention leads to the highest extraction of the carbon from the algae, thus optimizing the extraction of the alga organic substance. The yield in alga-extracted carbon (Got endogenous yield %) clearly shows the meliorative result.

[0071] Table 1

[0072] Further, the process allows to reduce the addition of basifying agent during the step of basic extraction of 75%, thus preventing the presence of residues of said agent in the final extract, which will have hence a composition more strictly related to the organic matter originating from the alga.

[0073] For example, the ratio between the alga-extracted carbon and the cation of the used base (K+in the case of KOH) is higher in the extracts obtained according to the invention. Table 2

[0074] Given the efficacy in maximizing the carbon extraction and the lower use of reagents amount, the production of the extract is more economic, i.e. extracting a kilogram of carbon from the alga can cost 45% less than the traditional chemical method.

[0075] Further, the enzymatic hydrolysis subsequent to the basic extraction, has the further advantage that can concentrate the extract up to 45-50% in dry matter without thickenings or j edification.

[0076] The separation of the broth from the sludge occurs by centrifugation and / or filtration.

[0077] The concentration of the hydrolyzed extract can occur by concentration under vacuum or direct osmosis or a combination of the previous technologies.

[0078] Object of the present invention are also the hydrolysates from algae, obtainable, or obtained, by the process of the invention, and their use as plant bio-stimulants.

[0079] Said hydrolysate from algae, preferably obtainable, or obtained, from the process of the invention, is characterized by a ratio between endogenous carbon (i.e. the alga-extracted total carbon) and metallic cation of the used base in the step of basic hydrolysis, preferably potassium, ranging from 0.75 to 1.85, preferably of 0.96 or 1.55.

[0080] Further, since the enzymatic step can be carried out at a sub-acid pH, i.e. pH less than 7.0, more preferably equal to 6.7, such pH value characterizes even the extract. Therefore, the thus obtained extract, at the end of the enzymatic hydrolysis process does not need further pH correction.

[0081] Said hydrolysate from algae obtainable, or obtained, by the process of the invention, can be used as plant bio-stimulant.

[0082] Finally, the sludge obtainable, or obtained, at the end of step g) or h) of the process of the invention, can be used as such or dried, as a soil improver, or it can be used for the formulation of organo-mineral fertilizers.

[0083] The term “bio-stimulant” according to the present invention means a substance that, when applied to plants, is able to improve the use efficiency of nutrients, the tolerance to abiotic stress, and / or the qualitative characteristics of crops. This action occurs independently from the nutrients content of the applied product, and it is due, instead, to the whole of bioactive molecules present therein.

[0084] All the embodiments according to the present invention can be combined with each other.

[0085] The following Examples further illustrate the invention.

[0086] Examples

[0087] Definitions and analytical methods

[0088] DM is the dry matter.

[0089] K+(%) is the potassium cation expressed as w / w percentage on the extract.

[0090] The dry matter is determined by drying in an oven at the temperature of 105 °C until a constant weight is reached.

[0091] Ctot is the total carbon content.

[0092] The method for the determination of the total carbon content is based on the complete combustion of the sample in the presence of oxygen. To obtain a complete oxidation, the sample is carried by a helium flux through a column filled with a catalyst, chromium oxide (CnCh). The thus formed substances, CO2 and H2O, pass through a filter withholding water. CO2 is separated by column chromatography and the analysis of combustion products is carried out by gas chromatography (elemental analysis).

[0093] The carbon in the extracts was reported as dry matter percentage.

[0094] The extraction yield of the carbon from alga endogenous Ctot % was calculated on the carbon of the sample, as follows endogenous Ctot % = (Ce- Ca) x 100 / Cmpwherein Ce(g) is the carbon present in the extract, Ca(g) is the carbon contributed by the organic acidity correctors (for example, citric acid) and Cmpis the carbon (g) present in the starting material.

[0095] Example 1 - Hydrolysate of the invention from Macrocystis pyrifera

[0096] The following steps are carried out: i) 100 g of dried algal biomass of Macrocystis pyrifera having DM of 98.7%, Ctot of 30.4% and K+(% on DM) of 7.4% were provided; ii) rehydrating for two hours said algal biomass; iii) the algal biomass was subjected to basic extraction by: a. addition of 25 g of KOH as basifying agent, having a DM of 100%; and b. addition of 2000 ml of water and a pH higher than 10 is reached. The step of basic extraction occurs at a temperature of 80 °C, for 4 hours. iv) the obtained algal biomass is subjected to a cycle of enzymatic hydrolysis at 45 °C for 2 hours, by: d. pH adjustment by addition of 45.75 g of 65% w / w citric acid as basicity corrector, having DM of 65% and Got of 37.5%, until a pH of 6.7 is reached; e. addition of 0.15 grams of Kelp Hydrolase as polysaccharide-degrading enzyme; g. centrifugation of the suspension and subsequent squeezing of the sludge by a filterpress for separation of:

[0097] 2090 g of liquid phase (broth) having: and 42 g of solid phase (sludge) having DM SS (%) = 30; and h. concentration of the broth (hydrolysate from algae) until a 40% dry matter concentration is reached by evaporation under vacuum.

[0098] Example 2 - Hydrolysate prepared according to the state of the art from

[0099] Macrocystis pyrifera

[0100] The following steps are carried out: i) 1000 g of fresh algal biomass of Macrocystis pyrifera having DM 9.87%, Got of 3.04% and K+(% on DM) of 7.4% were provided; iii) the algal biomass was subjected to a basic extraction by: a. addition of 107 g KOH as basifying agent, having a DM of 100%; and b. addition of 2857 ml of dilution water and 59.5 grams of KC1. A pH higher than 10 is reached.

[0101] The basic extraction step occurs at a temperature of 80 °C, for 6 hours.

[0102] The obtained material was centrifuged, then the sludge squeezed by a filter-press, thus obtaining:

[0103] 3650 grams of liquid phase (broth) having: and 373.5 grams of solid phase (sludge) having DM (%) = 30.0

[0104] The obtained extract is concentrated by evaporation under vacuum up to the reachable maximum, specifically up to 8.0% DM. At higher concentrations jellification and / or precipitation phenomena are observed.

[0105] Comparing the hydrolysate of the state of the art with the one of the invention, according to Example 1, is evident that: the yield in terms of alga-extracted Got (%) (Got endogenous yield (%)), as well as the amount in Got (%) of the extract is higher in the case of the hydrolysate of the invention, while the potassium ion content (K+%) is lower, with respect to the state of the art.

[0106] This characterizes the dry matter of the extract mostly in the alga-extracted organic substance; this is evident from the ndogenous / Ktot ratio which is higher with respect to the hydrolysate of the state of the art. Further, the hydrolyzed extract according to the invention can be concentrated up to higher DM % values, for example 40 % DM.

[0107] Example 3 - Hydrolysate of the invention from Durvillaea antarctica

[0108] The following steps are carried out: i) 100 g of dried algal biomass of Durvillaea antarctica having DM of 87%, Got of 30.4% and K+of 1.48% were provided; ii) rehydrating for two hours said algal biomass; iii) the algal biomass was subjected to basic extraction at a temperature of 80 °C, for 4 hours, by: a. addition of 25 g of KOH as basifying agent, having a DM of 100%; and b. addition of 2000 ml of dilution water and a pH higher than 10 is reached. iv) The algal biomass is subjected to a cycle of enzymatic hydrolysis at 45 °C for 2 hours, by: d. pH adjustment by 42.8 g of 65% w / w citric acid as basicity corrector, having DM of 65% and Got of 37.5%, until a pH of 6.7 is reached; e. addition of 0.15 grams of Kelp Hydrolase as polysaccharide-degrading enzyme; g. centrifugation of the suspension and subsequent squeezing of the sludge by a filterpress for separation of: 2080 grams of liquid phase (broth) having: and 33.8 grams of solid phase (sludge) having DM (%) = 30; and h. concentration of the broth (hydrolysate from algae) until a 40% dry matter concentration is reached by evaporation under vacuum.

[0109] Example 4 - Hydrolysate prepared according to the state of the art from Durvillaea antarctica

[0110] The following steps are carried out: i) 100 g of dried algal biomass of Durvillaea antarctica having DM of 87%, Ctotof 30.4% and K+of 1.48% were provided; ii) said algal biomass was rehydrated in water for two hours; iii) the algal biomass was subjected to basic extraction, by: a. addition of 107 g of KOH as basifying agent, having a DM of 100%; and b. addition of 2957 ml of dilution water and 47.2 grams of KC1. A pH higher than 10 is reached.

[0111] The step of basic extraction occurs at a temperature of 80 °C, for 6 hours.

[0112] The obtained material was centrifuged, then the sludge squeezed by a filter-press, thus obtaining:

[0113] 2936 grams of liquid phase (broth) having: and 275.2 grams of solid phase (sludge) having DM (%) = 30.0%.

[0114] The obtained extract was concentrated by evaporation under vacuum up to the reachable maximum, specifically up to 8.0% DM. At higher concentrations j edification and / or precipitation phenomena are observed.

[0115] Comparing the hydrolysate of the state of the art with the one of the invention, according to Example 3, it is evident that: the yield in terms of alga-extracted Got (%) (Got endogenous yield (%)), as well as the amount in Got (%) of the extract is higher in the case of the hydrolysate of the invention, while the potassium ion content (K+%) is lower, with respect to the state of the art.

[0116] This characterizes the dry matter of the extract mostly in the alga-extracted organic substance; this is evident from the ndogenous / Ktot ratio which is higher with respect to the hydrolysate of the state of the art.

[0117] Further, the hydrolyzed extract according to the invention can be concentrated up to higher DM% values, for example 40% DM.

[0118] Example 5 - Enzymatic hydrolysis of Durvillaea antarctica by alginate hydrolase

[0119] The alginic acid degrading-enzyme, alginate hydrolase, specifically Kelp Hydrolase, was tested in the enzymatic hydrolysis of Durvillaea antarctica, by the steps of: iv) enzymatic hydrolysis at 45 °C for 24 hours at pH 6.7, by: c. pre-conditioning for two hours at pH 8.7 at 60 °C; d. pH adjustment by 167 ml of 50% w / w phosphoric acid as basicity corrector, until a pH of 6.7 is reached; e. addition of 600.3 milligrams of Kelp Hydrolase; and g. separation of the broth from the sludge by centrifugation at 3750 rpm x 10 min for separation of 2515.34 grams of liquid phase or broth having: h. concentration of the broth (hydrolysate from algae) until a 40% dry matter concentration is reached by evaporation under vacuum.

[0120] By the use of the alginate hydrolase enzyme, for example Kelp Hydrolase, it was possible not only to obtain the separation of the liquid phase (broth) from the solid phase (sludge) of the sample by centrifugation, but also to characterize it and demonstrate a high yield in terms of Gotai contained in the hydrolysate, therefore resulting as an enzyme suitable for the enzymatic hydrolysis of the process of the invention, in addition to allowing the production of a concentrated extract with up to 40% DM.

[0121] However, the yields of the process to obtain hydrolysates from algae by enzymatic hydrolysis alone are still lower, with respect to those reported for the process of the invention, comprising both a basic hydrolysis step, and an enzymatic hydrolysis step, according to Example 3.

[0122] Example 6 - Comparison between the enzymatic hydrolysis of Macrocystis pyrifera by alginate lyase and by alginate hydrolase

[0123] The two polysaccharide-degrading enzymes were tested in the enzymatic hydrolysis of Macrocystis pyrifera.

[0124] The first enzyme alginate lyase was tested by the below described steps: iv) the algal biomass is subjected to a cycle of enzymatic hydrolysis at 45 °C for 24 hours at pH 8.0 by: c. pre-conditioning at pH 8.7 at 60 °C for 2 hours; d. pH adjustment by 2 ml of 50% w / w phosphoric acid as basicity corrector, until a pH of 8.0 is reached; e. addition of 355.32 milligrams of alginate lyase; and g. separation of the broth from the sludge by centrifugation at 3750 rpm x 10 min to obtain 2070.73 grams of liquid phase or broth having: h. concentration of the broth (hydrolysate from algae) until a 20% dry matter concentration is reached by evaporation under vacuum.

[0125] The second enzyme, alginate hydrolase, for example Kelp hydrolase, was used in the step of enzymatic hydrolysis of Macrocystis pyrifera by the below described steps: iv) The algal biomass is subjected to a cycle of enzymatic hydrolysis at 45 °C for 24 hours at pH 6.7, by: c. pre-conditioning for 2 hours at 60 °C, at pH 8.7; d. pH adjustment by 6 ml of 50% w / w phosphoric acid as basicity corrector, until a pH of 6.7 is reached; e. addition of 444.15 milligrams of Kelp hydrolase; and g. separation by centrifugation at 3750 rpm x 10 min to obtain 2837.49 grams of liquid phase or broth having: h. concentration of the broth (hydrolysate from algae) until a 40% dry matter concentration is reached by evaporation under vacuum.

[0126] By the use of the enzyme alginate hydrolase, for example Kelp hydrolase, a high yield in terms of alga-extracted Ctotai was observed, therefore resulting as an enzyme suitable for the enzymatic hydrolysis of the process of the invention.

[0127] Furthermore, the use of alginate hydrolase allows to concentrate the extract up to 40% DM.

[0128] However, the yields of the process to obtain hydrolysates from algae by enzymatic hydrolysis alone are still lower, with respect to those reported for the process of the invention, comprising both a basic hydrolysis step, and an enzymatic hydrolysis step, according to Example 1.

Claims

CLAIMS1. A process for the preparation of hydrolysates from algae comprising the steps of: i) starting from an algal biomass; ii) optionally rehydrating said algal biomass; iii) subjecting the algal biomass to basic extraction; iv) subjecting the thus obtained product to one or more cycles of enzymatic hydrolysis wherein at least one polysaccharide-degrading enzyme is used in each cycle; v) separating the thus obtained product by centrifugation and / or filtration to obtain: a. a liquid phase (hydrolyzed extract); and b. a solid phase (sludge); vi) recovering the hydrolyzed extract; and vii) concentrating the hydrolyzed extract; wherein at least one polysaccharide-degrading enzyme is alginate hydrolase.

2. The process according to claim 1, wherein the algal biomass consists of brown algae, preferably brown algae of the species Macrocystis pyrifera or Durvillaea antarctica or combinations thereof.

3. The process according to claim 1 or 2, wherein the polysaccharide-degrading enzyme is a mixture comprising alginate hydrolase and optionally alginate lyase, preferably is a mixture comprising alginate hydrolase and alginate lyase.

4. The process according to any one of claims 1 to 3, wherein the basic extraction of step iii) comprises the following steps: a) addition of a basifying agent; and b) addition of dilution water.

5. The process according to claim 4, wherein the basifying agent is selected from NaCCh and KOH, preferably it is KOH.

6. The process according to claim 4 or 5, wherein the basifying agent has a concentration ranging from 0.2% w / w to 2.0% w / w with respect to the total weight of the reaction mixture, more preferably of 0.5% w / w.

7. The process according to any one of claims 1 to 6, wherein the enzymatic hydrolysis of step iv) comprises the following steps: c) optional pre-conditioning at basic pH; d) pH adjustment by a basicity corrector and temperature adjustment; e) addition / inoculation of at least one polysaccharide-degrading enzyme; f) optional enzymatic inactivation; andg) centrifugation and / or filtration for separation of the liquid phase (broth) from the solid phase (sludge) and subsequent optional squeezing of the sludge; and h) broth concentration.

8. The process according to claim 7, wherein step c) of pre-conditioning occurs at a temperature ranging from 45 to 70 °C, preferably at 60 °C and at a pH from 6.5 to 9.0, preferably at 8.7, more preferably for 2 hours.

9. A hydrolysate of algal origin obtainable by the process according to anyone of claims 1-8.

10. Use of a hydrolysate of algal origin according to claim 9 as plant bio-stimulant.

11. Use of a sludge obtainable at the end of step g) or h) of the process of claim 7, as such or dried, as a soil improver or for the formulation of organo-mineral fertilizers.

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