Microbiological process for the production of a fermented extract of the olive mill wastewater

Abstract

The present invention relates to a biotechnological process for the production of a OMWW fermented extract comprising the following steps: a) providing and mixing OMWW and microalgae Chlorella, thus obtaining a starting material; b) inoculating the starting material with a lactic acid bacterium and / or a yeast, and c) fermenting the starting material. The fermented OMWW extract obtained has the nutritional and functional profiles that enrich the nutritional characteristics of the product contained it, for example wheat bread, introducing notable biological and antioxidant compounds. Simultaneously, the products of the invention exhibited enhanced antifungal properties and extended shelf life especially.

Description

[0001] P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0002] "MICROBIOLOGICAL PROCESS FOR THE PRODUCTION OF A FERMENTED EXTRACT OF THE OLIVE MILL WASTEWATER (OMWW)"

[0003] ** ** ** ** **

[0004] DESCRIPTION

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to a fermented extract of the olive mill wastewater (OMWW) and a microbiological process for obtaining the same.

[0007] The present invention originates in the field of biotechnological processes for the production of nutritional, dietary or food products.

[0008] In particular, the present invention relates to a biotechnological process for the production of a fermented extract of the olive mill wastewater having a high content of poliphenolic compounds.

[0009] PRIOR ART DISCUSSION

[0010] The request for sustainable food production and waste valorisation have become a prominent priority in the modem era. As the global population continues to grow, the concerns surrounding food security, resource scarcity, and environmental degradation necessitate innovative solutions that maximize the potential of agricultural wastes (Barral-Martinez, M.; Fraga-Corral, M.; Garcia-Perez, P.; Simal- Gandara, J.; Prieto, M.A. Almond By-Products: Valorization for Sustainability and Competitiveness of the Industry. Foods 2021 ,10, 1793).

[0011] Among these underutilized resources, olive-mill wastewater (OMWW), a by-product generated during olive oil extraction, holds immense promise due to its abundant availability and rich bioactive content (Zahi, M. R., Zam, W., & El Hattab, M. (2022), State of knowledge on chemical, biological and nutritional properties of olive mill wastewater. Food Chemistry, 132238.).

[0012] OMWW is inherently enriched with phenolic compounds which are renowned for their antioxidants and potential health benefits (Delgado, A. M., Issaoui, M., & Chammem, N. (2019), Analysis of main and healthy phenolic compounds in foods. Journal of AOAC International, 102(5), 1356-1364). The scientific community has actively advocated for the utilization of solvent extraction techniques, selective resins, membrane filtration, or enzymatic applications to extract these compounds, intended for nutraceutical and food applications (Foti, P., Occhipinti, P. S., Russo, P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0013] N., Scilimati, A., Miciaccia, M., Caggia, C., ... & Romeo, F. V. (2023), Olive mill wastewater fermented with microbial pools as a new potential functional beverage. Molecules, 28(2), 646). From a biotechnological point of view, the inhibitory and phytotoxic activity of phenols, low pH, and high salinity create difficult conditions for the growth and metabolic activity of microorganisms involved in fermentation (Yalgmkaya, S., & Kilig, G. B. (2019). Isolation, identification and determination of technological properties of the halophilic lactic acid bacteria isolated from table olives; Journal of food science and technology, 56(4), 2027-2037). Consequently, the potential of microbial activity in the valorization of OMWW remains constrained (Foti, P., Occhipinti, P. S., Russo, N., Scilimati, A., Miciaccia, M., Caggia, C., ... & Romeo, F. V. (2023). Olive mill wastewater fermented with microbial pools as a new potential functional beverage. Molecules, 28(2), 646).

[0014] It is still felt the need hence of a valorised use of the OMWW.

[0015] The main object of the present invention is hence to recycle the OMWW and to provide products capable to exploit the high contents of the nutritional and effective elements and substances present in OMWW.

[0016] SUMMARY OF THE INVENTION

[0017] The inventors surprisingly found out a microbiological fermentation process and starting materials that allowed to obtain a fermented extract of the olive mill wastewater (OMWW) showing a synergy of diverse components.

[0018] The microbiological fermented process of the invention starting from specific inventive starting materials allowed to overcome the inhibitory effects of OMWW, especially when Lactobacilli or yeasts were used to perform the fermentation.

[0019] Therefore, in the first aspect the invention relates to a microbiological fermentation process for the production of a OMWW (olive mill wastewater) fermented extract comprising the following steps: a) providing and mixing OMWW and microalgae Chlorella, thus obtaining a starting material; b) inoculating the starting material with a lactic acid bacterium, a yeast or a mixture thereof, and c) fermenting the starting material.

[0020] The inventors surprisingly found out that the addition of the microalgae Chlorella , P024061WG-01 Notarbartolo & Gervasi S.p.A. preferably Chlorella vulgaris, as crucial starting material not only avoid the inhibitory effects of the OMWW, but also was capable to thrive the OMWW thus generating effective microbial metabolites as it will be evident from the experimental part.

[0021] Without being bound to any theory the inventors deem that the interaction between the proteins of the microalgae Chlorella, preferably Chlorella vulgaris, and the polyphenols of the starting material OMWW generate complexes, that yeasts and lactobacilli, with their key enzymes, can break down generating smaller peptides, free aminoacids and polyphenols, thus enhancing their bioavailability and bioactivity. According to the inventors, in fact, the glycosidases were capable to break the bond between polyphenols and sugar molecules in glycosides, releasing free polyphenols characterizing the final fermented extracted so obtained.

[0022] For the scope of this invention, for Chlorella is meant any suitable species belonging to the genus Chlorella.

[0023] In a preferable version of the embodiment Chlorella is Chlorella vulgaris.

[0024] Therefore, in a further aspect the invention relates to a fermented OMWW extract obtainable by the microbiological process according to the invention.

[0025] Advantageously the lactic acid bacillus and / or the yeast of step b) can be one or more yeasts and / or one or more lactic acid bacteria (LAB) strains isolated from fresh apples, apple by-products, and fermented vegetables (sauerkraut).

[0026] In a preferred and advantageous embodiment, the yeast is selected from Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ.

[0027] In a further preferred and advantageous embodiment the lactic acid bacterium is a species selected from Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ, Lacticaseibacillus paracasei PF6 deposited with accession number DSM 19463 on MRS at the DSMZ, and Lactiplantibacillus plantarum POM1 deposited with accession number DSM 34000 on MRS at the DSMZ.

[0028] The inventors have hence found out that the above reported new lactic acid bacteria P024061WO-01 Notarbartolo & Gervasi S.p.A. and yeast strains and mixtures thereof were particularly suitable as inoculating material of the process of the invention.

[0029] In another aspect the invention relates to a strain of a yeast selected from the group consisting of Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ.

[0030] In a still another aspect the invention relates to a lactobacillus strain that is Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ.

[0031] The inventors have hence found out that the above reported new Lactobacilli and yeast strains and mixtures thereof were particularly suitable as inoculating material of the process of the invention.

[0032] The fermented extracts of the invention showed the following concentrations of total polyphenols (23133-27861 pg / L), total amino acids (294-1577 mg / L), whose ranges were considered among the strains used.

[0033] Preferably and as it will be clear from the experimental part, among the polyphenol hydroxytyrosol emerged as the predominant phenolic compound in Raw OMVWV- CHL (14793.18 ± 47.27 pg L’1), followed by tyrosol (2044.17 ± 7.83 pg L’1), p- coumaric acid (917.49 ± 56.98 pg L’1), ferulic acid (430.435 ± 9.93 pg L’1), and caffeic acid (284.205 ± 2.98 pg L’1).

[0034] The fermented OMWW extract of the invention showed also a high antifungal activity.

[0035] In view of the distinct nutritional and functional profiles of the final extract of the OMWW as it will be clear from the experimental part, the fermented OMWW extract is used for the preparation of supplements, food and nutritional products.

[0036] The invention hence relates to a composition comprising the fermented OMWW extract and an edible carrier.

[0037] In a preferred and advantageous aspect, the fermented OMWW extract is suitable for preparing wheat bread.

[0038] The fermented OMWW extract, or a product contained it, can be used for a cosmetic P024061WO-01 Notarbartolo & Gervasi S.p.A. use in the treatment of cutaneous aging, of skin and of hair.

[0039] As used herein, “hair” means scalp, head, facial and / or body hair, including the scalp, eye lashes, browns, mustache, beard, ear, nasal, chest, pubic and the like.

[0040] As used herein “edible carrier” means one or more solid or liquid compounds that are suitable for preparing edible product

[0041] As used herein “functional food” means any modified food or food ingredient that can provide a benefit or protection against a problem or a physiological condition, in addition to the traditional nutrients that are contained in it.

[0042] As used herein “nutraceutical product” means a product isolated or purified from edible substances. A nutraceutical is such when it is shown that it has a physiological benefit or that it provides protection against a problem or physiological disorder.

[0043] As used herein “dietary or food supplement” means a product that contains a vitamin, mineral, plant extract, amino acid, metabolite, extract, concentrate or mixtures of these ingredients.

[0044] In a still another aspect the invention relates to the fermented OMWW extract or a product contained it for use in the prevention and / or treatment of a skin disorders resulting from DNA damage produced by UV radiation.

[0045] Advantageously, the nutritional and functional profiles of the extract of the invention enrich the nutritional characteristics of the product contained it, for example wheat bread, introducing notable biological and antioxidant compounds preferably hydroxytyrosol, hydrocaffeic acid, luteolin, rutin, and tyrosol. The products of the invention exhibited enhanced antifungal properties and extended shelf life especially. This dual enhancement, combined with favorable textural and flavor attributes, positioned these products as a promising advancement in sustainable food production, waste management, and the improvement of dietary quality.

[0046] BRIEF DESCRIPTION OF THE FIGURES

[0047] The features and advantages of the present invention will be evident from the accompanying drawings, wherein:

[0048] - Figure 1 shows the concentration of glucose (g L’1), fructose (g L’1), lactic acid (g

[0049] L’1), acetic acid (g L ), and ethanol (g L’1) in Raw-, Unstarted- and Started- Chlorella vulgaris (CHL), Olive Mill Wastewater (OMWW), and their mixture (OMWW-CHL), P024061WO-01 Notarbartolo & Gervasi S.p.A. according to Example 2. Fermentation was carried out at 30°C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16, Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6 and Lactiplantibacillus plantarum P0M1. Different letters indicate significant differences (P < 0.05).

[0050] - Figure 2 shows the total proteins (%) and total peptides (%) in Raw-, Unstarted- and Started-Chlorella vulgaris (CHL), Olive Mill Wastewater (OMWW), and their mixture (OMWW-CHL) according to Example 3. Fermentation was carried out at 30 °C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16, Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6, and Lactiplantibacillus plantarum POM1 . Different letters indicate significant differences (P < 0.05).

[0051] - Figure 3 shows hyphal radial growth inhibition against Penicillium roqueforti DPPMAF1 , Penicillium paneum CBS101032, Aspergillus versicolor CBS117286, and Eurotium herbariorum CBS 117336 of Raw-, Unstarted- and Started-Chlorella vulgaris (CHL), Olive Mill Wastewater (OMWW), and their mixture (OMWW-CHL), which were incubated at 30 °C for 48 h according to the test of Example 6. Fermentation (Started sample) was carried out with single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, and Saccharomyces cerevisiae AY16, Lacticaseibacillus paracasei PF6, Leuconostoc mesenteroides GSL1 , and Lactiplantibacillus plantarum POM1. The percentage of growth inhibition was calculated from mean values as follows: Percentage of inhibition = [(mycelial growth under control conditions - mycelial growth in the presence of samples) / mycelial growth under control conditions] x 100.

[0052] These results showed the antifungal activity and the high nutritional and functional profiles of fermented OMWW extract obtained with the process of the invention.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] According to a general aspect of the invention, a microbiological process is provided for the production of a fermented OMWW extract.

[0055] According to a general aspect, the invention relates a microbiological fermentation process for the production of a OMWW fermented extract comprising the following steps: P024061WO-01 Notarbartolo & Gervasi S.p.A. a) providing and mixing OMWW and microalgae Chlorella, thus obtaining a starting material; b) inoculating the starting material with a lactic acid bacterium, a yeast or a mixture thereof, and c) fermenting the starting material.

[0056] The first step a) of the microbiological process of the invention relates to the provision and the mixture of OMWW and microalgae Chlorella.

[0057] The OMWW were preferably filtered before the mixing with Chlorella of step a).

[0058] For the scope of this invention, for Chlorella is meant any suitable species belonging to the genus Chlorella.

[0059] For Chlorella biomass is meant the product resulting from cultivation (reproduction) of the unicellular algae (microalgae) Chlorella spp.

[0060] Reproduction phase can be carried out in open systems, such as raceways and ponds or closed systems, such as photobioreactors and fermenters, both under specific environmental conditions (light, salinity, pH nutrients, temperature etc.) that could be changed for influencing the growth rate and the composition / nutritive profile of the biomass and the resulting products. Closed systems offer better control over the culture conditions and minimize contamination. Through specific conditions, darkness for example, is possible to reduce the content of chlorophyll for obtaining less greenish (pale yellow and off white) Chlorella products. Those products show better taste and are generally more suitable for food applications.

[0061] For Chlorella powder is meant the product resulting from the drying of Chlorella biomass by any technique (evaporation, spry drying, freeze drying etc.).

[0062] For Chlorella extract is meant the output obtained by the treatment of Chlorella biomass or Chlorella powder with a suitable solvent.

[0063] Suitable solvents are water, ethanol, glycerine and their mixtures, but for the purpose of the inventions is meant any suitable solvent capable of dissolving a specific fraction or component from the biomass or the powder with the purpose of concentrating it.

[0064] In a preferable version of the embodiment the microalgae Chlorella is Chlorella vulgaris.

[0065] Other useful Chlorella species that cane be used in the present invention are C. P024061WO-01 Notarbartolo & Gervasi S.p.A. pyrenoidosa', C. zofingiensis', C. stigmatophora, C. autotrophica, but not limited to these.

[0066] Products resulting from the harvesting of Chlorella biomass could be treated for destroying the cell-wall by many ways. This additional treatment is done for improving bioavailability or water solubility of the constituents especially when Chlorella is intended to be used for nutritional purpose, since humans cannot digest algae cell-wall in their natural state. Therefore, Chlorella cell walls are broken down in most dietary supplements.

[0067] Chlorella microalgae, preferably Chlorella vulgaris microalgae, is the crucial active ingredient of the process of the invention.

[0068] The species Chlorella vulgaris microalgae was studied and showed a higher total free amino acid concentration (636.57 ± 41.21 mg L-1) compared to the OMWW (103.79 ± 5.5 mg L’1), and the amino acid compositions were notably distinct as shown in Table 1 . Table 1. Quantification of the total (TFAA) and individual free amino acids (mg L-1) of Chlorella vulgaris suspension (CHL) and Olive mill wastewater (OMWW).

[0069] Amino acids CHL OMWW

[0070] Asp 52.2 ± 0.46 40.00 ± 2.24

[0071] Thr 21.56 ± 0.42 0.00 ± 0.00

[0072] Ser 23.66 ± 1.73 1.30 ± 0.08

[0073] Glu 233.86 ± 17.82 39.84 ± 0.35

[0074] Gly 32.00 ± 2.52 0.00 ± 0.00

[0075] Ala 151 .49 ± 8.48 12.63 ± 3.4

[0076] Vai 18.73 ± 1.02 4.73 ± 0.81

[0077] Cys 0.08 ± 0.00 0.09 ± 0.05

[0078] Met 3.71 ± 0.17 0.00 ± 0.00 lie 6.82 ± 0.03 2.53 ± 0.06

[0079] Leu 15.85 ± 1.58 0.76 ± 0.11

[0080] Tyr 9.33 ± 1.60 0.00 ± 0.00

[0081] Phe 11.25 ± 2.62 0.00 ± 0.00

[0082] Lys 24.99 ± 3.05 0.00 ± 0.00 P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0083] His 5.8 ± 0.27 0.00 ± 0.00

[0084] Arg 18.48 ± 0.31 1.91 ± 0.17

[0085] Pro 6.77 ± 0.28 0.00 ± 0.00

[0086] Total 636.57 ± 41 .21 103.79 ± 5.5

[0087] The mixing step a) is preferably carried out as follows: OMWW resulting from the olive oil processing is collected and frozen at -20°C. After few days, frozen OMWW is transferred to the fermentation plant. At the fermentation plant, OMWW is thawed and then centrifugated (more preferably on lab scale 12,000 rpm for 10 min) for removing the suspended solids. Then, the supernatant (further on called olive waste substrate) is separated and sterilized (more preferably 121 °C for 15 min on lab scale) for degrading particles and facilitating the subsequent filtration step. The olive waste substrate is then filtered several times with decreasing pores-sized filters reaching preferably 0.22 pm (Millipore Corporation, Bedford, MA, USA on lab scale). In an advantageous and preferred embodiment to 95 parts (w / w) of the filtered olive waste substrate is added 5 parts of C. vulgaris. The mixing step a) is carried out preferably at room temperature. Then, the mixture is more preferably sterilized at 121 °C for 15 min (on lab scale) and cooled before use.

[0088] The two main ingredients of the step a) were hence characterized by a specific qualification and quantification of the amino acids and together constitute the starting material to be used for step b).

[0089] The Chlorella microalgae, preferably Chlorella vulgaris, is added to OMWW preferably in an amount from 3% to 7% with respect to the total amount of the starting material. According to a preferred embodiment, the addition of Chlorella microalgae, preferably Chlorella vulgaris, to the OMWW substrate occurred in an amount of 5% with respect to the total amount of the starting material.

[0090] This advantageously led to pH increase in the mixed substrate OMWW-CHL, which was the preferred starting material for the inoculation step b) and fermentation of step c).

[0091] Step b) provides for inoculating the starting material with a lactic acid bacterium and / or a yeast.

[0092] Advantageously the lactic acid bacillus and / or the yeast of step b) can be one or P024061WO-01 Notarbartolo & Gervasi S.p.A. more yeasts and / or one or more lactic acid bacteria (LAB) strains isolated from fresh apples, apple by-products, and fermented vegetables (sauerkraut).

[0093] In accordance with preferred embodiments, the microbiological process requires selected microbial strains. An array of yeasts was isolated from OMWW, preferably M. caribbica and L fermentati, identified as starters due to their indigenous nature, potentially affording heightened performance compared to non-indigenous strains (Table 2).

[0094] Table 2. Selected yeast and Lactobacilli strains.

[0095] Type Species Code Sources

[0096] YEAST Saccharomyces cerevisiae AYI6 Apple by-product

[0097] YEAST Meyerozyma caribbica 5Y OMWW

[0098] YEAST Lachancea fermentati 9Y OMWW

[0099] Percorino

[0100] LAB Lacticaseibacillus paracasei PF6 cheese

[0101] LAB Leuconostoc mesenteroides GSL1 Apple

[0102] LAB Lactoplantibacillus plantarum POM1 Apple

[0103] In a preferred and advantageous embodiment, the yeast is selected from Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ.

[0104] In a further preferred and advantageous embodiment the lactic acid bacterium is a species selected from Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ, Lacticaseibacillus paracasei PF6 deposited with accession number DSM 19463 on MRS at the DSMZ, and Lactiplantibacillus plantarum P0M1 deposited with accession number DSM 34000 on MRS at the DSMZ.

[0105] Regarding the inoculum step, a different cell density was considered depending by the LAB or yeast inoculum. In particular, all the three LAB were prepared as cell suspension (ca. 9.0 Log CFU mL’1) and when inoculated in the mixture they P024061WO-01 Notarbartolo & Gervasi S.p.A. achieved a final cell density of ca. 7 Log CFU mL’1, while yeasts were prepared as cell suspension (ca. 7.0 Log CFU mL-1) and when inoculated in the mixture they reached a final cell density of ca. 5 Log CFU mL-1.

[0106] The inventors have hence found out that the above reported new Lactobacilli and yeast strains and mixtures thereof were particularly suitable as inoculating material of the process of the invention.

[0107] In another aspect the invention relates to a strain of a yeast selected from the group consisting of Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ

[0108] In a still another aspect the invention relates to a lactobacillus strain that is Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ,.

[0109] The step c) provides for the fermentation of the starting material. The fermentation is preferably carried at a temperature in the range from 25 to 35°C. The fermentation step was preferably performed for a time range from 36 to 52 hours.

[0110] The modification of the OMWW with Chlorella microalgae, preferably Chlorella vulgaris, affected positively the growth of microorganisms as it will be also clear from the experimental part.

[0111] The process of the invention effectively and surprisingly allowed the obtainment of the extract owing to the starting material, the OMWW-CHL, preferably with 5% of CHL, that was associated with an elevated rate of sugar consumption, leading to the consequential synthesis of microbial metabolites, and presenting a notable contrast to single chlorella or OMWW (Figure 1 ).

[0112] Glucose and fructose underwent predominant conversion by yeasts to ethanol, with S. cerevisiae and L fermentati displaying superior performance compared to M. caribbica. The notable emergence of mannitol synthesis was observed with L fermentati. The pattern of metabolites produced by Lactic acid bacilli was closely linked to the type of the substrate and the process of the invention showed completely different results of the starting material of the invention with respect to P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0113] Chlorella Vulgaris microalgae that was subjected to a solitary fermentation.

[0114] The preferred microbial metabolites of L plantarum and L paracasei were lactic acid and traces of acetic acid, whereas Leuc. mesenteroides, apart from lactic acid, followed additional metabolic strategy employing fructose as an alternative extracellular electron acceptor to produce mannitol, acetic acid, and ethanol.

[0115] According to preferred embodiments, the hydrolysis of protein was species and substrate dependent. When yeasts were only able to considerably decrease protein level in mixed substrate, Lactic acid bacilli, particularly L paracasei, demonstrated stronger proteolytic activity in both chlorella and mixed OMWW-CHL (Figure 2). While the alterations observed in peptide modification did not consistently mirror protein changes, they confirmed the high proteolytic activity of L paracasei. Furthermore, they revealed the presence of supplementary enzymatic activities capable of peptide-to-amino acid transformations, notably observed in S. cerevisiae and L plantarum. Indeed, with the exception of L. paracasei, all microorganisms exhibited substantial reductions in amino acid content, particularly S. cerevisiae and L. plantarum (Table 3).

[0116] P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0117] Table 3. Total and individual free amino acids (mg L’1) of Raw- Unstarted- and Started- mixture of Chlorella vulgaris and Olive Mill wastewater (OMWW-CHL). Fermentation was carried out at 30 °C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16,

[0118] 5 Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6,and Lactiplantibacillus plantarum POM1 . Different letters indicate significant differences (P < 0.05). o P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0119] Table 3 (continued)

[0120] Surprisingly and preferably the strains above cited according to a preferred embodiment were able to counteract the hostile conditions caused by the OMWW 5 and so allow the fermentation step c).

[0121] Indeed, the strains as starters utilized amino acids as substrate for energy generation, maintenance of cellular redox equilibrium, and biosynthetic processes. The fermented extracts of the invention showed the following concentrations of total polyphenols (23133-27861 pg / L), total amino acids (294-1577 mg / L), whose ranges io were considered among the strains used.

[0122] Preferably and as it will be clear from the experimental part, among the polyphenol hydroxytyrosol emerged as the predominant phenolic compound in Raw OMVWV- CHL (14793.18 ± 47.27 pg L’1), followed by tyrosol (2044.17 ± 7.83 pg L’1), p- coumaric acid (917.49 ± 56.98 pg L’1), ferulic acid (430.435 ± 9.93 pg L’1), and 15 caffeic acid (284.205 ± 2.98 pg L’1) P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0123] In some preferred embodiments, the substantial release of ferulic and caffeic acids in mixed OMWW-CHL substrates proved catalytic reactions that break specific bonds, while luteolin reduction implied formation of a stable protein-phenolic complex (Table 4). Table 4. Quantification of phenolic compounds (pg L’1) by LC-ESI-MS / MS in methanol / water soluble extract (MW-SE) obtained from Raw-, Unstarted- and Started- mixture of Chlorella vulgaris and Olive Mill Wastewater (OMWW-CHL). Fermentation was carried out at 30°C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16, Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6, and Lactiplantibacillus plantarum POM1 . Different letters indicate significant differences

[0124] (P < 0.05). P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0125] Table 4. continued a’bdifferent letters indicate significant differences (P < 0.05).

[0126] Fermentation significantly affected phenolic profiles, with different microorganisms yielding varied outcomes, but the total amounts of the phonolic substances were above 23000 pg / L. Specifically, in one embodiment, L plantarum exhibited a distinctive capability to metabolize ferulic and caffeic acids, potentially attributed to the presence of carboxylase and reductase enzymes, confirmed by the production of dihydrocaffeic acid upon caffeic acid metabolism. p-Coumaric acid was also metabolized by S. cerevisiae AYI6 and Leuc. mesenteroides GSL1 in others embodiments. This metabolic path typically yields phenolic acid derivatives with enhanced functional and biological attributes compared to their original precursors. L paracasei induced p-coumaric acid production through esterase-mediated ester bond cleavage. Esterase activity in L plantarum accounted for the hydrolysis of phenolic acid esters, liberating chlorogenic and caftaric acids. High tannase activity in L plantarum, L paracasei, and S. cerevisiae led to increased gallic acid content through tannin hydrolysis. Yeasts and LAB possess distinct metabolic pathways enabling them to utilize and modify 4-hydroxybenzaldehyde, yielding derivatives P024061WG-01 Notarbartolo & Gervasi S.p.A. such as 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol, along with glycosides and amino acid conjugates.

[0127] According to some embodiments, the synergistic action between OMWW and Chlorella microalgae, preferably Chlorella vulgaris during the fermentation step c) triggers surprisingly the synthesis of bioactive molecules, organic acids, and other secondary metabolites.

[0128] The fermented OMWW extract of the invention showed also a high antifungal activity.

[0129] In view of the molecule characterization profile of the extract of the invention inherent antifungal properties have been showed (Figure 3). Individually, OMWW and Chlorella exhibited varying degrees of inhibition against all fungal indicators. The antifungal potential against P. roqueforti and E. herbariorum was imposed by the combined use of OMWW and Chlorella, but fermentation subsequently reinstated and enhanced this antifungal activity. M. carribica 5Y and L fermentati 9Y exerted substantial inhibitory potential against multiple indicator fungi. S. cerevisiae AYI6 showed amplified antifungal effects in the mixed OMWW-CHL substrate against E. herbariorum. L paracasei PF6 and Leuc. mesenteroides GSL1 exhibited antifungal activity against P. paneum and A. versicolor, while L plantarum POM1 specifically against A. versicolor.

[0130] In view of the distinct nutritional and functional profiles the fermented OMWW extract is used for the preparation of supplements, food and nutritional products.

[0131] The invention hence relates to a composition comprising the fermented OMWW extract and an edible carrier.

[0132] In a preferred and advantageous aspect, the fermented OMWW extract is suitable for preparing wheat bread.

[0133] The compositions described herein are preferably suitable for oral administration.

[0134] The compositions for oral administration may be in solid or liquid form.

[0135] For being properly used in solid dosage forms the aqueous medium has to be processed with the purpose of obtaining a dry powder, with a low water content. The water removal could be done by all the techniques available such as spray drying, freeze drying, convection and vacuum oven drying. For stabilizing the powder, suitable carriers could be added, before or after the water removal process, with the P024061WO-01 Notarbartolo & Gervasi S.p.A. purpose of improving the technology characteristics of the powder such as flowability, moisture absorption and for reducing agglomeration. Silica and maltodextrin are the most used excipients, but the present invention can rely on every ingredient which use is allowed in the field where the invention is intended of being applied. Loss on drying should be lower than 5%.

[0136] In the case of the solid form, they contain the fermented OMWW obtained from the process described herein as a biologically active component and one or more physiologically acceptable excipients.

[0137] Typical solid form compositions comprise tablets, granules, capsules, powders, extemporaneous dosage forms, candies, chewing gums, jellybeans of different composition and shape.

[0138] The present invention applies to any solid, semi-solid and liquid dosage form which can become an edible carrier for administering the invention.

[0139] The present invention applies to all dosage forms in which the preparation is diluted before administration or used as such, being in solid, semi-solid or liquid form.

[0140] The tablets generally comprise a suitable carrier.

[0141] In this case, suitable excipients contained in the formulation are cellulose derivatives such as the purified and partially depolymerized cellulose such as microcrystalline cellulose or other derivatives such as hydroxymethyl cellulose, hydroxypropylmethyl cellulose (hypromellose), methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and it sodium and calcium salts, ethyl hydroxyethylcellulose, , and mixtures thereof. Other suitable excipients are starch and modified starch.

[0142] Further examples of suitable excipients comprise polymers belonging to the lactam family such as pyrrolidone and derivatives thereof, for example polyvinylpyrrolidone, polyvinylpolypyrrolidone and mixtures thereof, inorganic salts such as calcium or dicalcium phosphate, calcium carbonate, magnesium carbonate, glidants such as precipitated and fumed silica, magnesium oxide, lubricants such as magnesium stearate, triacylglycerols and mixtures thereof.

[0143] Tablets could be left uncoated as they result from tableting or coated using suitable excipient mixtures. For this purpose, film formers such as cellulose derivatives; plasticizers such as polyethylene glycols, stearic acid, glycerol, triethyl citrate; fillers P024061WO-01 Notarbartolo & Gervasi S.p.A. such as microcrystalline cellulose and colorants, from natural or synthetic sources are extensively used.

[0144] In the case of the liquid form, the composition contains the liquid fraction separated at the end of the step b) of fermentation as a biologically active component and one or more physiologically acceptable excipients.

[0145] Typical compositions in liquid form comprise solutions, emulsions, suspensions, syrups.

[0146] The fermented OMWW extract obtained with the process of the invention contained in the composition of the invention may be present in variable amounts, preferably comprised in the range from 0.0001 % to 100% by weight; more preferably from 0.001 % to 50% by weight, still more preferably from 0.1 % by weight to 20% by weight, typically from 0.5 to 5% by weight.

[0147] According to certain embodiments, the composition of the invention further comprises one or more active substances such as vitamins, minerals, micronutrients and other active substances.

[0148] According to some embodiments, the composition for oral administration is a functional food, a nutraceutical composition, a dietary product, a supplement or a nutritional product.

[0149] The composition may be marketed in conventional pharmaceutical containers such as blisters filled with soft / hard capsules or tablets; in some embodiments hard or soft capsules contain a solid dispersed in a food grade oil or other compatible liquid; sachets of thermo-welded aluminium of type sachet or stick pack containing a solid component or a powdery or granulated mixture or a dispersion of the solid component in a suitable liquid; containers for the extemporaneous reconstitution of a solid in a liquid, vials or bottles containing the dispersion of a solid in a liquid.

[0150] Other useful containers are flexible packaging like those known as stand-up pouch or “Doypack” generally made using multi-layers materials such as PE / EVOH / PE.

[0151] The fermented OMWW extract, or a product contained it can be used for a cosmetic use in the treatment of cutaneous aging, of skin, of hair.

[0152] In a still another aspect the invention relates to the fermented OMWW extract or a product contained it for use in the prevention and / or treatment of a skin disorders resulting from DNA damage produced by UV radiation. P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0153] According to other aspects, the present invention provides the use of the fermented OMWW extract or of the product contained for treatment by oral intake or cosmetic use of the skin in particular as anti-aging treatment or to reduce skin aging, for hair treatment.

[0154] The fermented OMWW extract obtained with the process of the invention has cellular antioxidant activity and is used in preventing or treating diseases that give rise to a non-physiological production of free radicals, typically skin diseases such as tumours, precancerous or actinic diseases or redness states such as consequent to sunburns, skin blemishes, roughness or skin aging. Furthermore, the fermented OMWW extract of the invention finds application in maintaining an individual's organism in physiological health conditions.

[0155] EXPERIMENTAL PART

[0156] Example 1 - Fermentation process

[0157] Methods: OMWW was thawed and subjected to centrifugation (12,000 rpm for 10 min) to remove suspended solids. Afterward, the collected supernatant was sterilized at 121 °C for 15 min even to degrade particles and facilitating the subsequent filtration step. Finally, the olive waste substrate was filtered several times with decreasing pores-sized filters reaching 0.22 pm (Millipore Corporation, Bedford, MA, USA). Subsequently, three distinct substrates were formulated: (i) suspension of 5% C. vulgaris in water (CHL); (ii) 95% filtered OMWW and 5% water (OMWW); (iii) mixture of 95% filtered OMWW and 5% C. vulgaris (OMWW-CHL). Yeasts and lactic acid bacteria were inoculated at an initial cell density of ca. 5 and ca. 7 Log CFU mL-1, respectively. The following microorganisms were inoculated: L paracasei PF6, L plantarum POM1 (9.10 ± 0.02 Log CFU mL-1 ) and Leuc. mesenteroides GSL1. The yeasts were Saccharomyces cerevisiae AYI6, Meyerozyma caribbica 5Y and Lachancea fermentati 9Y.

[0158] Fermentation was carried out at 30 °C for 48 h. Samples of microalgae and OMWW were taken before (Raw-CHL, -OMWW, and -OMWW-CHL) and after fermentation. Samples without microbial inoculum were incubated under the same condition (Unstarted-CHL, -OMWW, and -OMWW-CHL). Yeasts and LAB were enumerated by plate count Malt Extract Agar (MEA) (Oxoid) with 0.1 % of chloramphenicol, and on De Man, Rogosa and Sharpe (MRS) agar (Oxoid) containing 0.1 % of P024061WO-01 Notarbartolo & Gervasi S.p.A. cycloheximide (Sigma Chemical Co.). All plates were incubated at 30°C for 48 h. The values of pH were measured by a Foodtrode electrode (Hamilton).

[0159] Results: Due to thermal treatment, no microbial growth was observed in Raw and Unstarted samples, regardless of the substrates used. Starting from initial inoculum of ca. 5 Log CFU mL’1, all three yeasts showed almost the same capability to grow in different substrates, with slight preferences observed for the mixed substrate OMWW-CHL, resulting in cell densities ranging from 7.18 ± 0.09 to 7.62 ± 0.34. Among LAB, all three strains were able to grow and adapt in all substrates expect the one containing only OMWW, where they showed final cell densities lower than their initial inoculum size (ca. 6 Log CFU mL’1). Compared to substrate containing only C. vulgaris (CHL), in mixed substrate OMWW-CHL, a higher cell count was found for L paracasei PF6 (9.23 ± 0.19 Log CFU mL’1) and L plantarum POM1 (9.10 ± 0.02 Log CFU mL’1). Conversely, Leuc. mesenteroides GSL1 had an opposite preference, showing higher adaptability when it was cultivated in CHL (7.77 ± 0.25 Log CFU mL’1) compared to mixed substrate (7.17 ± 0.25 Log CFU mL’1).

[0160] The initial pH of Raw-CHL substrate was 6.13 ± 0.01. Upon its lactic fermentation a slight but not significant (P > 0.05) reduction was found among the starters, except for L plantarum PR1 (5.18 ± 0.06). Likewise, in the OMWW whose pH was 5.23 ± 0.02, none of the three LAB strains showed any appreciable acidification. When OMWW and C. vulgaris were combined (OMWW-CHL), the acidifying capacity of LAB significantly (P < 0.05) increased. In comparison to the raw substrate of pH 5.5 ± 0.05, L plantarum PR1 (3.82 ± 0.01 ) showed the lowest significant (P < 0.05) pH (3.75 ± 0.01 ), followed by and Leuc. mesenteroides GLS1 (4.6 ± 0.22).

[0161] Example 2 - Content of sugars, organic acids, and ethanol

[0162] Methods: Perchloric acid (5%, v w1) was added to Raw, Unstarted and fermented samples of Chlorella vulgaris (CHL), Olive Mill Wastewater (OMWW), and their mixture (OMWW-CHL), as precipitating agent. Fermentation was carried out at 30°C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16, Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6, and Lactiplantibacillus plantarum POM1. The resulting water-soluble extracts (PW-SE) were kept incubating at 4°C overnight, followed by centrifugation at 10,000 rpm for 10 min and filtration through a Millex- P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0163] HA filters with a pore size of 0.22 pm (Millipore Co.). Concentrations of glucose, fructose, mannitol, lactic acid, acetic acid, and ethanol were determined by HPLC (High-Performance Liquid Chromatography) system Ultimate 3000 (Dionex, Germering, Germany) equipped with an Aminex HPX-87H column (ion exclusion, Biorad Los Angeles, California), a Perkin Elmer 200a refractive index detector and UV detector operating at 210 nm. The elution was carried out at a temperature of 70°C, with a flow rate of 0.6 mL min-1, using H2SO4 5 mM as the mobile phase. Sugars, organic acids, and ethanol standards were purchased from Sigma-Aldrich (Steinheim, Germany).

[0164] Results: The HPLC analysis revealed glucose (8.20 ± 0.02 g L’1) and fructose (2.94 ± 0.02 g L’1) as the predominant sugars in OMWW, with comparatively lower concentrations in CHL (1.12 ± 0.01 g L-1and 0.85 ± 0.01 g L’1, respectively) (Figure 1 ). No significant (P > 0.05) differences in sugar consumption were found between the Raw and Unstarted samples among the three substrates. In OMWW, all yeast and LAB strains significantly (P < 0.05) reduced glucose and fructose, with Lachancea fermentati 9Y showing the highest reduction. The same strains showed significant (P < 0.05) sugar reduction in CHL and mixed OMWW-CHL substrate, except for L paracasei PF6, which did not significantly decrease glucose in CHL. The most notable reduction occurred in OMWW-CHL, where S. cerevisiae AYI6 and Lachancea fermentati 9Y almost completely metabolized glucose, and these strains, along with Leuc. mesenteroides GSL1 and L paracasei PF6, similarly caused a substantial fructose consumption. Mannitol was found only in OMWW and mixed OMWW-CHL when Leuc. mesenteroides GSL1 and L fermentati 9Y were inoculated. The main microbial metabolites quantified in the three substrates were lactic acid, acetic acid and ethanol. While L plantarum PR1 accumulated a small amount of lactic acid in OMWW, L paracasei PF6 and Leuc. mesenteroides GSL1 showed contrasting patterns in the other two substrates. Leuc. mesenteroides GSL1 caused the highest lactic acid concentration in CHL (1 .46 ± 0.01 g L-1) and the lowest in OMWW-CHL (7.19 ± 0.93 g L’1), whereas L. paracasei PF6 showed an opposite trend. After the lactic fermentation, a slight but significant increase in acetic acid was found especially in CHL and OMWW-CHL. Ethanol by yeasts was found in all substrates, with S. cerevisiae AYI6 exhibiting the highest production in CHL followed P024061WO-01 Notarbartolo & Gervasi S.p.A. by Lachancea fermentati 9Y in OMWW and consequently by both strains in OMWW- CHL. Among LAB starters, only Leuc. mesenteroides GSL1 had a limited capacity to produce ethanol.

[0165] Example 3 - Total protein and peptides determination

[0166] Methods: Total protein concentration was determined in Raw, Unstarted and fermented samples of Chlorella vulgaris (CHL), Olive Mill Wastewater (OMWW), and their mixture (OMWW-CHL) using Bradford assay. Fermentation was carried out at 30 °C for 48 h by single cultures of Meyerozyma caribbica 5Y, Lachancea fermentati 9Y, Saccharomyces cerevisiae AY16, Leuconostoc mesenteroides GSL1 , Lacticaseibacillus paracasei PF6, and Lactiplantibacillus plantarum POM1. Two mL of samples were centrifuged at 10,000 rpm for 10 min, and then filtered using a 0.45 pm filter (Millipore Corporation, Bedford, MA, USA). 96-well microplate reader (Tecan Infinite 200, Italy) was prepared by adding in each well 2.5 pL of extract, 197.5 pL of MilliQ water, and 50 pL of Bradford reagent. After 10 min, the absorbance was measured at 595 nm and protein quantification was calculated by constructing a calibration curve of Bovine Serum Albumin (BSA) (Sigma-Aldrich) as standard.

[0167] The total peptide concentration was determined in (PW-SE) extracts of all the samples using o-phthalaldehyde (OPA) method. Briefly, OPA reaction mix was prepared by combining the following reagents and diluting to a final volume of 100 mL with distilled water: 50 mL of 100 mM sodium tetraborate; 5 mL of 20% (w w1) sodium dodecyl sulfate; 540 pL of thiolactic acid, and 2.5 mL of OPA solution (50 mg mL-1OPA dissolved in ethanol 96%). In 96-well microplate reader, 730 pL of reaction mix was added together with 18.25 pL of the sample. After 2 min the absorbance was measured at 340 nm and peptide quantification was determined by the calibration curve of Tryptone (Biolife) as a reference.

[0168] Results: Protein hydrolysis and peptides release were monitored throughout the fermentation across the three substrates (Figure 2). The initial protein content in CHL and OMWW was estimated at 3.00 ± 0.34 and 2.85 ± 0.03%, respectively. In OMWW, minor variations were observed among the strains, where none of them caused significant changes when compared to the Raw and Unstarter control samples. On the contrary, when LAB were used, especially L paracasei PF6 (1.60 P024061WG-01 Notarbartolo & Gervasi S.p.A.

[0169] ± 0.17%), showed significant hydrolysis of protein in CHL substrate when compared to the two controls. When the two substrates were mixed (OMWW-CHL), the three yeasts also showed significant (P < 0.05) reduction, with L paracasei PF6 consistently achieving the highest proteolytic activity (1.90 ± 0.13%). By reviewing peptides, various trends were surfaced. No significant (P > 0.05) changes were observed between fermented and Raw- and Unstarted-OMWW. The only exception was when Meyerozyma caribbica 5Y was inoculated in CHL. Additionally, S. cerevisiae AYI6 and L plantarum POM1 in OMWW-CH. Since the mixture of C. vulgaris and OMWW resulted as the best substrate for the fermentation.

[0170] Example 4 - Total and individual free amino acids quantification

[0171] Methods: To determine the total and individual free amino acid (FAA) in Raw, Unstarted and fermented samples, a Biochrom 30 Amino acid Analyzer combined with a Li-cation exchange column was employed. First, 50 mg of sulfosalicylic acid was transferred into a 1 .5 mL tube and incubated at 4 °C for 1 h. After incubation, 1 mL of the samples were added into the tubes and the mixture was incubated again at 4 °C for 1 h. To recover the aminoacidic fraction, a centrifugation step (10,000 rpm for 10 min) followed by a filtration step through a 0.22 pm filter (Millipore Corporation, Bedford, MA, USA) was performed. The resulting samples were then injected into the Biochrom 30 Amino Acid Analyzer from Biochrom Ltd. (Cambridge Science Park, England).

[0172] Results: The CHL showed higher total free amino acid concentration (636.57 ± 41.21 mg L’1) compared to the OMWW (103.79 ± 5.5 mg L’1), and the amino acid compositions were notably distinct ( see Table 3 above). Compared to Raw OMWW- CHL, the incubation of OMWW-CHL without the microbial inoculum (Unstarted OMWW-CHL) did not have a significant (P > 0.05) effect on the amino acid profile and concentration (see Table 3 above). Conversely, after fermentation, Thr, Ser, lie, and Lys were significantly utilized by all microbial strains (see Table 3 above). Notably, L paracasei PF6 stood out as the only strain capable of significantly (P < 0.05) synthesizing the highest amounts of Asp, Gly, Ala, Cys, (Leu , Phe, Arg , and Pro). L plantarum POM1 completely utilized Vai, Phe, and His. Glu, being the most abundant amino acid (261.79 ± 1.33 mg L-1), remained relatively stable in most of the fermented samples, except when L plantarum POM1 and S. cerevisiae AYI6 P024061WQ-01 Notarbartolo & Gervasi S.p.A. were used which in turn caused noticeable (P < 0.05) reduction (ca. 60 and 70%, respectively). A similar trend was also found for Ala wherein S. cerevisiae AYI6 and L fermentati 9Y significantly (P < 0.05) reduced its content compared to Raw sample. Compared to Raw- (712.9 ± 7.9 mg L’1) and Unstarted- (712.38 ± 5.5 mg L’1) OMWW-CHL, total concentration of FAA decreased in almost started samples (30 - 50%), with S. cerevisiae AYI6 causing the highest reduction (ca. 80%). L paracasei PF6 showed slightly higher but not significant values for total free amino acids.

[0173] Example 5 - Phenolic compounds quantification

[0174] Methods: Phenolics analysis was conducted employing methanol water-soluble extracts (MW-SE). The extraction procedure involved blending 5 mL of samples with an equal volume of pure methanol, followed by prolonged stirring at room temperature for 24 h. stirring conditions. Afterward, the solution was subjected to centrifugation (10,000 rpm for 10 min), filtration through 0.22 pm filters, and evaporation under a nitrogen flow. The residue obtained was subsequently reconstituted in a methanol solution (80%) containing the internal standard trans- Cinnamic-d5 Acid at a concentration of 10 ppm. The analysis of 45 distinct free phenolic compounds was carried out using a targeted LC-MS / MS approach. This analytical method was based on a modified and validated version of the procedure previously established by Tlais et al. (2021 ) by using a UHPLC Dionex 3000 (Thermo Fisher Scientific, Germany) equipped with a Waters Acquity HSS T3 column (1.8 pm, 100 mm x 2.1 mm) (Milford, MA, USA) and coupled to a TSQ Quantum ™ Access MAX Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Germany) with an electrospray source. Target phenolics were detectable under multiple reaction monitoring (MRM) modes and the compounds were identified based on their reference standard, retention time, qualifier, and quantifier ion. The management of the chromatographic system and data acquisition was by Xcalibur software version 4.1 (Thermo Fisher Scientific, Germany).

[0175] Results: 3-Hydroxytyrosol emerged as the predominant phenolic compound in Raw OMWW-CHL (14793.18 ± 47.27 pg L’1), followed by tyrosol (2044.17 ± 7.83 pg L’1), p-coumaric acid (917.49 ± 56.98 pg L’1), ferulic acid (430.435 ± 9.93 pg L’1), and caffeic acid (284.205 ± 2.98 pg L-1) (see Table 4 above). Other identified phenolics P024061WQ-01 Notarbartolo & Gervasi S.p.A. spanned from a minimum of 19.645 ± 0.34 (for luteolin) to a maximum of 104.42 ± 10.35 pg L’1(for quercetin). As expected, the absence of microbial inoculum during incubation did not exert a statistically significant (P > 0.05) effect on the phenolic composition of the mixed substrate. Conversely, the inoculum of cultures yielded substantial alterations in the phenolic compound profile compared to Raw and Unstarted samples. Notably, L plantarum POM1 stood out as the most potent inducer (P < 0.05) of gallic acid release, followed by L paracasei PF6 and S. cerevisiae AYI6.Over the 48 h incubation period, all starter cultures facilitated significant (P < 0.05) liberation of 3-hydroxytyrosol, with Leuc. mesenteroides GSL1 and L plantarum POM1 showcasing heightened efficacy (ca. 50%). Analogous increasing trends were found for tyrosol, where Leuc. mesenteroides GSL1 maintained its metabolic prominence (P < 0.05). Likewise, the augmentation of rutin concentration was a recurrent observation (P < 0.05) across most microbail strains, except for L fermentati 9Y. Compared to the two controls, caftaric and chlorogenic acid increased only when L plantarum POM1 was inoculated. Caffeic and ferulic acids showed similar trend, expect being significantly utilized by L plantarum POM1 . 4-Hydroxybenzaldehyde was markedly (P < 0.05) metabolized by all strains, especially by Leuc. mesenteroides GSL1 . Hydrocaffeic acid exclusively featured in PR1 fermented OMWW-CHL. Regarding p-coumaric acid only L. paracasei PF6 caused a significant (P < 0.05) increase of concentration, whereas L. plantarum PR1 showed its complete utilization. Intriguingly, the release of luteolin was confined exclusively (P < 0.05) to LAB strains.

[0176] Example 6 - In vitro antifungal activity

[0177] Methods: Aspergillus versicolor CBS 117286, Eurotium herbariorum CBS117336, Penicillium paneum CBS101032, and Penicillium roqueforti DPPMAF1 , obtained from the Culture Collection of Centraal bureau voor Schimmelcultures (Utrecht, Holland), were used as indicator microorganisms. These selected species correspond to some of most relevant spoilage fungi in baked goods (Lavermicocca et al., 2000, Suhr and Nielsen, 2004). They were grown in Potato Dextrose Agar (pH 5.6) (PDA, Oxoid Laboratories, Hampshire, UK) at 25 C for 24-72 h. Hyphal radial growth rate assay was used to determine the in vitro antifungal activity of Raw, Unstarted and fermented mixed substrate, as previously described by (Quiroga, P024061WO-01 Notarbartolo & Gervasi S.p.A.

[0178] Sampietro, & Vattuone, 2001 ) with some modifications. Briefly, each sample was added (16%, v v1final concentration) to Potato Dextrose Agar (PDA) medium (Oxoid Ltd, Basingstoke, Hampshire, England) and poured into mini-Petri dishes. Control plates contained PDA alone. A 5mm 0 of fresh mycelia of indicator microorganism was placed as mold indicator in the center of the mini-Plates containing the culture medium. The hyphal radial growth rate was measured after 6-7 days of incubation at 25 °C and the related inhibition percentage was calculated as follows:

[0179] Growth inhibition (%) = [(mycelial growth under control conditions - mycelial growth in the presence of LMW-WSE) / mycelial growth under control conditions] x 100.

[0180] Results: Antifungal effect of the fermented OMWW-CHL was observed for all the microbial strains tested to a different extent on the different molds (Figure 3). Generally, the least inhibited mold was P. roqueforti showing inhibition rates of approximately 15%, where M. caribbica 5Y and Leuc. mesenteroides GSL1 didn’t exert any influence. On the contrary, P. paneum, A. versicolor and E. herbariourum were more susceptible to the fermented OMWW-CHL. In particular, the mixture substrates fermented by M. caribbica 5Y and S. cerevisiae AYI6 showed the best antifungal activity especially on E. herbariourum (28% of inhibition). All mixture substrates fermented by starters showed significant inhibition of A. versicolor (23%) expect mixture substrates fermented by S. cerevisiae AYI6 (13%).

[0181] (Original in Electronic Form)

[0182] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0183] (This sheet is not part of and does not count as a sheet of the international application) 3 / 4

[0184] PCT

[0185] (Original in Electronic Form)

[0186] (This sheet is not part of and does not count as a sheet of the international application)

[0187] FOR RECEIVING OFFICE USE ONLY (Original in Electronic Form)

[0188] (This sheet is not part of and does not count as a sheet of the international application)

[0189] FOR INTERNATIONAL BUREAU USE ONLY

Claims

P024061WO-01 Notarbartolo & Gervasi S.p.A.CLAIMS1 . A microbiological fermentation process for the production of a OMWW (olive mill wastewater) fermented extract comprising the following steps: a) providing and mixing OMWW and microalgae Chlorella, thus obtaining a starting material; b) inoculating the starting material with a lactic acid bacterium and / or a yeast, and c) fermenting the starting material.

2. The microbiological fermentation process according to claim 1 , wherein microalgae Chlorella is Chlorella vulgaris.

3. The microbiological fermentation process according to claim 1 or claim 2, wherein the lactic acid bacillus and / or the yeast of step b) can be one or more yeasts and / or one or more lactic acid bacteria (LAB) strains isolated from fresh apples, apple byproducts, and fermented vegetables (sauerkraut).

4. The microbiological fermentation process according to anyone of claims 1 to 3, wherein the yeast is selected from Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ.

5. The microbiological fermentation process according to anyone of claims 1 to 3, wherein the lactic acid bacterium is a species selected from Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ, Lacticaseibacillus paracasei PF6 deposited with accession number DSM 19463 on MRS at the DSMZ, and Lactiplantibacillus plantarum POM1 deposited with accession number DSM 34000 on MRS at the DSMZ.

6. The microbiological fermentation process according to anyone of claims 1 to 5, wherein the step c) of fermentation is carried out a temperature in the range from 25 to 35°C, preferably for a time range from 36 to 52 hours.

7. A fermented OMWW extract obtainable by the process according to anyone of claims 1 to 6.

8. The fermented OMWW extract of claim 7 having a concentration of total polyphenols from 23133 to 27861 pg / L and a concentration of total amino acids from28P024061WG-01 Notarbartolo & Gervasi S.p.A.294 to 1577 mg / L.

9. A composition comprising the fermented OMWW extract of claim 7 or 8 and an edible carrier.

10. A product selected from a supplement, a functional food, a nutritional product comprising the fermented OMWW extract of claim 7 or 8 and suitable excipients.11 . The product of claim 10 that is a wheat bread.

12. A cosmetic use of the fermented OMWW extract of claim 7 or 8 or of the composition of claim 9 or of the product of claim 10 or 11 in the treatment of cutaneous aging, of skin and of hair.

13. A fermented OMWW extract according to claim 7 or 8 or a composition of claim 9 or a product of claim 10 or 11 for use in the prevention and / or treatment of a skin disorders resulting from DNA damage produced by UV radiation.

14. A strain of a yeast selected from Meyerozyma caribbica 5Y deposited with accession number DSM 35112 on Malt extract (ME) at the DSMZ, Lachancea fermentati 9Y deposited with accession number DSM 35113 on Malt extract (ME) at the DSMZ and Saccharomyces cerevisiae AY16 deposited with accession number DSM 35114 on Malt extract (ME) at the DSMZ.

15. A strain of the lactic acid bacterium that is the species Leuconostoc mesenteroides GSL1 deposited with accession number DSM 35111 on MRS at the DSMZ.

Images