EP4739143A1

EP4739143A1 - Method of obtaining a fermented olive pomace paste, fermented olive pomace paste, saccharomyces cerevisiae and uses thereof

Info

Publication number: EP4739143A1
Application number: EP23765319.1A
Authority: EP
Inventors: Helena Maria NETO FERREIRA DE SOUSA; Maria Beatriz PRIOR PINTO OLIVEIRA; Josman DANTAS PALMEIRA; Rita CARNEIRO ALVES
Original assignee: Requimte Rede De Quimica E Tecnologia; Universidade do Porto
Current assignee: Requimte Rede De Quimica E Tecnologia; Universidade do Porto
Priority date: 2023-07-06
Filing date: 2023-08-28
Publication date: 2026-05-13
Also published as: WO2025008670A1

Abstract

The present invention relates to a fermented olive pomace paste comprising Saccharomyces cerevisiae with accession no. DSM 34718, with nutritive value and advantageous synbiotic action, as well as the method of obtaining such fermented olive pomace paste and its use as a synbiotic food ingredient or dietary supplement with antioxidant and anti-inflammatory effect. The present invention further relates to the Saccharomyces cerevisiae with accession no. DSM 34718 and its use as a probiotic.

Description

METHOD OF OBTAINING A FERMENTED OLIVE POMACE PASTE, FERMENTED OLIVE POMACE PASTE, SACCHAROMYCES CEREVISIAE AND USES THEREOF

The present application claims the benefit of priority to the Portuguese provisional patent application no. 118803, filed July 6, 2023, the Portuguese provisional patent application no. 118861, filed August 8, 2023, and the Portuguese provisional patent application no. 118880, filed August 11, 2023, the contents of which are incorporated herein by reference.

The present invention is encompassed in the human food sector and relates to a fermented olive pomace paste comprising Sac c haromyces cerevisiae with accession no. DSM 34718, as well as the method of obtaining such fermented olive pomace paste and its use as a synbiotic food ingredient or dietary supplement with antioxidant and anti-inflammatory effect. The present invention further relates the Saccharomyces cerevisiae with accession no. DSM 34718 and its use as a probiotic.

Waste and by-product generation have an impact on the environmental, economic, and social sectors. Several biomaterials are not utilized and are deposited in landfills where, due to microbial decomposition, there is an increase in greenhouse gas (GHG) emission and leachate production¹.

The negative economic effects are brought on by the expenses associated with processing solid waste in landfills, since it may be difficult to manage large amounts of various degradable materials². According to estimates from the Food and Agriculture Organization (FAO), nearly a third of all food meant for human consumption is lost or wasted globally.

Nonetheless, one in nine people still lacks adequate nutrition, which equates to almost a billion tons of food and $940 billion in economic losses each year. 40% of food in the US is lost or wasted every year, costing the economy an estimated $218 billion. Food makes up 24% of the solid waste dumped in American landfills, which are the country's third-largest source of methane emissions connected to people. Reducing this food waste offers chances to improve resource and energy conservation, boost productivity and economic efficiency, and combat climate change³.

Each year, the European Union (EU) produces close to 59 million tons of food waste (131 kg/person), with a market value estimated at 132 billion euros⁴. Around 10% of the food made available to EU customers (at retail, food services, and residences) may be wasted, according to Eurostat's estimations. Meanwhile, 36.2 million individuals nationwide cannot afford a decent dinner every other day⁵. The social impact is due to an ethical and moral dimension within the definition of global food security, since between 702 and 828 million people suffer from hunger⁶.

The upcycling, valorization, and usage of these by-products is a priority, aiming at responsible consumption and production given that the global production of plant-based products is constantly growing and generates enormous waste⁷.

The circular economy and residue valorization concepts have come to the forefront to reduce waste, conserve resources, improve production sector efficiency, and value by-products to create value-added products that will help the economy become more sustainable and solve environmental issues. One of the greatest challenges is to valorize residues from the agri-food industry when they still have advantageous nutritional properties.

In this context, one of the biggest residue-making industries is the olive oil sector. It leads to the production of by-products (e.g., olive pomace paste and olive leaves) and wastes (e.g., wood and wastewater), representing an important environmental issue in the Mediterranean areas, where they are generated in huge quantities in short periods of time⁸.

According to the International Olive Council, about 3,000,000 tons of olive oil are produced every year, but olive oil represents only 20% of the fruit. Consequently, 80% of the olive remains as olive pomace (OP)⁹ ^, ¹⁰ and more than 12,000,000 tons of this by-product are available each year as an alternative ingredient (after stone removal) that can be valorized¹¹.

Due to major environmental problems and the associated cost of disposing of olive oil by-products, specifically the OP, there is a stimulus to value it¹². Environmental concerns are due to negative physical, chemical, and biological effects on soil; potential phytotoxicity to crops; and potential risk to groundwater¹³. Inhibition of soil microbial activity may in turn reduce soil fertility by inhibiting key processes in nutrient cycling responsible for the formation of labile forms of macro and micro elements; thus, the release of olive mill waste (OMW) into the environment is not recommended¹ ³ ^-1 ⁵. Therefore, the possibility of OMW composting has been highlighted in order to transform its chemical profile and turn the phytochemical properties into fertilizer through fermentation⁸.

Olive oil extraction is represented in (an adaptation from Albuquerque et al.¹⁶). The production begins with defoliation and washing, followed by milling, where the oil is extracted from the olive. The malaxation step allows the olive oil drops to assemble and facilitate the separation from the aqueous phase. At last, the OP and the oil are separated by horizontal centrifugation step, followed by a vertical centrifugation step to remove all the remaining impurities¹⁷.

The raw OP contains crushed hull, skin, pulp, water, and residual oil¹⁸. It is composed of small amounts of crude protein and a high percentage of dietary fiber, mainly lignin (27%), followed by cellulose (15%) and hemicellulose (10%) (11). Nunes et al. (2021) verified that an OP extract can be considered an all-in-one advantageous ingredient since it presents a mixture of lipidic and hydrophilic bioactive compounds usually not present in other plant extracts. In addition, the authors also observed OP antibacterial activity against gram-positive and gram-negative bacteria¹⁹.

The olive pomace paste (OPP) microbiota is subsequently influenced by the OPP chemical composition, which is in turn influenced by the weather, olive cultivar region, extraction method, and growing conditions.

Previous studies have revealed that the microbiome of OPP is similar to that of other oil by-products like olive mill wastewater (OMWW) and is composed primarily of bacteria and yeast. Proteobacteria were found to be the most prevalent microorganism, followed by Actinobacteria (Streptomyces), Firmicutes (Staphylococcus), and Acidobacteria, according to Vivas et al.²⁰.

Furthermore, members of Pseudoxanthomonas, Hydrocarboniphaga and Stenotrophomonas (Gammaproteobacteria) were detected, with Comamonas (Betaproteobacteria) as the main microbial group. The cultivar seems to have a significant influence on the fungus population. The dominant yeasts were Pichia caribbica (syn. Meyerozyma caribbica), Pichia holstii (syn. Nakazawaea holstii), and Zygosaccharomyces fermented (syn. Lachancea fermenta), which were followed, to a lesser extent, by Zygosaccharomyces florentinus (syn. Zygotorulaspora florentina), Lachancea thermotolerans (syn. Kluyveromyces thermotolerans), Saccharomyces cerevisiae, and Saccharomyces rosinii (syn. Kazachstania rosinii) ^20-22.

Probiotics are live microorganisms showing health benefits with a multifactorial mode of action.⁴¹ The introduction of probiotics, prebiotics, or synbiotics into human diet is favorable for the intestinal microbiota and can be provided by functional synbiotic food⁴².

The fermented product will act as a synbiotic food once some nutritional components can stimulate the proliferation of certain strains in the gut, functioning as a prebiotic. The typical bitterness of olive pomace, mainly due to phenolic compounds, is counterbalanced by fermentation products by analogy with fermentation of table olives.

Currently, OP is mainly used to recover the residual oil by solvent extraction²³. It is possible to recover the stone fragments that can be used as fuel for heating the kilns or to produce activated carbon^24,25. Nevertheless, the OP derived from the two-phase decanter and the pitted one are difficult to manage for the oil extraction because more time and energy are necessary for pomace dehydration²⁶. Thus, researchers have been focusing their findings on sustainable uses of olive pomace involving the extraction of the molecules of interest.

The direct use of OP has been mainly proposed for non-edible purposes, such as clay bricks. Since wet OP forms pores, it allows the production of construction materials with insulation properties²⁷. Due to its adsorption characteristics, OP has also been used as pollutant remover from soil, being effective against pollutants such as heavy metals and triazinic herbicides.

Its chemical properties allow its use as a conditioner and fertilizer²⁸. Additionally, OP is a natural source of phenolic compounds, and several studies are focused on the development of new extraction methods to improve the extraction yield^29-31.

In view of the above, the present invention unexpectedly proposes a fermented olive pomace paste comprising Sac c haromyces cerevisiae with accession no. DSM 34718, with nutritive value and advantageous synbiotic action.

The fermented olive pomace paste is obtained by fermentation, one of the most commonly used processes in the food industry, which keeps the essential qualities of the product, maximizes nutrients with bioactive characteristics, and lengthens the food's shelf life^32,33. Antimicrobial, antioxidant, and anti-inflammatory bioactivities are produced during the fermentation of plant by-products, which are important for human health³⁴.

It is also an object of the present invention the use of the fermented olive pomace paste as high-added nutritional value food ingredient or dietary supplement. OPP has already been used as a main source of nutrients for enzyme production using solid-state fermentation by Aspergillus species^35,36. Also, due to its nutritional value, OPP is also used as a feed supplement for animal production³⁷.

The present invention discloses, in a first aspect, a method of obtaining a fermented olive pomace paste comprising the steps of:

a. providing a fresh olive pomace from the harvested and processed olives used for olive oil production;

b. spontaneous fermenting the fresh olive pomace at temperatures varying from 4 to 37 ºC and time intervals varying from 24 hours to 6 months;

c. collecting the fermented olive pomace paste,

wherein the population of the strain Saccharomyces cerevisiae with accession no. DSM 34718, is predominant when the fermentation step arrives at phase 4,

wherein the fermentation reduces the bitterness; and

with the proviso that the fermentation occurs solely by the action of the wild microbiota present in the fresh olive pomace.

In a second aspect, the present invention discloses a fermented olive pomace paste comprising carbohydrates, olive fat, proteins, vitamins, fiber, phenolic compounds and Saccharomyces cerevisiae with accession no. DSM 34718,

wherein the moisture content of the fermented olive pomace paste ranges from 55 - 78 % of dry weight;

wherein the crude protein level of the fermented olive pomace paste ranges from 5 - 10 g / 100 g of dry weight; and

wherein the dietary fiber level of the fermented olive pomace paste ranges from 40 - 60 % of dry weight,

wherein the total carbohydrates level of the fermented olive pomace paste ranges from 65 - 90 % of dry weight,

wherein all dry weight ranges relate to the dry weight of said fermented olive pomace paste.

In a third aspect, the present invention discloses the use of the fermented olive pomace paste as a synbiotic food ingredient or dietary supplement with antioxidant and anti-inflammatory effect.

In a fourth aspect, the present invention discloses a Saccharomyces cerevisiae strain, which was deposited under the accession no. DSM 34718.

In a fifth aspect, the present invention relates to the use of the Saccharomyces cerevisiae as a probiotic.

Olive oil production is an agricultural sector typical of Mediterranean countries that faces serious problems with the management of by-products due to phytotoxicity and olive pomace (OP) is the most representative one.

OP contains a high level of phenolic antioxidants, which are beneficial for our health, but an emergent problem to sustainable agriculture due to its environmental burden.

Phenolic antioxidants inhibit the growth of microorganisms, which may be found in the environment, in foods and animals. As only a minimal phenolic fraction passes into the oil phase, the majority of phenolics remain in OP.

The OP is essentially dumped in landfills, which can cause problems for nearby crops and aquatic life.

The present invention herein proposes the transformation of an environmentally toxic by-product of olive oil production in a fermented olive pomace paste with nutritive value and advantageous synbiotic action, thus contributing to environmental protection and healthy food production.

Not only the fermented olive pomace paste is use as high-added nutritional value food ingredient or dietary supplement, but it also constitutes a new approach to olive oil production waste management, relevant in terms of sustainable development, environmental protection and the promotion of economic alternatives for this productive traditional food chain.

The present invention aims to achieve the Sustainable Development Goals towards a green and circular economy and zero waste, while promoting the discovery of a new functional food ingredient or dietary supplement, valuable in terms of well-being through food.

More specifically, the present invention advantageously transforms an agroindustry by-product (olive pomace) into a healthy food ingredient or dietary supplement (fermented olive pomace paste) with a positive impact on gut microbiota biodiversity. This approach will allow the exploitation of the advantageous components of the olive pomace instead of its environmentally negative impacting destination.

The fermentation of olive pomace allows obtaining a valuable food-grade ingredient, valorising the solid waste of olive oil production. The method of obtaining the fermented olive pomace paste uses the Sac c haromyces cerevisiae present in the olive pomace’s microbial endogenous ecosystem.

During fermentation, the enzymatic activity of the raw material and the metabolic activity of microorganisms can change the nutritive and bioactive properties of food matrices with beneficial consequences for human health³⁸.

Thus, another advantage of the present invention relates to the Sac c haromyces cerevisiae with accession no. DSM 34718, comprised in the fermented olive pomace paste and which is responsible for the alteration of organoleptic characteristics, reducing bitterness in a time dependent way.

In order to facilitate an understanding of the principles according to the embodiments of this invention, reference will be made to the illustrated embodiments in the Figures and the language used to describe them.

It should also be understood that there is no intention to limit the scope of the invention to the content of the Figures and that modifications to the inventive features illustrated herein, as well as additional applications of the illustrated principles and embodiments, which would normally occur to a person skilled in the art having possession of this description, are considered within the scope of the claimed invention.

Fig.1

is a representation of olive oil extraction, adapted from Albuquerque et al.¹⁶.

Fig.2

graphically represents OPP wild fermentation at room temperature growth curve, difference between microorganism concentration in PCA and SAB.

Fig.3

graphically represents the prebiotic potential assay results using L. fermentum.

Fig.4

graphically represents the prebiotic potential assay results using L. paracasei.

Fig.5

graphically represents the prebiotic potential assay results using L. plantarum.

Fig.6

graphically represents the prebiotic activity assay results using L. fermentum.

Fig.7

graphically represents the comparison of prebiotic potential assay and activity results - L. fermentum.

Fig.8

graphically represents the prebiotic activity assay results using L. paracasei.

Fig.9

graphically represents the comparison of prebiotic potential assay and activity results - L. paracasei.

Fig.10

graphically represents the prebiotic activity assay results using L. plantarum.

Fig.11

graphically represents the comparison of prebiotic potential assay and activity results - L. plantarum.

Fig.12

graphically represents the prebiotic activity evaluation with 40% of OPP - L. fermentum, L. plantarum and L. paracasei.

b. spontaneous fermenting the fresh olive pomace at temperatures varying from 4 to 37 ºC and time intervals varying from 24 hours to 6 months; and

c. collecting the fermented olive pomace paste,

wherein the population of the strain Saccharomyces cerevisiae with accession no. DSM 34718, is predominant when the fermentation step arrives at phase 4 (day 32),

wherein the fermentation reduces the bitterness; and

with the proviso that the fermentation step occurs solely by the action of the wild microbiota present in the fresh olive pomace.

The method of the present invention occurs by batch fermentation of the olive pomace in a sterile container, in order to create a boundary that keeps all foreign microorganisms out.

Thus, the method of the present invention occurs by using closed sterile containers to collect the fresh olive pomace (step a) and by using a clean and sterile fermenter (step b), wherein the fermenter is previously sparged with steam. A 0.2-micron sterile filter can be also used to ensure that no foreign organism can make it into the system.

In a preferred embodiment of the present invention, the fermentation step (step c) of the fresh olive pomace is maintained for 4 days at a temperature that ranges from 20 to 21 ºC. Also, for comparison and shelf-life analysis, the fermentation was maintained for 6 months.

The fermented olive pomace paste from step c can be freshly used (directly or fractioned) or further lyophilized.

The present invention further discloses, in a second aspect, a fermented olive pomace paste comprising carbohydrates, olive fat, proteins, vitamins, fiber, phenolic compounds and Saccharomyces cerevisiae with accession no. DSM 34718,

wherein the moisture content of the fermented olive pomace paste ranges from 55 - 78 %;

wherein the protein level of the fermented olive pomace paste ranges from 5 - 10 g / 100 g of dry weight; and

In one embodiment of the present invention, the olive fat comprises fatty acids selected from the group consisting of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, alpha-linolenic acid, 9-eicosenoic acid, docosanoic acid, lignoceric acid, isomers thereof and combinations thereof. In a preferred embodiment of the present invention, 70 - 78 % of the olive fat is oleic acid.

In one embodiment of the present invention, the proteins are amino acids selected from the group consisting of aspartic acid, glutaric acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, lysine, hydroxyproline, proline and combinations thereof.

In one embodiment of the present invention, the vitamin is vitamin E, wherein the vitamin E is present in the form of alpha-tocopherol, beta-tocopherol and gamma-tocopherol.

In one embodiment of the present invention, the fiber is selected from the group consisting of insoluble, soluble fiber and combinations thereof. More specifically, the fiber consists of the solid component of the paste, wherein insoluble and soluble fiber are both relevant in terms of potential prebiotic action.

It is also important to emphasize that the dietary fiber level is very dependent on the type of the olive pomace paste. For example, in case the olive pomace paste comprises olive pits, the dietary fiber level is higher.

In one embodiment of the present invention, the phenolic compounds are selected from the group consisting of hydroxytyrosol, tyrosol, oleuropein, verbascoside, elenolic acid, catechol, rutin and combinations thereof.

Dietary polyphenols and their metabolites promote intestinal health modulating the gut microbial composition in a positive manner, in which the growth of beneficial microbes is stimulated, whereas the pathogens are inhibited.

In preferred embodiments of the present invention, the fermented olive pomace paste further comprises limonene as a volatile compound contributing to the aroma and taste.

In preferred embodiments of the present invention, the fermented olive pomace paste comprises:

– 65 - 90 % dry weight of carbohydrates;

– 2 - 20 % dry weight of olive fat with 70 - 78% oleic acid;

– 5 - 10 % dry weight of proteins;

– 2 - 10 mg /100 g dry weight of vitamin E;

– 40 - 60 % dry weight of dietary fiber;

– 15-50 mg / g dry weight of total phenolic compounds expressed in gallic acid equivalents; and

– 6 x 10⁶ - 14 x 10⁶ CFU / ml of Saccharomyces cerevisiae with accession no. DSM 34718,

After fermentation, colony forming units (CFU) count shows a microbiota enriched in Sac c haromyces cerevisiae with accession no. DSM 34718, which is associated to the production of gas and acidification.

The Saccharomyces cerevisiae with accession no. DSM 34718 has a significant impact on the flavor, texture, and preservation of the fermented olive pomace paste of the present invention.

The microbiota has an important role in the alteration of organoleptic characteristics of the fermented olive pomace paste. More specifically, the fermentation process reduces the oleuropein and increases the hydroxytyrosol contents, obtaining an improvement in antioxidant and nutritional levels, reducing bitterness, and providing a pleasant aroma.

In a third aspect, the present invention discloses the use of the fermented olive pomace paste as a food ingredient or dietary supplement, wherein the fermented olive pomace paste is used fresh and directly, i.e., with no further treatment or separation.

It is also possible to use fractions of the fermented olive pomace paste. For example, antioxidants can be extracted from the fermented olive pomace paste and be provided as a particular supplement for food or cosmetics. Also, essential amino acids can be obtained from the fractionated portion.

In a fourth and fifth aspects, the present invention relates to the Saccharomyces cerevisiae with accession no. DSM 34718, which can be further isolated from the fermented olive pomace paste and used as a probiotic.

The synbiotic effect associated with the prebiotic action of this food ingredient and the probiotic action of Saccharomyces cerevisiae will create a valuable fermented food able to promote healthy impact in intestinal microbiota with the consequent value in biodiversity and postbiotic effects associated to the metabolites generated in the gut microbiome related to the gut-brain axis and intestinal health.

Examples

In order to evaluate the characteristics of the fermented olive pomace paste of the present invention, olive pomace paste (OPP) samples are natural, plant-based, and chemical-free. More specifically, the OPP was obtained from one of the major Portuguese olive oil production regions - Trás-os-Monte. The OPP microbiota is influenced by the OPP chemical composition, which is in turn influenced by the weather, olive cultivar, geographical region, extraction method and growth conditions.

The total fermentation process occurred for six months under batch conditions, and it was evaluated by sample collection at 48 hours of fermentation, 96 hours, 8 days, 16 days, 32 days, and at the end of the process (six months) in order to analyze the microbiological profile over time.

The microbiological analysis aims to identify the changes during the fermentation process, verify the time of optimal growth of the microorganisms, their diversity, and determine if the microbiota changes with time.

The prebiotic effect was also assessed using monocultures of L. fermentum, L. paracasei, and L. plantarum. Such assessment aims to determine if the fermented olive pomace paste of the present invention fulfill the three main requisites of a prebiotic substance, which are: (i) not to be digested by endogenous enzymes in the human gut; (ii) to be selectively fermented by specific genera / species of gut microbiota; and (iii) to increase specific microorganisms that confer benefits regarding human health⁴⁰.

The fermentation process occurred wildly - using the own microbiota of OPP without dilution.

To be able to evaluate the microorganisms, selective and differential media were chosen, such as MacConkey agar (Liofilchem, Italy) - to verify the presence of gram-negative bacteria; Slanetz-and-Bartley agar (Liofilchem, Italy) - to verify the presence of Enterococci; Manitol Salt Agar (MSA) (Liofilchem, Italy) - to verify the presence of Staphylococci; Chromagar Orientation(CHROMagar, France) - in order to see the microbial variety; Man, Rogosa and Sharpe Agar (MRS) (Liofilchem, Italy) - to verify the presence of Lactobacilli; Sabouraud Dextrose Agar (SAB) (Liofilchem, Italy) - to verify the presence of yeast; 100 µL of each sample were inoculated in each medium and incubated at 37 ºC for 48 hours.

The sample was accompanied for 32 days, wherein 5 g were collected, and then 100 µL from the sample were spread directly in the medium. In order to analyze the microbiota diversity without interference, 1 g of OPP was diluted in 9 mL of sterile saline solution, making dilutions of 10⁰ to 10^-8, and then 100 µL of each dilution were spread in plate count agar (PCA) (Liofilchem, Italy) and incubated at 37 ºC for 48 hours.

According to previous studies and known protocols, yeasts are prevalent in OPP microbiota; therefore, the SAB was used to evaluate the CFU/mL of yeast present during the fermentation process - 1 g of OPP was diluted in 9 mL of sterile saline solution, making dilutions of 10⁰ to 10^-8, then 100 µL of each dilution were spread in SAB and incubated at 37 ºC for 48 hours.

The CFU/mL was calculated using Equation 1 below:

(1)

Prebiotic activity assay

The prebiotic potential assay was performed using MRS broth and a monoculture of standard probiotics: Lactobacillus fermentum ATCC ® 9338, Lactobacillus paracasei subsp. paracasei ATCC ® BAA-52, and Lactobacillus plantarum subsp. plantarum NCTC ® 13644.Growth behavior of the different strains is dependent on the typical energetic metabolism of each, that influences the interactions with the environment, conditioning different responses in terms of prebiotic potential determination.

Controls

Three controls were performed: the negative control was done using 9 mL of MRS broth without glucose (Liofilchem, Italy) and 1 mL of 0.3 McFarland monoculture suspension, and two positive controls were done using, respectively, glucose and inulin - reference prebiotic, both at 2% concentration w/v.

The controls were made by adding 0.2 g of glucose to 9 mL of MRS broth, followed by the sterilization of the solution by a cellulose filter (0.22 µm of pore size). The same steps were followed regarding inulin. The initial pH was measured using a pHmeter (Mettler Toledo) in a sterile environment to avoid contamination.

Timepoints and sample concentration

Timepoints were chosen according to the growth curve obtained earlier in the study, namely: T0 - without fermentation; T2 - two-day fermentation; T4 - four-day fermentation and T32 - 32-day fermentation. In order to evaluate the OPP fermentation in more complex ways, for each timepoint selected, a sample of 2.5%, 5%, and 10% w/v was evaluated.

In each fermentation timepoint, 10 g of OPP was collected and sterilized by autoclave. In a sterile environment (using flame), the OPP was weighted using an analytical balance: 0.25 g (2.5%), 0.50 g (5%) and 1.00 g (10%). The MRS broth was added - 9 mL - to the weighted sample. The initial pH was measured using a pHmeter (Mettler Toledo) in a sterile environment to avoid contamination. The same procedure was performed on the samples after simulated gastrointestinal digestion in vitro (SGDI).

Cell suspension

The cell suspension was made using a McFarland cytometer (DEN-1) to measure the optical density (OD). Fresh CFU of the selected microorganism monoculture were carefully added to a saline solution until 0.3 McFarland. The cell suspension was added (1 mL) to each control, and to each weighted sample with MRS broth. The controls and each timepoint sample were submitted to an incubation of 48 hours at 37 ºC.

Assay evaluation

In order to evaluate the growth of each microorganism in the controls and samples and be able to compare the results, a serial dilution (10⁰ – 10^-7) using saline solution was performed, followed by the pour plate method of 100 µL in MRS agar (Scharlau, Spain) done in triplicate.

The Petri dishes were incubated for 48 hours at 37 ºC and then dishes with a CFU quantity between 30 and 300 were counted. The final pH was measured, using a pHmeter (Mettler Toledo) in order to determine if there was any acid fermentation by decreasing pH.

OPP Simulated Gastrointestinal Digestion in vitro (SDGI)

In order to be able to evaluate the actual prebiotic effect of OPP, a simulated gastrointestinal digestion in vitro (SGDI) was performed according to known protocols.

Oral Phase

To simulate the mastication of solid food, a manual mincer is used. A paste-like consistency is produced by adding an electrolyte stock solution with simulated salivary fluid (SSF) to the minced food. The final ratio of food to SSF needs to be 50:50 (w/v). Human salivary α-amylase is added, and the final concentration is 75 U/mL, followed by CaCl₂, which needs to be at 0.75 U/mL in the final mixture, and the necessary quantity of water in order to dilute the SSF solution. The contact time with the enzyme is 2 minutes at 37 ºC and pH 7, therefore the pre-warming of the reagents until this temperature is necessary.

Gastric phase

Five parts of the oral bolus is mixed with four parts of simulated gastric fluid (SGF) of 50:50 (v/v) after the addition of other recipients and water. Porcine pepsin is added to achieve 2000 U/mL as well as gastric lipase at 60 U/mL in the final digestion mixture, followed by CaCl₂to achieve 0.075 mM in the final digestion mixture. To reduce the pH to 3, HCl 1M is added. Finally, the necessary amount of water is added to the mixture to dilute the stock solution of SGF. The digestion time is 2 hours at 37 ºC, with mixing. The pH may need to be readjusted with 1M HCl during digestion.

Intestinal phase

After the addition of additional receivers and water, the ultimate ratio of gastric chyme to simulated intestinal fluid (SIF) is 50:50 (v/v) by combining 5 parts of chyme with 4 parts of electrolyte stock solution from the SIF. In order to neutralize the gastric mixture to pH 7.0, the addition of 1M NaOH is needed. Bile salts (10mM) are added, followed by trypsin in pancreatin (100 U/mL). The solution incubation occurs for 2 hours at 37 ºC and pH 7, while mixing.

Prebiotic activity assay after SDGI

The prebiotic activity assay after the SDGI was performed using the same monocultures mentioned above and same negative and positive controls.

As regards the timepoints, due to the limited possibility of performing the SGDI, the 48-hour fermentation time sample was discarded; therefore, three samples were selected (T0, T4 and T32). For each time selected, the concentration of OPP to evaluate was 40% w/v, i.e, 4.00 g.

The samples were treated as indicated above. Cells suspension and assay evaluation were as disclosed above as well.

Statistical analysis

One-way analysis of variance (ANOVA) was the statistical method employed to compare the results. The same assay, carried out with the same monoculture, yielded triplicate findings for the negative control and the selected sample. These results were submitted to data analysis using Excel, and statistical data was gathered.

Results

Fermentation Process

When the initial OPP microbiology was examined, it was feasible to confirm that it contained a significant number of microorganisms. Since yeast only makes up a portion of the OPP microbiota, the results of the PCA were 9.2x10⁴ CFU/mL and the SAB were 3.3x10⁴ CFU/mL (see Table 1 below).

There was a lack of growth in MAC, MSA, and SLZ; hence, once bacteria such as Enterobacteriaceae, Staphylococcus, and Enterococcus were not detected.

These results show a predominance of yeast growth related to the predominant isolated yeast - Saccharomyces cerevisiae.

Table 1: Information regarding OPP T0 microbiological analysis

	T0
Volume (mL)	0,1
Dilution	1 x10²
pH	4.75
PCA (CFU)	92
PCA (CFU/mL)	9.2 x10⁴
SAB (CFU)	33
SAB (CFU/mL)	3.3 x10⁴
MAC (CFU)	0
UTI (CFU)	50
MSA (CFU)	0
MRS (CFU)	> 300
SLZ (CFU)	0

The sample inoculated at room temperature had its exponential growth phase between T0 and the second day, reaching its greater microorganism density on the second day.

In , a significant decrease after the second day and a growth after the eighth day until day 16 can be verified. This could be due to the acid environment that inhibits the growth of several microorganisms, selecting the ones capable of development in these conditions, the growth is due to the amount of nutrients still available.

This environment change is important since the stipulated conditions are not favorable to pathogenic strains and are extremely favorable to strains with probiotic effects, such as Lactobacillus and yeast strains.

Fermented olive pomace paste characterization:

The fermented olive pomace paste of the present invention was assessed to verify characteristics of moisture content and components amounts in different time points.

The results are disclosed in Table 2 below.

Table 2: Chemical composition of raw olive pomace and fermented olive pomace.

Parameter	Raw olive pomace	Fermented olive pomace
Moisture (g/100 g fw)	55-78	55-78
Total ash (g/100 g dw)	5-10	5-10
Total ash (g/100 g fw)	1-5	1-5
Total protein (g/100 g dw)	5-10	5-10
Total protein (g/100 g fw)	1-5	1-5
Essential amino acids (mg/g dw)	15-30	15-30
Essential amino acids (mg/g fw)	3-14	3-14
Non-essential amino acids (mg/g dw)	25-40	25-40
Non-essential amino acids (mg/g fw)	6-18	6-18
Total carbohydrates (g/100 g dw)	65-90	65-90
Total carbohydrates (g/100 g fw)	16-41	16-41
Total dietary fiber (g/100 g dw)	40-60	40-60
Total dietary fiber (g/100 g fw)	10-27	10-27
Total fat (g/100 g dw)	2-20	2-20
Total fat (g/100 g fw)	0.5-9	0.5-9
*Oleic acid (C18:1n9c) (relative % of total fatty acids)*	70-78	70-78
*Linoleic acid (C18:2n6c) (relative % of total fatty acids)*	7-11	7-11
Total vitamin E (mg/100 g dw)	1-8	2-10
Total vitamin E (mg/100 g fw)	0.2-3.6	0.5-4.5
Total phenolic compounds (mg GAE/g dw)	15-50	15-50
Total phenolic compounds (mg GAE/g fw)	3-23	3-23
DPPH ^• scavenging activity (mg TE/g dw)	10-60	10-60
DPPH ^• scavenging activity (mg TE/g fw)	2-27	2-27
Ferric Reducing Antioxidant Power (mmol FSE/g dw)	0.2-0.6	0.2-0.6
Ferric Reducing Antioxidant Power (mmol FSE/g fw)	0.05-0.3	0.05-0.3
Hydroxytyrosol (mg/g dw)	2-5	3-6
Hydroxytyrosol (mg/g fw)	0.5-2.3	0.7-3
Tyrosol (mg/g dw)	0.1-0.4	0.3-2
Tyrosol (mg/g fw)	0.02-0.2	0.05-1

fw – fresh weight, dw – dry weight, GAE – gallic acid equivalents, TE – trolox equivalents, FSE – ferrous sulfate equivalents

This table shows that the main chemical changes that occur are an increase in the content of vitamin E and hydroxytyrosol, both of which have beneficial effects on health (antioxidant and anti-inflammatory effect). All other nutritional and chemical parameters are maintained.

It is of note that the fermentation further transforms the phenolics related to bitterness in hydroxytyrosol and tyrosol. Thus, the fermentation of the olive pomace reduces the bitterness depending on the microbiota present and the length of fermentation.

The phenolic compounds and DPPH inhibitor compounds show the antioxidant activity of the product. In addition, the fermented olive pomace maintains its richness in minerals (total ashes), protein (with the presence of essential amino acids), and dietary fiber.

It is important to note that the ranges shown in Table 2 may vary depending on the olive pomace paste (olive cultivar, its geographic origin and extraction method) and whether the pits are present or absent. The pit can be removed before or after fermentation, thus results may vary accordingly (for example, the pit is a source of fiber and phenolic compounds).

Prebiotic activity potential assay

The prebiotic activity potential assay results were different for each probiotic used. The assay performed with Lactobacillus fermentum showed a better development of the microorganism showing an increase with the OPP concentration, regardless of fermentation time ( ).

There was a greater CFU per volume in the OPP’s inoculum, verifying the nutritional richness of this substance regarding L. fermentum. The relationship between microorganism growth and OPP fermentation time is inverse; there is a decrease in CFU/mL of L. fermentum in inoculum with longer fermentation time.

This result may be due to the utilization of the substrates that this bacterium uses to ferment since the fermentation process needs carbohydrates in order to happen. Therefore, the longer the fermentation, the higher the depletion in substrate availability over time.

The assay performed with Lactobacillus paracasei showed a better development of the microorganism with a decrease in OPP concentration depending on fermentation time ( ).

Although Lactobacillus paracasei developed better in the OPP inoculum when compared to the negative control, it was verified that there was a greater CFU per volume in the inoculums with 5% OPP concentration to T0 than 10% and in the inoculum with 2.5% OPP concentration to T2 and T4 than the ones with 5% and 10% OPP.

One hypothesis to justify this result is that it may be due to the presence of a substance or substances that inhibit this microorganism from developing, so the higher the concentration of the OPP, the greater the concentration of this substance. Therefore, the evaluation of chemical composition over time is important.

Olives are rich in phenolic compounds; oleuropein, elenolic acid, verbascoside, and demethyloleuropein are the most prevalent ones. Previous studies verified the inhibition effect that oleuropein has on pathogenic bacteria when the concentration is higher than 200 µg/mL and verified that reducing the substrate in their test medium resulted in an inhibition of L. plantarum, Pediococcus cerevisiae, Lactobacillus brevis, and Leuconostoc mesenteries growth by oleuropein.

The abundance of substrates in the OPP T0 5% sample may overcome the concentration of inhibitor, which does not occur in the OPP T0 10% sample. Elenolic acid and the aglycone of oleuropein were inhibitory to lactic acid bacteria (97, 98). The relationship between microorganism growth and OPP fermentation time is inverse; there is a decrease in CFU/mL of L. paracasei in inoculum with longer fermentation time.

Phenolic compounds are known for their antioxidant activity, prebiotic effect, anticancer activity, prevention of cardiovascular and neurodegenerative diseases, and many other benefits. Therefore, it may seem incoherent to discuss the negative influence that these compounds may have on beneficial bacteria. However, the positive or negative effects depend not only on the molecule but also on its concentration.

Fermentation is already an approach that proposes to change the chemical profile of the OPP, mainly in order to make it more palatable to human since, due to its phenolic content, the OPP has a bitter taste, while simultaneously improving the microorganism profile to make it a probiotic food.

The results of L. plantarum prebiotic potential activity assay show a similar relationship between microorganism development and OPP concentration ( ). With the concentration increase, a depletion of CFU per unit of volume can be assessed. Therefore, a similar difference in profile regarding the growth in OPP T32, i.e., the CFU per unit of volume, does not have significant changes regarding the OPP concentration.

Due to the process of wild fermentation, there is a decrease in substrate content and the creation of metabolites, such as acids and gases that change the environment, providing different conditions over time that change the type of phenolic compounds in an advantageous way once hydroxityrosol and tyrosol show an enhancement with fermentation progression.

Prebiotic activity assay (after SGDI)

In order to confirm the prebiotic activity, the test component must reach the intestine after being exposed to the conditions that the gastrointestinal tract provides, such as the stomach acidic, digestive enzymes, and bile acid.

The prebiotic activity assay was performed after submitting each of the FOPP timepoint sample to SGDI. The Lactobacillus fermentum results showed a greater CFU per milliliter of the microorganism in the inoculums with 5% OPP than in the 2.5% and 10% inoculums, as illustrated in and .

The remaining substances, after the SGDI, are the ones that the human body cannot break down and absorb, components such as dietary fiber (DF) and phenolic compounds. The decrease in microbial growth could be due to a higher concentration of phenolic compounds, overcoming the abundance of DF in the sample.

Higher CFU per milliliter in the OPP DIG T4 inoculum can be verified. The statistical analysis using one-way (ANOVA) to compare the negative control result (average = 8.30E4 CFU/mL) with the OPP DIG T0 10% (average = 4.33E5 CFU/mL) - which was the sample with less CFU per milliliter - (p<0.05), therefore, a significant growth, verifying a prebiotic effect.

The prebiotic activity assay performed with Lactobacillus paracasei confirms the inhibition profile assessed in the prebiotic potential assay ( and ). The inoculums from OPP DIG T0, OPP DIG T4 and the sample OPP DIG T32 10% had less growth than the negative control; the antibacterial properties of phenolic compounds may have caused an inhibition that outweighed the availability of substrate.

The OPP DIG T32 2.5% sample had higher growth, followed by the 5% concentration. The number of CFU per milliliter in OPP DIG T32 5% had statistical irrelevancy compared to the negative control (p > 0.05), therefore, there was no prebiotic activity confirmed regarding the assay with L. paracasei.

However, OPP DIG T32 2.5% was statistically different from the negative control (p < 0.05). Therefore, regarding L. paracasei, the only sample that had a prebiotic effect was the OPP at 2.5% concentration that was fermented for 32 days.

The prebiotic activity assay performed with Lactobacillus plantarum had a different profile compared to the prebiotic potential assay ( and ) . The CFU per milliliter of L. plantarum in OPP DIG T0 inoculums increased along with the concentration.

The OPP DIG T4 had higher growth in the 5% sample, followed by 10% and then 2.5%. The only sample that had a CFU per milliliter higher than the negative control (average = 4.97E8 CFU/mL) was the OPP DIG T0 10% (average = 1.33E9 CFU/mL): the difference was statistically relevant (p < 0.05). Hence, for L. plantarum, there is a prebiotic effect of non-fermented OPP at 10% concentration.

After the GDSI, each sample was liquid and had a different aspect from the pre-GDSI sample, which was a paste; therefore, the distribution of DF and phenolic compounds may differ. An exploratory assay using OPP DIG at 40% concentration was performed to evaluate the microorganism response. The results obtained can be assessed in .

The L. fermentum inoculums evaluated had a significant growth of almost 3 logs of difference from the negative control. A one-way ANOVA was used to compare the OPP DIG 40% inoculums with the negative control. The sample without fermentation had a statistical difference (p > 0.05), as did the inoculums with fermentation, 4 days and 32 days.

The number of CFU per milliliter was higher in OPP DIG T4 40% (1.22E8 CFU/mL), followed by OPP DIG T32 40% (1.14E8 CFU/mL), than in OPP DIG T0 40% (1.08E8 CFU/mL). Hence, it was verified that for L. fermentum the fermentation process enhances the OPP prebiotic activity.

The inoculums in which Lactobacillus paracasei was used as monoculture, the quantity of CFU per milliliter was lower than the one used in the negative control. Therefore, no prebiotic activity was verified in the assay using 40% of each sample (T0, T4 and T32) submitted to GDSI using monoculture of L. paracasei.

The inoculums OPP DIG T4 40% (average = 9,60E8 CFU/mL) and OPP DIG T32 40% (average =1,34E9 CFU/mL) regarding Lactobacillus plantarum’s assay had a quantity of CFU per milliliter significantly higher than the negative control, and the quantity of OPP DIG T0 40% (average = 6,17E8 CFU/mL) was also higher but not statistically different. Therefore, the fermentation time of L. plantarum increased the prebiotic effect of the OPP.

Through these exploratory assays, the inhibitory action of the OPP on L. paracasei could be assessed. Additionally, the OPP prebiotic effect in L. fermentum was verified, as well as in L. plantarum, depending on the fermentation timing and concentration conditions.

The quantity of CFU/mL in the inulin and glucose controls by the pour plate method showed similar counting; however, there was a greater pellet in the glucose control when compared to the inulin and comparing to the remaining inoculums in every assay. A higher pH depletion in the glucose control was also detected.

The similar quantity of CFU per volume may be due to cellular death by acidification of the environment; therefore, performing the same protocol using a buffer is required.

According to the literature, the standard protocol used to evaluate a substance’s prebiotic effect is the evaluation of the prebiotic activity score, which can

be assessed by the measurement of the optic density difference.

Due to the OPP’s physical properties, the initial solution (medium with the OPP) is already turbid, so the usage of OD could not be assessed due to results inaccuracy. The protocol used in this study had an exploratory approach and was based on Slizewska et al article³⁹.

Afterall, the use of fermentation to obtain value compounds is a solution to improve the reutilization of olive pomace from the agroindustry. Fermentation enhances olive pomace microbiota composition in beneficial components able of putative prebiotic action. Fermentation products will contribute to the gut microbiome, impacting positively the faecal microbiota composition.

As food safety indicators, culturomic and / or molecular evaluations were performed to assess the presence of E. coli, STEC, K. pneumoniae, Citrobacter spp, E. cloacae, Salmonella spp, L. monocytogenes, E. faecium and E. faecalis. All results were negative, demonstrating the potential of using OPP for the food industry due to its microbiological safety.

The above-described subject matter is provided as an illustration of this invention and should not be interpreted in a limiting sense. The terminology used to describe preferred embodiments of this invention should not be construed to limit the invention to those particular embodiments.

As used in the description, definite and indefinite articles, in their singular form, are intended to encompass plural forms unless the context of the description explicitly indicates otherwise.

The indefinite articles "a" or "an" should generally be interpreted as "one or more" unless the sense of a singular embodiment is clearly defined in a specific situation.

It will be understood that the terms "comprise" and "include," when used in this description, specify the presence of stated features, elements, components, steps, and operations but do not preclude the presence or addition of one or more other features, elements, components, steps, and operations.

As used throughout this patent application, the term "or" is used in an inclusive sense rather than an exclusive sense unless the exclusive sense is clearly defined in a specific situation. In this context, a phrase such as "X utilizes A or B" should be interpreted as including all relevant inclusive combinations.

All changes, as long as they do not modify the essential characteristics of the following claims, should be considered within the scope of protection of this invention.

A sample of the Sac c haromyces cerevis i ae comprised in the fermented olive pomace paste of the present invention has been deposited with the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH on July 25, 2023 and received the accession no. DSM 34718.

The deposit was performed on the same terms as those laid down in the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure of April 28, 1977 and the characteristics of the biological material are herein disclosed.

The citation list is as follows:

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Claims

A method of obtaining a fermented olive pomace paste characterized by comprising the steps of:
a. providing a fresh olive pomace from the harvested and processed olives used for olive oil production;
b. spontaneous fermenting the fresh olive pomace at temperatures varying from 4 to 37 ºC and time intervals varying from 24 hours to 6 months; and
c. collecting the fermented olive pomace paste,
wherein the population of the strain Saccharomyces cerevisiae with accession no. DSM 34718, is predominant when the fermentation step arrives at phase 4,
wherein the fermentation reduces the bitterness; and
with the proviso that the fermentation occurs solely by the action of the wild microbiota present in the fresh olive pomace.
The method, according to claim 1, characterized by the fact that it occurs by batch fermentation of the olive pomace in a sterile container.
The method, according to any of claims 1 and 2, characterized by the fact that the fermented olive pomace paste from step c is used fresh or further lyophilized.
A fermented olive pomace paste characterized by the fact that it is obtained by the method disclosed in any of the claims 1 to 3, the fermented olive pomace paste comprising carbohydrates, olive fat, proteins, vitamins, fiber, phenolic compounds and Saccharomyces cerevisiae with accession no. DSM 34718,
wherein the moisture content of the fermented olive pomace paste ranges from 55 - 78 % of dry weight;
wherein the crude protein level of the fermented olive pomace paste ranges from 5 - 10 g / 100 g of dry weight; and
wherein the dietary fiber level of the fermented olive pomace paste ranges from 40 - 60 % of dry weight,
wherein the total carbohydrates level of the fermented olive pomace paste ranges from 65 - 90 % of dry weight,
wherein all dry weight ranges relate to the dry weight of said fermented olive pomace paste.
The fermented olive pomace paste, according to claim 4, characterized by the fact that the olive fat comprises fatty acids selected from the group consisting of palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linoleic acid, arachidic acid, alpha-linolenic acid, 9-eicosenoic acid, docosanoic acid, lignoceric acid, isomers thereof and combinations thereof.
The fermented olive pomace paste, according to any of claims 4 and 5, characterized by the fact that the proteins are amino acids selected from the group consisting of aspartic acid, glutaric acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, lysine, hydroxyproline, proline and combinations thereof.
The fermented olive pomace paste, according to any of claims 4-6, characterized by the fact that the vitamin is vitamin E, wherein the vitamin E is present in the form of alpha-tocopherol, beta-tocopherol and gamma-tocopherol.
The fermented olive pomace paste, according to any of claims 4-7, characterized by the fact that the fiber is selected from the group consisting of insoluble, soluble fiber and combinations thereof.
The fermented olive pomace paste, according to any of claims 4-8, characterized by the fact that the phenolic compounds are selected from the group consisting of hydroxytyrosol, tyrosol, oleuropein, verbascoside, elenolic acid, catechol, rutin and combinations thereof.
The fermented olive pomace paste, according to any of claims 4-9, characterized by the fact that it further comprises limonene.
Use of the fermented olive pomace paste as disclosed in any of claims 4-10, characterized by the fact that it is as a synbiotic food ingredient or dietary supplement with antioxidant and anti-inflammatory effect.
Use, according to claim 11, characterized by the fact that the fermented olive pomace paste is used fresh, directly or fractioned.
Saccharomyces cerevisiae characterized by the fact that a representative sample of said strain was deposited under the accession no. DSM 34718.
Use of the Saccharomyces cerevisiae as defined in claim 13, characterized by the fact that it is as a probiotic.