Protein hydrolysate from residual Escherichia coli cells from the fermentation process.

Abstract

The invention relates to a protein hydrolysate preparation from residual Escherichia coli cells from the fermentation process for the production of chemical intermediates or polyhydroxyalkanoates, a procedure for obtaining a protein hydrolysate preparation.

Description

[0001] PROTEIN HYDROLYSATE OF RESIDUAL ESCHERICHIA COL1 CELLS FROM A FERMENTATION PROCESS

[0002] DESCRIPTION

[0003] The present invention relates to a protein hydrolysate of Escherichia coli cells left over from a fermentation process for the production of chemical intermediates or polyhydroxyalkanoates, the process for obtaining it, the use of this protein hydrolysate as a biostimulant, and the biostimulants comprising it.

[0004] The environmental impact of cultivation systems, the need to reduce production costs and, consequently, to decrease chemical fertiliser applications to the soil, have become important objectives in the agricultural sector. Farmers are therefore faced with the need to optimise the application of fertilisers, which have been an efficient tool for increasing crop yields in recent decades. An alternative to chemical fertilisers is now biostimulants based on protein hydrolysates.

[0005] Protein hydrolysates are obtained by the hydrolysis of proteins and contain a mixture of peptides, oligopeptides and free amino acids. Such hydrolysates can have biostimulant activity on plants, for example by improving the uptake and assimilation of nutrients (e.g. nitrate nitrogen and iron), stimulating endogenous plant defence responses to biotic and abiotic stresses (such as salinity, drought and temperature extremes) and influencing product quality (for example by favouring higher antioxidant content, higher protein level and lower nitrate content).

[0006] The type of action they exert on plants depends on the composition of the protein hydrolysates. Specific amino acids can be associated with a certain effect on plants. Glutamic acid, for example, boosts plants’ resistance to various environmental stresses, intervening in resistance mechanisms in adverse situations and promoting the assimilation of inorganic nitrogen. Proline accelerates recovery time from abiotic stress and reduces its effect by strengthening cell walls (Hayat et al., 2012, Plant Signal Behav. 7(11): 1456-1466) and increases pollen fertility (Mattioli et al., 2018, BMC Plant Biol. 18( 1) :356). Glycine and alanine stimulate plant growth by supporting chlorophyll synthesis, chelating micronutrients, improving pollen fertility or resistance to low temperatures (Mikula K. et al., 2022, Environ Sci Pollut Res). Arginine is a precursor of cytokinins involved in cell growth, axillary bud growth and leaf senescence (Winter et al. 2015. Front Plant Sci. 6:534). Alanine, valine, leucine, isoleucine and methionine improve fruit quality as flavour precursors. Tyrosine enhances aerial growth, CO2 uptake and efficient water use (Alfosea-Simon M. et al., 2020, Sustainability 12:22). The composition of protein hydrolysates strongly depends on the origin of the raw material from which they are derived and the process used to produce them. The most common protein hydrolysate products on the market are obtained from raw materials of animal or plant origin. Raw materials of animal origin are residues from leather processing (e.g. collagen) and from the fish industry, and typically undergo chemical hydrolysis at high temperatures and in a strongly acidic or alkaline environment. However, the hydrolysates obtained in this way present some safety risks, linked to possible biological contamination that can be vectors of potentially dangerous infections for humans, such as Bovine Spongiform Encephalopathy (BSE) in cattle. Furthermore, there is an increasing demand in the Agri-Food supply chain for eco-certified and sustainable sources that are alternatives to animal ones, also because of growth in the world population of vegetarian and vegan consumers, who are very mindful of the entire food production chain.

[0007] On the other hand, protein hydrolysates produced from raw materials of plant origin, for example those derived from leguminous plant seeds, often have higher production costs, both because the protein component in the biomass is lower than in animal sources, and because to obtain them land has to be taken away from agricultural crops for food, making their production process less sustainable.

[0008] A solution is therefore offered by protein hydrolysates of microbial origin, which do not present the same safety risks as hydrolysates of animal origin and have advantages for sustainability, both environmental and economic. Microorganisms have a high growth rate, so large amounts of microbial biomass can be obtained in a short time, with a high protein content, typically 43 to 85% by dry weight. This microbial biomass can undergo hydrolysis with high yields, resulting in protein hydrolysates that can be effectively used as biostimulants for plants.

[0009] The microbial biomass may be a by-product of a fermentation process, for example one for the production of chemical intermediates or polyhydroxy alkanoates. At the end of a fermentation process the residual microbial biomass present in the fermentation broth is generally deactivated or killed, for example by thermal means, separated from the fermentation broth and finally disposed of as a waste by-product in landfill or incinerated. Instead of being a waste by-product, this microbial biomass can advantageously be made use of by subjecting it to hydrolysis and obtaining a protein hydrolysate rich in peptides, oligopeptides and free amino acids.

[0010] One example of a protein hydrolysate derived from microbial biomass can be found in Xu et al. (Bioengineered, 2020, 11:1, 1-10), where they describe obtaining a protein hydrolysate from Escherichia coli cells resulting from a fermentation process for tryptophan synthesis. The microbial biomass obtained from the fermentation process is first pre-treated to increase the permeability of the cell membrane to enzymes, and then subjected to enzyme hydrolysis. The hydrolysate obtained is not however used as a plant biostimulant, but as a source of nitrogen for the fermentation medium to produce the tryptophan itself. Although it is rich in certain amino acids such as arginine, leucine and lysine, this protein hydrolysate does not in fact contain - or contains small amounts of - aspartic acid, serine, cysteine, proline, glutamic acid and glycine, which play an important part in promoting plant tolerance to stress and are therefore important components of bio stimulants.

[0011] Document US 2012 / 129695 describes a biostimulant for plants comprising a protein hydrolysate of residual cells from a fermentation process for the production of amino acids.

[0012] Patent application WO 2021 / 240470 describes a biostimulant comprising a protein component resulting from the hydrolysis of a biomass obtained from a dedicated culture of methanotrophic bacteria.

[0013] Similarly, documents WO 2021 / 055366 and WO 2018 / 144965 describe biostimulants comprising protein hydrolysates produced from a microbial biomass consisting of microorganisms capable of converting gaseous substrates (th, CO2 or syngas) into high-protein biomass.

[0014] It has now surprisingly been found that, subjecting residual Escherichia coli cells from a fermentation process for the production of chemical intermediates or polyhydroxyalkanoates to hydrolysis, yields a protein hydrolysate that is particularly suitable for use as a plant biostimulant. This protein hydrolysate comprises proline and cysteine, and one or more amino acids chosen from aspartic acid, glutamic acid, serine, alanine, glycine, threonine, valine and leucine.

[0015] According to a first aspect, the present invention therefore relates to a protein hydrolysate of residual Escherichia coli cells from a fermentation process for the production of chemical intermediates or polyhydroxy alkanoates.

[0016] These chemical intermediates are preferably selected from the group that comprises, or consists of, dialcohols, monoalcohols, hydroxy acids, monoacids and diacids, amino acids and amides. Examples of dialcohols are ethanediol, propanediol, butanediol and hexanediol. Examples of monoalcohols are butanol and ethanol. Examples of hydroxy acids are lactic acid. Examples of monoacids are lactic, acetic and formic acids. Examples of diacids are succinic acid, fumaric acid and furandicarboxylic acid. One example of an amide is caprolactam.

[0017] Preferably, said chemical intermediates are dialcohols (diols) such as 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,6-hexanedioL Even more preferably said diols are 1,3-butanediol, 1,4-butanediol or mixtures thereof. 1,4-butanediol is however preferred.

[0018] These polyhydroxyalkanoates (PHA) are preferably selected from the group comprising, or consisting of: polyhydroxybutyrate, polyhydroxybutyrate- valerate, polyhydroxybutyrate- propanoate, polyhydroxybutyrate-hexanoate, polyhydroxybutyrate-decanoate, polyhydroxybutyrate-dodecanoate, polyhydroxybutyrate -hexadecanoate, polyhydroxybutyrate-octadecanoate, poly 3-hydroxybutyrate-4-hydroxybutyrate. More preferably said polyhydroxyalkanoates are selected from the group consisting of polyhydroxybutyrate (PHB), polyhydroxybutyrate- valerate (PHBV) and polyhydroxybutyrate- hexanoate (PHBH).

[0019] According to a preferred embodiment, said Escherichia coli cells are residues from a fermentation process for the production of butanediol, preferably 1,3-butanediol, 1,4-butanediol or a mixture thereof.

[0020] According to an even more preferred embodiment, said Escherichia coli cells are residues from a fermentation process for the production of 1,4-butanediol.

[0021] These Escherichia coli cells are a residue cells that, if not used to obtain protein hydrolysates, would be a waste by-product of the fermentation process.

[0022] The protein hydrolysate according to the invention is particularly suitable for use as a plant biostimulant.

[0023] A second aspect of the present invention is therefore the use of said protein hydrolysate as a bio stimulant for plants.

[0024] A third aspect of the present invention is a plant biostimulant comprising said protein hydrolysate.

[0025] The present invention will be described in more detail below.

[0026] The protein hydrolysate according to the present invention preferably comprises at least 20% by weight of dry matter, preferably 20 to 90%, more preferably 30 to 90%, even more preferably from 40 to 90% by weight.

[0027] The dry matter of the protein hydrolysate is understood to be the weight of the residual portion of the protein hydrolysate after removal of the water it contains and can be determined, for example, according to ASTM El 756 - 08.

[0028] The protein hydrolysate according to the present invention preferably comprises 115 to 600 g / L, preferably 150 to 600 g / L, more preferably 200 to 500 g / L, even more preferably 200 to 480 g / L, of total amino acids. The protein hydrolysate according to the present invention preferably comprises 50 to 99% of total amino acids, preferably 60 to 90%, respect to the dry weight of the hydrolysate.

[0029] The protein hydrolysate according to the present invention advantageously comprises 5 to 65% by weight of free amino acids, respect to the total amino acids.

[0030] Preferably, it comprises 10% by weight or more of free amino acids and 65% or less, more preferably 55% or less, even more preferably 35% or less of free amino acids, respect to the total amino acids.

[0031] The protein hydrolysate according to the present invention advantageously comprises one or more amino acids chosen from proline, aspartic acid, glutamic acid, serine, alanine, glycine, threonine, cysteine, valine and leucine.

[0032] The protein hydrolysate according to the present invention preferably comprises at least 0.5%, preferably at least 1%, more preferably 2 to 15%, more preferably 2 to 10%, even more preferably 2.7 to 10% by weight, respect to the % of total amino acids, of proline.

[0033] The protein hydrolysate according to the present invention preferably comprises at least 5%, preferably 8 to 20%, even more preferably 9 to 15%, respect to the % of total amino acids, of alanine.

[0034] The protein hydrolysate according to the present invention preferably comprises at least 3%, preferably 4 to 15%, even more preferably 5 to 10% by weight, respect to the % of total amino acids, of glycine.

[0035] The protein hydrolysate according to the present invention preferably comprises at least 3.5%, preferably 4 to 12%, even more preferably 4 to 10% by weight, respect to the % of total amino acids, of threonine.

[0036] The protein hydrolysate according to the present invention preferably comprises at least 4%, preferably 5 to 15%, even more preferably 8 to 15% by weight, respect to the % of total amino acids, of aspartic acid.

[0037] The protein hydrolysate according to the present invention preferably comprises at least 5%, preferably 8 to 20%, even more preferably 9 to 15% by weight, respect to the % of total amino acids, of glutamic acid.

[0038] The protein hydrolysate according to the present invention preferably comprises at least 3%, preferably 3 to 15%, even more preferably 3 to 8% by weight, respect to the % of total amino acids, of serine.

[0039] The protein hydrolysate according to the present invention preferably comprises at least 0.1%, preferably 0.2 to 5%, even more preferably 0.3 to 4% by weight, respect to the % of total amino acids, of cysteine. The protein hydrolysate according to the present invention preferably comprises at least 3%, preferably 4 to 15%, even more preferably 5.5 to 13% by weight, respect to the % of total amino acids, of valine.

[0040] The protein hydrolysate according to the present invention preferably comprises at least 3%, preferably 4 to 16%, even more preferably 6.5 to 15% by weight, respect to the % of total amino acids, of leucine.

[0041] The protein hydrolysate according to the present invention advantageously comprises peptides or amino acids with a molecular weight of less than or equal to 1060 Da in amounts of at least 50%, preferably at least 70%, more preferably at least 80% and even more preferably at least 85% by weight, respect to the total amino acids.

[0042] The protein hydrolysate according to the present invention advantageously comprises peptides or amino acids with a molecular weight of less than or equal to 572 Da in amounts of at least 30%, preferably at least 40%, more preferably at least 50% and even more preferably at least 60% by weight, respect to the total amino acids.

[0043] The protein hydrolysate according to the present invention advantageously comprises peptides with a molecular weight greater than or equal to 1060 Da in quantities lower than 35%, preferably lower than 20%, more preferably lower than 15% by weight, with respect to the total amino acids.

[0044] The protein hydrolysate according to the present invention advantageously comprises peptides with a molecular weight greater than or equal to 5700 Da in quantities lower than 1%, preferably lower than 0.7% by weight, with respect to the total amino acids. Even more preferably, the protein hydrolysate of the invention does not comprise peptides with a molecular weight greater than 5700 Da.

[0045] The protein hydrolysate according to the present invention advantageously comprises peptides with a molecular weight greater than or equal to 6500 Da in quantities lower than 0.7%, preferably lower than 0.5% by weight, with respect to the total amino acids. Even more preferably, the protein hydrolysate according to the present invention does not comprise peptides with a molecular weight greater than 6500 Da.

[0046] With regard to the macronutrient content, the protein hydrolysate according to the present invention preferably comprises 2 to 20% by weight, preferably 3 to 10% by weight, of organic nitrogen, measured by the Kjeldahl method.

[0047] The protein hydrolysate according to the invention preferably comprises quantities of less than 300 ppm, preferably less than 200 ppm, even more preferably 0.1 to 100 ppm of heavy metals such as Iron, Zinc, Copper, Manganese, Nickel, Lead or Chromium. The protein hydrolysate according to the present invention preferably has an ash content of advantageously less than or equal to 10%, preferably less than or equal to 8%, even more preferably less than or equal to 5% by weight.

[0048] If the Escherichia coli cells are a residue from a fermentation process for the production of butanediol, the protein hydrolysate of the invention comprises butanediol, preferably 1,3- butanediol, 1,4-butanediol or a mixture thereof.

[0049] The protein hydrolysate according to the invention may comprise up to 10%, more preferably 0.01 to 10%, more preferably 0.05 to 5%, more preferably 0.1 to 5%, even more preferably 0.1 to 1% by weight, respect to the total weight of the hydrolysate, of butanediol.

[0050] If the Escherichia coli cells are a residue from a fermentation process for the production of 1,4-butanediol, the protein hydrolysate may comprise up to 10%, more preferably 0.01 to 10%, more preferably 0.05 to 5%, more preferably 0.1 to 5%, even more preferably 0.1 to 1% by weight, respect to the total weight of the hydrolysate, of 1,4-butanediol.

[0051] If the Escherichia coli cells are a residue from a fermentation process for the production of

[0052] 1.3-butanediol, the protein hydrolysate may comprise up to 10%, more preferably 0.01 to 10%, more preferably 0.05 to 5%, more preferably 0.1 to 5%, even more preferably 0.1 to 1% by weight, respect to the total weight of the hydrolysate, of 1,3-butanediol.

[0053] The protein hydrolysate according to the invention may comprise up to 5%, preferably 0.01 to 5% of sugars. Said sugars are preferably glucose and fructose.

[0054] The protein hydrolysate according to the invention may comprise up to 15% by weight, preferably 8 to 1% of glycerol.

[0055] The protein hydrolysate according to the invention advantageously has a conductivity of less than 7.5 mS / cm, preferably less than 6.5 mS / cm, measured by a Conductivity Meter on a sample of the protein hydrolysate comprising 50 to 99% of total amino acids, respect to the dry weight of the hydrolysate, using a probe suitable for reading conductivity in the range 0.01-1000 mS / cm, such as Inlab 731-ISM (Mettler Toledo).

[0056] If the Escherichia coli cells are a residue from a fermentation process for the production of

[0057] 1.4-butanediol, such a fermentation process may be performed for example according to the process described in WO 2015 / 158716. In said document, 1,4-butanediol is synthesised by a fermentation process from a culture medium containing at least one sugar, preferably glucose and optionally one or more sugars other than glucose, in the presence of one or more suitable microorganisms.

[0058] The culture medium may comprise other substances necessary for growth and sustenance of the microorganism during the fermentation stage, such as elements like C, H, O, N, K, S, P, Fe, Ca, Co, Mn, Mg. Typically, the culture medium may include one or more components selected from the group consisting of sugars other than glucose, protein hydrolysates, proteins, amino acids, organic acids, vitamins, mineral salts, yeast extracts, and trace elements such as Cobalt, Calcium and Copper. Cobalt, Calcium and Copper can be dosed into the culture medium, for example, as salts such as Cobalt chloride, Calcium chloride and Copper chloride. Generally, the culture medium comprises at least one sugar, usually glucose and optionally one or more sugars other than glucose, in concentrations between 10 and 100 g / L. As the microorganism consumes one or more sugars during the fermentation stage of this process, it is generally necessary to reintroduce these sugars into a fermentation reactor. This reintroduction can be carried out in a continuous or discontinuous manner, as known to those skilled in the art. The components of the culture medium, including sugars, protein hydrolysates, proteins, amino acids, organic acids, vitamins, mineral salts, yeast extracts and trace elements are typically added in excess of the microorganism's actual needs and are not therefore fully utilised and remain in the broth at the end of fermentation in the form of impurities.

[0059] To limit the content of unused sugars and thus optimise the economy of the process, the supply of one or more sugars is advantageously interrupted or gradually decreased before the end of fermentation. With regard to other possible components of the culture medium, the culture medium generally contains salts, essential minerals, and antifoaming agents. The culture medium can be prepared in any way known to those skilled in the art, for example by mixing all its components together or by pre-mixing all the components with the exclusion of glucose and adding them later, individually or already pre-mixed in turn. It is also possible to use a commercially available culture medium as a starting point and suitably modify its composition at a later stage, for example when bringing the culture medium into contact with the microorganism with at least one metabolic pathway for the synthesis of 1,4-butanediol from a renewable source. During fermentation, the combination consisting of the microorganism and the culture medium comprising one or more sugars is maintained under conditions suitable for exploiting the metabolic pathway for the synthesis of 1,4-butanediol from renewable sources. Those skilled in the art will also be able to check the progress of the process during fermentation, for example by monitoring one or more parameters and, if necessary, acting upon them to restore the process to conditions suitable for the production of 1,4-butanediol.

[0060] If Escherichia coli cells are a residue from a fermentation process for the production of 1,3-butanediol, hereafter also referred to as “1,3-BG”, this fermentation process may be performed according to, for example, the process described in WO 2010 / 127319. Fermentation broth comprising 1,3-BG from renewable sources is generally obtained by a fermentation process from a culture medium known in the art to support the growth of one or more microorganisms having at least one metabolic pathway for the synthesis of 1,3-BG, under anaerobic or micro-aerobic conditions. Fermentation is carried out in batch, fed-batch or continuous mode. Anaerobic conditions are maintained by first blanketing the medium with nitrogen, while micro-aerobic conditions can also be achieved with a small hole for limited aeration. The pH of the medium is typically maintained at an optimum pH by the addition of a base, such as NaOH or other bases, or an acid.

[0061] With regard to the metabolic pathways for the synthesis of 1,3- and 1,4-butanediol, these may be present in the microorganism in their natural state or they may be artificially created, for example by altering, modifying, amplifying, eliminating or restricting metabolic pathways already existing in the microorganism, by inserting genetic material from one or more other organisms into the microorganism, by inducing spontaneous genetic mutations, by adding, during the process, chemical compounds that inhibit or stimulate said metabolic pathway, or otherwise by making use of any genetic engineering technique. Microorganisms having metabolic pathways for the synthesis of 1,4-butanediol are known to those skilled in the art and are, for example, described in Yim, H. et al., Nature Chemical Biology, Vol. 7, July 2011, pp. 445-452 and in patent applications WO 2008 / 115840, WO 2009 / 023493, WO 2010 / 030711, WO 2010 / 071697, WO 2010 / 141780, WO 2010 / 141920, WO 2011 / 031897, WO 2011 / 047101, WO 2011 / 066076, WO 2012 / 177943.

[0062] Microorganisms with metabolic pathways for the synthesis of 1,3-BG are known to those skilled in the art and are, for example, described in Kataoka, N. et al., Biosci. Biotechnol. Biochem., 2014, Vol 78, p. 695-700 and in patent application WO 2010 / 127319.

[0063] In cases where Escherichia coli cells are a residue from a fermentation process for PHA production, this is typically performed by inoculating the bacterial culture in a suitable medium to promote the production of cell biomass and, at a later stage, the growth conditions are possibly changed to induce the synthesis and accumulation of PHA in the form of intracellular inclusions. PHA synthesis is usually induced by subjecting the microorganism to a deficiency of macronutrients such as phosphorus, nitrogen and sulphur, and simultaneously to an excess of carbon sources.

[0064] Microorganisms with metabolic pathways for the synthesis of polyhydroxyalkanoates are known to those skilled in the art and are, for example, described in Lee, S.Y. et al. (Appl Environ Microbiol, 2003, Vol. 69(6): 3421-3426), Saranya, V. et al. (Braz J Microbiol., 2011, Vol. 42(3): 1109-1118), Chen, J. et al. (Microb Cell Fact, 2018, Vol. 17: 102), Zhuang, Q. (Microb Cell Fact, 2019, Vol. 18: 135).

[0065] At the end of the fermentation process, the microbial biomass present in the fermentation broth can optionally undergo a preliminary treatment, typically of a physical-chemical nature, in order to be deactivated or killed.

[0066] One example is a heat pretreatment, which is typically performed at a temperature of around 50°C to 80°C, preferably around 60°C to 70°C, at least around 60°C, or at least around 70°C, for a time of around 1 minute to 10 minutes, preferably 2 minutes to 5 minutes, 2 minutes to 3 minutes, at least 2 minutes or at least 2.5 minutes.

[0067] In the case of residual microbial biomass from a fermentation process for PHA production, pretreatment may also be applied in order to denature the proteins and weaken the cell wall, to facilitate subsequent extraction of the polymer. Examples are heat treatment, alkalinisation and freeze-drying of the cells.

[0068] At the end of the fermentation process, or after optional pre-treatment, the fermentation broth undergoes solid / liquid separation to separate a solid fraction comprising cells from a liquid fraction.

[0069] By fermentation broth is meant an aqueous solution comprising cells, residual culture medium and fermentation product.

[0070] The aforementioned solid / liquid separation may comprise one or more operations chosen from among: sedimentation, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis, flocculation and combinations thereof. Centrifuging and filtration operations are preferred.

[0071] The solid fraction including cells may optionally undergo one or more washing operations, followed by one or more solid / liquid separation operations.

[0072] Such washing operations may for example be performed using 1 or 2 volumes of water, respect to the volume of the solid fraction, and such solid / liquid separation operations may include centrifuging and filtration.

[0073] Where the Escherichia coli cells are a residue from a fermentation process for PHA production, the solid fraction comprising cells removed from the liquid fraction may optionally undergo additional treatments for extraction of the PHA produced. Such treatments may include extraction with solvent or bases such as sodium hydroxide, digestion with chemicals such as surfactants and / or sodium hypochlorite or enzymes, mechanical breakdown, breakdown by osmosis in a hypotonic environment or breakdown with supercritical fluid. At the end of the solid / liquid separation operations and, in the case of fermentation for PHA production, of the optional additional treatments mentioned above, the solid fraction comprising cells has a dry matter content of 5 to 20%, preferably 8 to 15% by weight, with respect to the total weight of the solid fraction. Said solid fraction has an organic nitrogen content of 0.5 to 3%, preferably 1 to 2% by weight, respect to the total weight of the solid fraction.

[0074] By dry matter is meant the weight of the residual portion of the solid fraction after removal of the water it contains and can be determined, for example, according to ASTM El 756 - 08.

[0075] The organic nitrogen content may be measured using the Kjeldahl method.

[0076] Said solid fraction comprising cells may undergo an optional protein extraction and solid / liquid separation step. Protein extraction may, for example, be performed by placing the solid fraction in an aqueous solution at a pH of 10 to 14, preferably 11 to 13, for a time of 1 to 4 hours, under conditions of gentle agitation, for example at 200 rpm, and at a temperature of 30 to 50°C. The extracted proteins are then separated from the cell residues by one or more solid / liquid separation operations, resulting in a liquid protein extract. These solid / liquid separation operations include centrifuging and filtration.

[0077] According to one embodiment, this solid fraction is subjected to protein extraction and solid / liquid separation, resulting in a liquid protein extract.

[0078] According to an alternative embodiment, this solid fraction is not subjected to protein extraction and solid / liquid separation.

[0079] The solid fraction or liquid protein extract is subjected to hydrolysis.

[0080] According to one embodiment, the solid fraction is subjected to hydrolysis, resulting in a hydrolysed cellular cream.

[0081] According to an alternative embodiment, the liquid protein extract is subjected to hydrolysis, resulting in the protein hydrolysate.

[0082] Hydrolysis may be performed by any known method suitable for lysing cellular protein components into free amino acids and / or low molecular weight peptides. It may be for example chemical or enzyme hydrolysis or a combination thereof. Enzyme hydrolysis is preferred.

[0083] Enzyme hydrolysis may be performed using any enzyme or mixture of enzymes capable of hydrolysing proteins into free amino acids and / or low molecular weight peptides.

[0084] Enzyme hydrolysis is performed by proteolytic enzymes, known as proteases, which are able to break down the peptide chain of proteins at specific points and hydrolyse them to free amino acids and / or low molecular weight oligopeptides. The proteases may be endoproteinases and / or exoproteinases.

[0085] In one embodiment, enzyme hydrolysis is performed using an alkaline protease. An alkaline protease is preferably used for hydrolysis of the protein extract.

[0086] In an alternative embodiment, enzyme hydrolysis is performed using a neutral protease. A neutral protease is preferably used for hydrolysis of the solid fraction including cells. In another alternative embodiment, enzyme hydrolysis is performed using an acid protease. According to a preferred embodiment, enzyme hydrolysis is performed in the presence of proteases obtained from thistle or safflower heads. For example, proteases from thistle or safflower heads are obtained by immersing thistle or safflower pistils in a phosphate buffer solution with a molarity of 10 to 50 mM, pH 4.5 to 5.5, using a ratio of 5 to 10% by weight to the volume of buffer solution. The resulting suspension is kept stirred at from 100 to 200 rpm, at a temperature of 30 to 50°C, for 60 to 240 minutes. The suspension is then subjected to suitable solid / liquid separation techniques, for example by one or more operations chosen from sedimentation, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis and combinations thereof. Preferably said solid / liquid separation operations comprise centrifuging and filtration operations. After solid / liquid separation, the enzyme extract is obtained in the liquid, which may be concentrated, for example by ultrafiltration.

[0087] Enzyme hydrolysis is performed by selecting appropriate amounts of reagents, reaction conditions and reaction sequences to achieve an adequate level of enzyme activity. Enzyme hydrolysis is for example performed by placing the solid fraction comprising cells or the protein extract in aqueous solution in contact with proteases in a weight ratio of 0.1 to 0.5 g / g, preferably 0.3 to 0.5 g / g of enzyme per 1 g of dry cell biomass or protein content of the extract. The resulting suspension may be incubated at a pH and temperature appropriate to the specific protease used, and for a period of time adequate to obtain the desired amount of proteolysis.

[0088] If a neutral protease is used, the suspension may be incubated at pH 5.5 to 8.5, preferably 6.5 to 8.

[0089] If an alkaline protease is used, the suspension may be incubated at pH 10 to 14, preferably 11 to 13.

[0090] If an acid protease is used, the suspension may be incubated at pH 1 to 4, preferably 2 to 3. The temperature is advantageously maintained at 40 to 70°C, preferably 50 to 60°C.

[0091] Agitation is advantageously set at 100 to 300 rpm, preferably 150 to 250 rpm.

[0092] Hydrolysis is advantageously prolonged for 48 to 96 hours, preferably 60 to 72 hours.

[0093] Enzyme hydrolysis may be carried out in any suitable device known to those skilled in the art, such as a reactor with temperature control and stirring. In cases where the solid fraction has undergone hydrolysis, enzyme hydrolysis is preferably followed by a subsequent solid / liquid separation step. This solid / liquid separation step makes it possible to separate a cell pellet from the hydrolysed protein hydrolysate in the liquid phase. Where the liquid protein extract has undergone hydrolysis, enzyme hydrolysis is optionally followed by a subsequent solid / liquid separation step. This solid / liquid separation step allows the enzymes used for enzyme hydrolysis and any additional solid residues to be removed from the protein hydrolysate.

[0094] Said subsequent solid / liquid separation step may comprise one or more operations chosen from sedimentation, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis and combinations thereof. Filtration operations are preferred, such as gauze filtration and / or microfiltration and / or ultrafiltration.

[0095] According to one embodiment, the subsequent solid / liquid separation step comprises gauze filtration and microfiltration operations.

[0096] Any ultrafiltration technique using any filter unit with semi -permeable membranes, for example of the tubular, hollow-fibre, spiral or plate-and-frame type and working using tangential flow or flow perpendicular to the membrane surface may be used for ultrafiltration. Tangential flow ultrafiltration is preferred. With regard to filter membranes, semi -permeable membranes of cellulose acetate, cellulose acetate derivatives such as cellulose acetobutyrate, and synthetic polymers, such as polypropylene, polyamides, polyimides, PVDF (polyvinyldene fluoride), PAN (polyacrylonitrile), PES (polyether sulfone) and ceramics can be used.

[0097] Preferably, PES membranes with a porosity of 1 kDa or less are used.

[0098] The choice of temperature, transmembrane pressure and other operating conditions at which the ultrafiltration phase is performed is determined primarily by the viscosity of the aqueous mixture fed and the type and porosity of the membrane used.

[0099] As ultrafiltration proceeds, the viscosity of the aqueous mixture and the transmembrane pressure naturally tend to increase and separation efficiency tends to decrease. This necessitates the use of gradually increasing pressures which, if too high, can damage the filter unit and compromise the efficiency of the process. To avoid the use of excessively high pressures, it is possible to resort to what is known as diafiltration, by feeding one or more aliquots of a refreshing solution that compensates for the part of the aqueous mixture permeated through the membrane. Diafiltration may be performed in both continuous and discontinuous regimes.

[0100] At the end of these solid / liquid separation operations following enzyme hydrolysis, the protein hydrolysate is obtained in the liquid phase. This protein hydrolysate may advantageously be concentrated by known techniques, for example by distillation, evaporation or reverse osmosis, until the desired protein concentration is reached.

[0101] According to one embodiment, the present invention therefore also relates to a process for obtaining a protein hydrolysate comprising the enzyme hydrolysis of residual Escherichia coli cells from a fermentation process for producing chemical intermediates or polyhydroxy alkanoates .

[0102] The enzyme hydrolysis is preceded and / or followed by a solid / liquid separation step.

[0103] This process for obtaining the protein hydrolysate includes the steps of: a) subjecting the fermentation broth to solid / liquid separation to separate a solid fraction comprising cells from a liquid fraction, b) subjecting this solid fraction to enzyme hydrolysis, resulting in a hydrolysed cellular cream, c) subjecting said hydrolysed cellular cream to solid / liquid separation, resulting in a cell pellet and a protein hydrolysate in the liquid phase.

[0104] Said enzyme hydrolysis (step b)) may optionally be preceded by one or more operations of washing the solid fraction, followed by one or more solid / liquid separation operations.

[0105] According to an alternative embodiment, the present invention relates to a process for obtaining a protein hydrolysate comprising the enzyme hydrolysis of a protein extract of residual Escherichia coli cells from a fermentation process for producing chemical intermediates or polyhydroxy alkanoates .

[0106] This process for obtaining the protein hydrolysate includes the steps of: i. subjecting the fermentation broth to solid / liquid separation to separate a solid fraction comprising cells from a liquid fraction, ii. subjecting this solid fraction to protein extraction and one or more solid / liquid separation operations, resulting in a liquid protein extract, iii. subjecting said liquid protein extract to enzyme hydrolysis, resulting in a protein hydrolysate.

[0107] Said protein extraction (step ii.) may optionally be preceded by one or more operations of washing the solid fraction, followed by one or more solid / liquid separation operations.

[0108] The protein hydrolysate from step iii. may optionally undergo further solid / liquid separation operations.

[0109] The protein hydrolysate according to the invention is particularly suitable for use as a plant biostimulant. This biostimulant may optionally include one or more additives and / or adjuvants that make the product suitable for field applications and enhance its properties in both fertigation and spray applications. Such additives and adjuvants may be selected from: carriers, adhesives, filming agents, wetting agents, penetrating agents, cold protectants, emulsifying agents, wetting agents, dispersing agents, stabilisers, preservatives, diluents, micronutrients, macronutrients and combinations thereof.

[0110] The carrier is selected from the group comprising but not limited to: lignite, bentonite, peat, vermiculite, charcoal, soil mixtures, manure or combinations thereof.

[0111] The cold protector is selected from the group comprising but not limited to: polyethylene glycol (PEG), glycerol, DMSO, polyvinyl alcohol, sodium alginate, gelatin, gellan or combinations thereof.

[0112] Adhesive adjuvants with film-forming and wetting power, used to allow the biostimulant active ingredient to remain on the leaf for as long as possible and penetrate into the cuticle, are selected from the group comprising but not limited to: xanthan gum, carboxymethylcellulose (CMC), gum arabic, polyvinylpyrrolidone (PVP), chitosan, alginate, modified or complexed starches or combinations thereof.

[0113] Wetting additives are selected from the group comprising but not limited to: alkoxylated polyol esters, ethoxylated and non-ethoxylated sorbitans, alkylpoly gluco sides or combinations thereof. These additives have the function of preventing the biostimulants from drying out and thus crystallising on the leaf, leaving them moist and thus able to act.

[0114] Penetrating agents as well as emulsifiers and wetting agents serve to promote the entry of the biostimulant into the leaf cuticle and are selected from the group comprising but not limited to: cationic surfactants, anionic surfactants, non-ionic surfactants, silicon-based surfactants and combinations thereof. These surfactants may be natural, semi- synthetic, synthetic or combinations thereof.

[0115] Examples of surfactants are ethoxylated and ethoxylated-propoxylated sorbitans, ethoxylated alcohols, block or star copolymers, partially hydrolysed and ethoxylated polyol esters, ethoxylated and / or propoxylated vegetable oils.

[0116] Dispersing additives, used to stabilise the formulation over time, are selected from the group comprising but not limited to: lignin sulfonates and block copolymers, ethoxylated tristyrylphenols, dioctyl sulfosuccinates and the like, naphthalene sulfonates and EO / PO block copolymers, star copolymers, xanthan gum or combinations thereof.

[0117] The stabiliser and preservative are selected from the group comprising but not limited to: potassium sorbate, sorbic acid, lactic acid, trehalose, sugars, mannitol, citric acid, polyglutamic acid, benzoic acid or sodium benzoate, propylene glycol, propionic acid, zinc sulfate, iron sulfate, copper sulfate, l,2-benzisothiazolin-3-one (BIT) and the like, 2, 2', 2"- (hexahydro- 1,3,5- triazine-l,3,5-triyl)triethanol and the like, pentane- 1,2-diol, 3-phenylpropan-l-ol and the like or combinations thereof.

[0118] The diluent is selected from the group comprising but not limited to: ionic buffer diluent, salt solution, water or combinations thereof.

[0119] The biostimulant according to the invention may be administered to plants together with solutions containing micro- and / or macronutrients or may be formulated together with them.

[0120] Examples of micronutrients are: iron, nickel, copper, zinc, boron, manganese, calcium, sodium, molybdenum and magnesium variously complexed such as with EDTA and other organic complexing agents.

[0121] Examples of macronutrients are nitrogen, potassium and phosphorus.

[0122] The biostimulant according to the invention may be in solid or liquid form. In the liquid form, which is the preferred form, the biostimulant is placed in contact with or applied to the plant at a concentration ranging from approximately 1 g / L to approximately 20 g / L.

[0123] This biostimulant may be applied to the soil, rhizosphere, aerial or non-aerial parts of the plant, selected from the group comprising roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruit, seeds and their combinations.

[0124] In some embodiments, the biostimulant is applied as a foliar spray solution at a dose of 1 to 20 g per litre of water.

[0125] In other embodiments, the biostimulant is applied directly to the soil by fertigation, with a dose of 1 to 20 g per litre of water.

[0126] The biostimulant according to the invention may be administered to agricultural, horticultural, floricultural, fruit crops or any combination thereof.

[0127] Among agricultural crops, the biostimulant is preferably applied to tomato, aubergine, pumpkin, cucumber, courgette, lettuce, cauliflower, strawberry and tobacco crops.

[0128] Application of the biostimulant results in an improvement in the performance of the plants to which it is administered. This performance improvement includes one or more of:

[0129] • an increase in the number, size, weight or quality of hypogeal or aerial biomass, for example roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits and / or seeds,

[0130] • increased photosynthetic activity or chlorophyll content,

[0131] • increase in protein, dietary fibre, P-carotene, essential oils and / or specific plant metabolites, • improved absorption or utilisation of nutrients or minerals.

[0132] In some embodiments, application of the biostimulant according to the invention promotes or improves plant growth, by even some 10% to 60% compared to plants that did not receive the administration. For example, application of the biostimulant according to the invention results in an increased number of roots and their length in tomato and aubergine plants, increased leaf growth and total biomass production in squash and cucumber plants.

[0133] In some embodiments, application of the biostimulant according to the invention confers greater plant tolerance to abiotic stresses, such as high salinity, promoting plant growth and chlorophyll synthesis and accelerating recovery time.

[0134] These effects on growth can be surprisingly enhanced if polyphenols are added to the biostimulant composition. Such polyphenols may be extracted from waste vegetable biomass, such as from thistle and safflower seeds, after pressing and extraction of the oil they contain. The following examples illustrate the present invention for non-limiting purposes.

[0135] ANALYTICAL METHODS used in the examples

[0136] Determination of nitrogen content.

[0137] Nitrogen content was measured using the Kjeldahl method.

[0138] Determination of protein content.

[0139] To assess the protein content of the solid fraction including cells, the experimental value of the organic nitrogen content measured by the Kjeldahl method was multiplied by the conversion factor 6.25.

[0140] Amino acid analysis: total and free amino acids

[0141] In order to quantify the total and free amino acids present in the hydrolysate, the hydrolysate was subjected to hydrolysis with 6N HC1 for 2.0 hours at 150°C inside flame-sealed vials. After hydrolysis, the sample was neutralised with sodium carbonate and subjected to amino acid analysis. The amino acid analysis was performed using ortho-p-phthalaldehyde (OPA) as derivatising agents for the determination of primary amino acids and fluorenyl- methyloxycarbonyl chloride (FMOC) for secondary amino acids. The derivatisation method was performed at room temperature and integrated into the RP-HPLC analysis using a Gemini 5 pm NX-C18 110 °A, 250x3 mm column. The mobile phases used were respectively: (A) 10 mM NaiHPCU, 10 mM NaiELO?, pH 8.2, (B) Acetonitrile: methanol: water (45:45:10, v:v:v). The detection parameters were: Signal A = 338 nm (amino acids derivatised by OPA, 10 nm bandwidth); Signal B = 262 nm (amino acids derivatised by FMOC derivatised amino acids, 10 nm bandwidth).

[0142] Pigment extraction and measurement of chlorophyll content For the measurement of chlorophyll content in plant tissue, the method proposed by Porra et al. (1989) was followed, with some modifications. Approximately 0.2 g of dry plant material was homogenised with a pestle with an aliquot of sand. Then 800 pL of cold 80% acetone in water was added and the material was further homogenised. The extract was centrifuged at 7000 rpm for 3 minutes. The supernatant was recovered and the pellet was treated again in the same way to extract the residual chlorophylls. Aliquots of the sample were analysed by spectrophotometric reading at 663 nm (preferential absorption chlorophyll a) and 648 nm (preferential absorption chlorophyll b). The following formulae were used for the calculation of chlorophyll Chi concentrations:

[0143] 1) Chi a = 12.25 x ABS 663 - 2.55 x ABS 648 (pg / mL);

[0144] 2) Chi b = 20.31 x ABS 648 - 4.9 x ABS 663 (pg / mL);

[0145] 3) Chi a+b = 17.76 x ABS648 + 7.34 x ABS663 (pg / mL).

[0146] Where pg are micrograms of dry plant material and mL are millilitres of extraction solution. Determination of conductivity

[0147] The conductivity was measured on samples of protein hydrolysates, after dilution to 1% by weight in distilled water, by the benchtop Electrical Conductivity Meter, model Seven Multi (Mettler Toledo), at 20 °C. The conductivity calibration was performed against air (point zero) and with a standard buffer of 12,8 mS / cm before each measure to ensure reliability, using a probe suitable for reading conductivity in the range 0.01-1000 mS / cm, such as Inlab 731-ISM (Mettler Toledo).

[0148] EXAMPLES

[0149] Example 1: Production of a hydrolysate from residual Escherichia coli cells from a fermentation process for the production of 1,4-butanediol.

[0150] Escherichia coli cells genetically modified for the production of 1,4-butanediol was subjected to fermentation using the process described in WO 2015 / 158716. At the end of fermentation, the fermentation broth was kept at 70°C for 30 minutes and then filtered, removing a solid fraction comprising cells with a dry matter content of 11% and total nitrogen content of 1.17%, by weight to total weight, and a protein content of 65.9%, by weight to dry matter.

[0151] 1 kg of this solid fraction was resuspended in 1 litre of water and the pH was adjusted to 7.5 by the addition of 10N KOH. The resuspended solid fraction was hydrolysed using BS-L enzyme (neutral protease supplied by Enzyme Supplies), employing an enzyme load of approximately 1:180 w / w, evaluated on the protein content of enzyme and substrate respectively. Enzyme hydrolysis was performed at 55°C for 18 hours, stirred at 200 rpm. At the end of the reaction, the liquid phase was separated from the cell pellet by centrifuging at 8000 rpm for 40 minutes and was then filtered through gauze. Finally, it was subjected to tangential flow filtration, using 2 Millipore membranes having a 1 kDa cut-off in series and 0.10 m2filter surface area each mounted on a Millipore system, and diafiltration.

[0152] The characteristics of the protein hydrolysate obtained are shown in Table 1.

[0153] Comparative Example 2: Production of a hydrolysate from Escherichia coli cells unable to produce 1,4-butanediol.

[0154] Cells of Escherichia coli ATCC 8739 (not genetically modified for 1,4-butanediol production) were subjected to fermentation as described in WO 2015 / 158716. After pre-treatment at a temperature of 70°C for 30 minutes, the solid fraction comprising cells was separated from the liquid fraction by subjecting the fermentation broth to filtration. This solid fraction had a dry matter content of 4.9% and a total nitrogen content of 0.63% by weight, respect to total weight, and a protein content of 61.19% by weight respect to dry matter.

[0155] The solid fraction was subjected to enzyme hydrolysis and solid / liquid separation, using the conditions described in Example 1.

[0156] The characteristics of the protein hydrolysate obtained are shown in Table 1.

[0157] Table 1. Characteristics of the protein hydrolysates obtained in Example 1 and Comparative Example 2.

[0158] As may be seen from the results in Table 1, using the same growth conditions, the use of Escherichia coli cells genetically modified to produce 1,4-butanediol resulted in a solid fraction comprising cells with a higher dry matter, organic nitrogen and amino acid content than Escherichia coli cells not modified to produce 1,4-butanediol.

[0159] Example 3: Leaf growth test and total biomass production in cucumber (Cucumis salivas).

[0160] Cucumber seeds were placed to germinate in pots containing universal compost and the seedlings were allowed to grow to the first true leaf stage. Every 3-4 days, starting from the first true leaf stage, 15 ml of a solution of the protein hydrolysate obtained in Example 1 was administered per pot / plant by fertigation, at a concentration of 5 g per litre of water. The first treatment was carried out on plants 1 week old (from germination), up to 3 weeks, for a total of 7 treatments. As a control, in the same manner as described above, other plants were given 15 ml of water instead of the protein hydrolysate.

[0161] Every week from the start of treatment, the length of the first (1FV), second (2FV) and third (3FV) true leaves was measured. The difference between leaf length measured before administration of the protein hydrolysate and that measured 3 weeks after administration (known as the “growth delta”) is shown in Table 2. The 4-week-old plants were taken and weighed (fresh weight). The same plants were also dried in an oven at 90°C to measure the dry weight (data in Table 2).

[0162] Comparative Example 4

[0163] The same procedure as in Example 3 was applied to cucumber seeds to which 15 ml of the protein hydrolysate solution from comparative Example 2 was administered per pot / plant, at a dilution of 14 g / L, in order to have the same dry matter content as in Example 3. The results are shown in Table 2.

[0164] Table 2 Results of leaf growth and total biomass production tests in cucumber.

[0165] The results for Example 3 showed that the protein hydrolysate obtained in Example 1, at the dose used, had a significant biostimulating effect on leaf growth of all 3 first true leaves, also leading to an increase in fresh weight and dry weight of the treated plants after 3 weeks, compared to the control. In contrast, the results for Comparative Example 4 showed that the hydrolysate treatment obtained in Comparative Example 2 produced no significant bio stimulatory effect on leaf growth, and even showed a slight inhibitory effect on third leaf development.

[0166] Example 5: Leaf growth test and total biomass production in pumpkin (Cucurbita moschatd). Pumpkin seeds were germinated in pots containing universal compost and the seedlings were grown to the first true leaf stage. Every 3-4 days from the first true leaf stage, 25 ml of the protein hydrolysate from Example 1 was administered per pot / plant by fertigation at a concentration of 5g / L in water. The first treatment was carried out on plants 1 week old (from germination) to 3 weeks old, for a total of 7 treatments. Each week from the start of treatment, the length of the first (1FV), second (2FV) and third (3FV) true leaves was measured. The difference between the leaf length measured before administration of the protein hydrolysate and that measured 3 weeks after administration (known as the “growth delta”) is shown in Table 3. The 4- week-old plants were taken and weighed (fresh weight). The same plants were also dried in an oven at 90°C to measure the dry weight. As a control, the same protocol was applied to pumpkin seeds that were given 25 ml of water instead of the protein hydrolysate. The results are shown in Table 3.

[0167] Table 3. Results of leaf growth and total biomass production tests in pumpkins.

[0168] The results shown in Table 3 demonstrate that administration of the protein hydrolysate obtained in Example 1 to pumpkin seedlings resulted in an increase in leaf size, up to 4 times larger than the control in the case of the third true leaf. In addition, the biomass weight doubled as a result of administration.

[0169] Example 6: Root elongation test in tomato (Solarium ly coper sicum).

[0170] 10 tomato seedlings, germinated and grown for 7 days on agar media (M&S substrate 2.2 g / L; sucrose 30 g / L; agar 8 g / L at pH 5.8) were removed, the roots removed and the seedlings transferred to new agar media containing the protein hydrolysate in Example 1 at a concentration of 0.3 g / L. As a control, in the same manner as described above, some seedlings were transferred to new agar media in the absence of the protein hydrolysate. After 5 days, the number and length of the roots were measured. The results are shown in Table 4.

[0171] Table 4. Results of elongation and root growth tests in tomato. The results obtained show that feeding tomato seedlings with the protein hydrolysate obtained in Example 1 resulted in the formation of more roots per plant, and these were also found to be longer than in the control.

[0172] Example 7: Root elongation test in aubergine (Solarium melongend).

[0173] 10 aubergine seedlings, germinated and grown for 7 days on agar media (M&S salts 2.2 g / L; sucrose 30 g / L; agar 8 g / L at pH 5.8) were removed, the roots removed and the seedlings transferred to new agar media containing the protein hydrolysate in Example 1 at a concentration of 0.3 g / L. As a control, in the same manner as described above, some seedlings were transferred to new agar media in the absence of the protein hydrolysate. After 5 days, the number and length of the roots were measured. The results are shown in Table 5.

[0174] Table 5. Results of elongation and root growth tests in aubergine.

[0175] The results obtained show that administration of the protein hydrolysate obtained in Example 1 to aubergine seedlings resulted not only in a greater number of roots per plant than in the control, but above all an increase in their length.

[0176] Example 8: Salt stress resistance in aubergine plants (Solarium melongena).

[0177] TenlO-day-old aubergine seedlings were treated by spraying 3 ml of a 10 g / L aqueous solution of the protein hydrolysate in Example 1. The application was repeated every 5 days for 20 days, for a total of 5 applications. 8 days after the start of treatment, the seedlings were subjected to salt stress by spraying with an aqueous solution of 100 mM NaCl for 14 days.

[0178] Samples of the epigeal part were pre-washed before salt stress, and 7 and 14 days after stress. Pigments were extracted from the samples and the chlorophyll content per mg of plant tissue was determined. As a control, the same protocol was applied to 10 aubergine seedlings that were given 3 ml of water instead of the protein hydrolysate. The results obtained are shown in Table 6.

[0179] Table 6. Chlorophyll content of aubergine plants subjected to salt stress. The results obtained show that the application of the biostimulant obtained in Example 1 to aubergine seedlings subjected to salt stress stimulated their chlorophyll synthesis, as early as 7 days after the stress and for at least 14 days after it, giving the plants greater tolerance. This effect was not visible in seedlings subjected to stress but only given water (control).

[0180] Example 9: Salt stress resistance in tomato plants (Solarium lycopersicum).

[0181] Ten 10-day-old tomato seedlings were treated by spraying 3 ml of a 10 g / L aqueous solution of the hydrolysate in Example 1. The application was repeated every 5 days for 20 days, for a total of 5 applications. 8 days after the start of treatment, the seedlings were subjected to salt stress by spraying with an aqueous solution of 100 mM NaCl for 14 days.

[0182] Samples of the epigeal part were pre-washed before salt stress, after 7 and 14 days of stress. Pigments were extracted from the samples and the chlorophyll content per mg of plant tissue was determined. As a control, the same protocol was applied to 10 tomato seedlings that were given 3 ml of water instead of the protein hydrolysate. The results are shown in Table 7.

[0183] Table 7. Chlorophyll content of tomato plants subjected to salt stress.

[0184] The results obtained show that treatment with the hydrolysate obtained in Example 1 produced a protective effect against salt stress, which caused a decrease in the chlorophyll content of the leaves. After 7 days of stress, in fact, unlike the control, the chlorophyll content did not decrease in the plants treated with the hydrolysate according to the invention (Example 9). After prolonged salt stress of 14 days, in the plants in Example 9, the chlorophyll content decreased in the leaves, but this decrease was less than that observed in the control treated with water alone, showing a bio stimulating effect of the hydrolysate in Example 1 in protecting against salt-induced leaf chlorosis.

[0185] Example 10

[0186] Escherichia coli cells genetically modified for the production of 1,4-butanediol were subjected to fermentation using the process described in WO 2015 / 158716. After pre-treatment at a temperature of 70°C for 30 minutes, the solid fraction comprising cells was separated from the liquid fraction by subjecting the fermentation broth to filtration. This solid fraction had a dry matter content of 12.9% and a total nitrogen content of 1.56% by weight, respect to total weight, and a protein content of 9.7% by weight respect to dry matter.

[0187] A sample of the solid fraction was subjected to enzyme hydrolysis util the content of free amino acids has reached about 30%, respect to the total amino acids, and then to a solid / liquid separation, using the conditions described in Example 1.

[0188] The characteristics of the protein hydrolysate obtained are shown in Table 8.

[0189] Comparative Example 11

[0190] A sample of the solid fraction separated in Example 10 was subjected to enzyme hydrolysis until the content of free amino acids has reached about 70% by weight, respect to the total amino acids, and then to a solid / liquid separation, using the conditions described in Example 1. The characteristics of the protein hydrolysate obtained are shown in Table 8.

[0191] Table 8. Characteristics of the protein hydrolysates obtained in Comparative Example 10.

[0192] Example 12

[0193] Lettuce plants (at BBCH stage 13-14, BBCH-scale is an universal coding system used to identify the phenological development stages of plants) were transferred to field and let them set for one day. Every 3-4 days, the plants (9 plants for each treatment) were treated by fertigation with only water (control) or with 100 ml of the protein hydrolysates obtained in Example 10 and 11 comparative, at a concentration of 5.5 g per L, for a total of 5 treatments.

[0194] Before each treatment, the plant diameter and the collar gauge were measured. The increase in diameter and the gauge (expressed as “growth delta”) calculated for plants treated with water and with the protein hydrolysate are shown in Table 9.

[0195] After 40-45 days from the first treatment, the plants were taken and the total weight (fresh weight) was calculated for each treatment. The results obtained are reported in Table 9 and clearly show an increase of about 36% of production, in terms of fresh weight, induced by the treatment with the protein hydrolysates of the Example 10, respect to the treatment with water or with the protein hydrolysates of the Example 11 comparative.

[0196] Table 9. Increase in plant growth (total diameter and collar gauge) after each treatment and fresh weight after 40-45 days from the first treatment.

Claims

CLAIMS1. Protein hydrolysate of residual Escherichia coli cells from a fermentation process to produce chemical intermediates or polyhydroxy alkanoates, comprising 50-99 % by weight of total amino acids, respect to the dry weight of the hydrolysate, characterized in that it comprises 5 to 65% by weight of free amino acids, respect to the total amino acids2. Protein hydrolysate according to claim 1, having a conductivity of less than 7.5 mS / cm, measured by a Conductivity Meter on a sample of the protein hydrolysate comprising 50 to 99% by weight of total amino acids, respect to the dry weight of the hydrolysate, using a probe suitable for reading conductivity in the range 0.01-1000 mS / cm.

3. Protein hydrolysate according to any of claims 1-2, comprising one or more amino acids selected from proline, aspartic acid, glutamic acid, serine, alanine, glycine, threonine, cysteine, valine and leucine.

4. Protein hydrolysate according to claim 3, comprising at least 3.5% by weight, respect to the % of total amino acids, of threonine.

5. Protein hydrolysate according to claim 3, comprising at least 3% by weight, respect to the % of total amino acids, of serine.

6. Protein hydrolysate according to any of claims 1-5, comprising butanediol.

7. Protein hydrolysate according to claim 6, comprising 1,3-butanediol, 1,4-butanediol or a mixture thereof.

8. Protein hydrolysate according to claim 6 comprising up to 10% by weight, respect to the total weight of the hydrolysate, of 1,4-butanediol.

9. Process for obtaining the protein hydrolysate according to any of claims 1-8, comprising enzyme hydrolysis of residual Escherichia coli cells from a fermentation process for producing chemical intermediates or polyhydroxy alkanoates.

10. Process according to claim 9, comprising the steps of: a) subjecting the fermentation broth to solid / liquid separation to separate a solid fraction comprising cells from a liquid fraction, b) subjecting this solid fraction to enzyme hydrolysis, resulting in a hydrolysed cellular cream, c) subjecting the hydrolysed cellular cream to solid / liquid separation, resulting in a cell pellet and a protein hydrolysate in the liquid phase.

11. Process for obtaining the protein hydrolysate according to any of claims 1-8, comprising enzyme hydrolysis of a protein extract of residual Escherichia coli cells from a fermentation process for producing chemical intermediates or polyhydroxy alkanoates.

12. Process according to claim 11, comprising the steps of: i. subjecting the fermentation broth to solid / liquid separation to separate a solid fraction comprising cells from a liquid fraction, ii. subjecting this solid fraction to protein extraction and solid / liquid separation, resulting in a liquid protein extract, iii. subjecting said liquid protein extract to enzyme hydrolysis, resulting in a protein hydrolysate.

13. Process according to any of claims 9-12-, in which said enzyme hydrolysis is performed in the presence of proteases extracted from thistle or safflower heads.

14. Use of the protein hydrolysate according to any of claims 1-8 as a plant biostimulant.

15. Plant biostimulant comprising the protein hydrolysate according to one or more of claims 1-8.