Recent Advances in Microbial Bioconversion as an Approach to Boost Hydroxytyrosol Recovery from Olive Mill Wastewater

Abstract

Olive mill wastewater (OMWW) is a highly complex matrix derived from olive oil extraction, containing phenolic compounds, lipids, minerals, and organic acids. Hydroxytyrosol (HT), an outstanding antioxidant and health-promoting phenolic compound, has garnered significant interest as a natural preservative and functional ingredient. Enzymatic hydrolysis, utilizing purified enzymes to cleave glycosidic or ester bonds, and microbial bioconversion, employing whole microorganisms with their intrinsic enzymes and metabolic pathways, are effective biotechnological strategies for fostering the release of HT from its conjugated forms. These approaches offer great potential for the sustainable recovery of HT from OMWW, contributing to the valorization of this environmentally impactful agro-industrial by-product. Processed OMWW can lead to clean-label HT-enriched foods and beverages, capitalizing on by-product valorization and improving food safety and quality. In this review, the most important aspects of the chemistry, technology, and microbiology of OMWW were explored in depth. Recent trends and findings in terms of both enzymatic and microbial bioconversion processes are critically discussed, including spontaneous and driven fermentation, using selected microbial strains. These approaches are presented as economically viable options for obtaining HT-enriched OMWW for applications in the food and nutraceutical sectors. The selected topics aim to provide the reader with a solid background while inspiring and facilitating future research and innovation.

1. Introduction

In recent decades, the recovery of phenolic compounds from various natural sources, particularly from agri-food by-products, has garnered significant interest across multiple research fields. These ubiquitous compounds feature unique chemical structures composed of one or more aromatic rings and numerous hydroxyl groups, which contribute to their remarkable reduction–oxidation properties [1]. In addition to their well-established antioxidant and antiradical activities, significant biological effects have been documented for phenolic compounds, which have found applications in the prevention and management of numerous oxidative stress-related disorders, such as cardiovascular diseases [2], obesity [3], type 2 diabetes (T2D) [4], skin disorders [5], cancer [6], and metabolic syndrome [7], among others.

Furthermore, olive polyphenols benefit the gastrointestinal system, protect low-density lipoproteins (LDLs) from oxidative damage, and support the immune system [8]. The main olive polyphenols include phenolic acids (such as ferulic and chlorogenic acid), secoiridoids (like oleuropein) and their derivatives (for example, hydroxytyrosol (HT) and tyrosol), flavonoids (such as luteolin and rutin), and lignans (such as pinoresinol). HT, a primary compound in virgin olive oil (VOO) and table olives, has attracted attention for its antioxidant properties and potential uses in the food industry as a preservative and functional ingredient [9,10]. Notably, its antioxidant and free radical scavenging activity exceeds that of butylated hydroxytoluene (BHT), a commonly used synthetic antioxidant [11]. Additionally, HT has been shown to reduce inflammation, fight cancer and obesity, promote wound healing, and exhibit neuroprotective, antibacterial, and antiviral effects [12]. However, producing high amounts of HT is challenging, as extracting it from natural sources through physical and chemical methods is often time-consuming and costly. Common techniques like resin chromatography and solid/liquid or liquid/liquid extraction are labor-intensive and yield low amounts [13]. Similarly, chemical synthesis and biocatalysis face limitations such as harsh reaction conditions, high energy use, and the need for hazardous organic solvents [12]. Alternatively, clean biotechnological methods have been developed to facilitate the release of HT from its conjugated forms—including oleuropein (OLE), HT glycosides, and verbascoside—found in olive oil industry by-products such as pomace, wastewater, stones, and leaves.

Olive mill wastewater (OMWW), comprising both the water content of the olive fruit (also known as olive vegetation water) and the processing water introduced during olive oil extraction, has been highlighted as one of the most highly concentrated sources of HT. Annually, approximately 30 million cubic meters of OMWW is generated in a short period, principally in Mediterranean countries [14], during olive oil extraction by traditional pressing or continuous centrifugation, which in turn includes both a two-phase and three-phase system (Figure 1).

OMWW contains most olive biophenols and HT at a higher (up to 100 times) content than VOO itself [16]. However, due to the high biological and chemical oxygen demands (COD), resulting from the high biodegradable and non-biodegradable organic matter content, OMWW cannot find direct application as a concentrated source of HT.

Untreated OMWW is classified as a hazardous pollutant with environmental implications for both soil and aquatic ecosystems [17]. When discarded outdoors, OMWW alters soil pH and produces undesirable phytotoxic effects, negatively affecting seed germination and the growth of both plants and beneficial soil microorganisms. Furthermore, the total suspended solids (TSS) and COD values in OMWW frequently exceed the maximum standard limits set by European countries (TSS < 35 mg/L; COD < 125 mg O₂/L) [18], underscoring the urgent need for management policies and innovative treatment technologies to address OMWW pollution effectively.

Many photocatalytic, physical, and chemical approaches have been broadly investigated for the removal and/or recovery of phenolic substances, to decrease the environmental impact of OMWW, turning it into a valuable and economically appealing matrix [19,20].

However, free HT is not easily recovered from its conjugated forms using filtration technologies, resin absorption, or solvent extraction [16]. These methods often require large amounts of toxic solvents, which makes them unsuitable for environmentally friendly extraction processes. Alternatively, biotechnological enzymatic treatments, including driven fermentation with selected microbial pools, could arise as viable strategies for directly producing HT from OMWW [16].

By integrating recent findings (over the last ten years) and highlighting future research directions, this review provides a critical and up-to-date overview of the sustainable recovery of HT from OMWW, with a particular focus on emerging biotechnological enzymatic treatments. Emphasis is placed on cost-effective fermentation processes driven by selected microbial consortia and on the key operational parameters that influence HT release. This review aims to bridge knowledge from phytochemistry, microbiology, and process technology to support the development of clean-label, HT-enriched products, enhancing consumer health while reducing OMWW environmental impact.

2. Phytochemical Characteristics of Olive Mill Wastewater (OMWW) and Target Bioactive Constituents

In this section, the phytochemical composition of OMWW is discussed in detail, emphasizing valuable nutrients, particularly polyphenols. These compounds are known for their numerous functional properties and are highly sought after in various industrial sectors, including food, nutraceuticals, and cosmetics.

Generally, untreated OMWW appears as a muddy, dark liquid composed of sugars, oily residues, minerals, and phenolic compounds. Notably, high concentrations of ions, mainly magnesium, potassium, nitrogen, iron, and calcium, are present [21]. All these minerals are essential for the human body, and deficiencies in any of them can cause various health problems [22]. According to Zahi and co-workers [23], potassium levels in OMWW range from 0.73 to 8.6 g/L, and calcium levels can reach up to 1.1 g/L. Based on this, properly treated OMWW could be utilized as a sustainable source of these minerals in the food industry, including the beverage sector [24,25]. Lastly, soluble dietary fibers, mainly pectin, have been recovered from OMWW and used as excellent gelling agents for both food and biomedical purposes [26,27].

Strictly focusing on polyphenols, it has been observed that, according to their hydrophilic nature due to the presence of multiple hydroxyl (OH) groups in their structure, approximately 53% of their initial content is transferred from the olive fruit to the aqueous phase, specifically OMWW, around 45% in the pomace and only 2% in VOO [21].

Overall, polyphenol representatives in OMWW encompass a range of simple molecules, such as phenolic acids, and compounds with more complex molecular structures, such as flavonoids. Usually, glycosylated derivatives, namely phenol aglycones conjugated with mono-, di-, or oligosaccharides, are also detected [28]. In general, the differences in terms of chemical structures account for the variability in their modes of action and health properties. The main polyphenolic classes identified in OMWW are presented in

Table 1. Key polyphenolic constituents in olive mill wastewater (OMWW) and their main biological properties. Average concentrations calculated from reported means are shown.

Bioactive Compound	Content Range a	Biological Properties	Reference
Secoiridoids and derivatives
Hydroxytyrosol	110.2–469.1	Antioxidant, anti-inflammatory, chemopreventive, antimicrobial, skin bleaching, cardioprotective, and antiatherogenic	[29,30,31,32,33]
Hydroxytyrosol glucoside	78–1522	Antioxidant, antimicrobial	[34,35,36]
Ligstroside	23.8–35.0	Antioxidant, anti-inflammatory	[37,38]
Oleuropein	1.5–36.1	Antioxidant, neuroprotective, cardioprotective, antiatherogenic, hypoglycemic, antimicrobial, and antiviral	[29,31,32]
Tyrosol	9.5–89.7	Antioxidant, anti-inflammatory, antidepressive-like activity, chemoprotective, skin-protective	[29,32,39]
Verbascoside	49.0–271.3	Antioxidant, neuroprotective, chemoprotective	[31,32,40,41]
Phenolic acids
4-hydroxyphenyl acetic acid	8.5–274.0	Antioxidant, hypoglycemic	[11,32,42]
Caffeic acid	12.9–321.0	Antioxidant, antimicrobial, wound healing promoting	[11,31,43]
Cinnamic acid	1.2–4.8	Antioxidant, anti-inflammatory	[39,44,45]
p-coumaric acid	117.0–298.0	Antioxidant, anti-inflammatory, chemopreventive	[11,46,47]
Ferulic acid	70.2–95.0	Antioxidant, antimicrobial, photoprotective, hypoglycemic	[11,46,48]
Homovanillic acid	3.2–56.9	Antimicrobial	[29,49]
Syringic acid	10.3–30.6	Neuroprotective, hepatoprotective, cardioprotective, hypoglycemic	[39,44,50]
Vanillic acid	1.5–7.0	Cardioprotective	[29,31,39,51]
Flavonoids
Apigenin	0.1–1.9	Antioxidant, chemopreventive, hypoglycemic, antimicrobial, and antiviral	[29,52,53]
Luteolin	13.7–15.2	Antioxidant, cardioprotective, anti-inflammatory, anticonvulsant	[32,54,55]
Luteolin-7-O-glucoside	8.7–26.6	Antioxidant, anti-inflammatory	[29,56]
Naringin	INq	Antioxidant, anti-inflammatory, anti-ulcerative	[52,57]
Naringenin	INq	Antioxidant, anti-inflammatory, anti-infective, cardioprotective	[52,58]
Rutin	7.2–32.4	Antioxidant, chemopreventive, hepatoprotective	[29,33,44]
Quercitrin	INq	Antioxidant, anti-inflammatory, antimicrobial, analgesic	[59,60]
Lignans
Pinoresinol	13.1–78.7	Antioxidant, anti-inflammatory, chemopreventive	[29,31,61]
Carotenoids b
β-cryptoxanthin	0.1–0.6	Protective against osteoporosis, chemopreventive	[62,63]
Zeaxanthin	0.4–1.9	Protective against ocular diseases	[62,64]
Tocopherols b
α-tocopherol	15.6–39.1	Antioxidant, anti-aging, skin-protective	[62,65]
γ-tocopherol	2.6–5.6	Antioxidant, anti-aging, skin-protective	[62,65]

^a Concentrations are reported as mg/L unless indicated otherwise. ^b Carotenoids and tocopherols are expressed in μg/g of dry matter; INq: Identified but not quantified.

. They include secoiridoids and derivatives, phenolic acids, flavonoids, lignans, carotenoids, and tocopherols [28].

It is generally accepted that the most abundant phenolic compounds in OMWW are HT, tyrosol, and OLE [33]. However, some studies reported very low HT concentrations [29,62]. Similarly, OLE, which is biosynthetically formed from the esterification of HT with elenolic acid, was not detected by Lesage-Meessen and co-workers [66], found in low amounts by Lafka and colleagues [67], and identified as a main constituent by Visioli and co-workers [68]. Such significant variations in qualitative/quantitative data can probably be explained by transfer, transformation, and partition phenomena that occur during VOO extraction [62]. Each step in the process—such as olive crushing, paste malaxation, and VOO separation—affects the final phytochemical profile of matrices derived from Olea europaea L. [69]. This was demonstrated by Klen and colleagues, who, for the first time, in a single study, combined the phenolic constituents of all matrices involved in processing a Slovenian olive variety (Istrska belica cv): VOO, destoned fruits, stones, paste, pomace, and OMWW [59]. Using UPLC-DAD-ESI-QTOF-HRMS analysis, they detected eighty different phenols, including both native fruit compounds and their technological derivatives. Interestingly, apigenin aglycone was found in all olive by-products, including OMWW, but not in the native fruit, where only its glycosylated derivatives were present. According to the authors, the presence of new phenolic compounds in olive by-products could be ascribed to their technologically induced formation and/or release from any of the fruit compartments [59]. Similarly, rutin, luteolin, luteolin glycosides, quercitrin, and verbascoside were found in OMWW and other by-products but not in VOO [59].

Within the framework of the circular economy, the recovery of bioactive compounds from OMWW is a key focus. Several targeted molecules with excellent bioactive properties can be extracted from the OMWW produced during a specific VOO production process. However, a single olive polyphenol pathway from the fruits to the paste and final by-products (pomace or OMWW) cannot be established. Additionally, factors such as olive cultivar, harvesting time, seasonal variation, and storage duration strongly influence OMWW’s phytochemical profile and must be considered when developing effective valorization strategies.

3. Microbial Communities in OMWW

Microbial communities naturally found in OMWW consist of bacteria, yeasts, and molds [70,71], with densities ranging between 10⁵ and 10⁶ colony-forming units per milliliter (CFU/mL) [21]. The main microbial communities together with their functional properties are reported in Table 2. More specifically, the culturable fraction comprises a limited number of bacterial groups, including Firmicutes, Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. Among them, Gammaproteobacteria and Betaproteobacteria significantly prevail, constituting the most abundant identified communities [17]. In a study conducted by Tsiamis and colleagues [72], culture-dependent and -independent approaches (PhyloChip microarray) were applied to characterize the bacterial community of OMWW derived from three Greek olive cultivars: Kalamon, Xondroelia, and Ladoelia. The results revealed a common dominance of Proteobacteria (α, β, γ), Firmicutes, Actinobacteria, and Bacteroidetes, suggesting a shared basic microbiological structure among the varieties. Nevertheless, significant differences emerged among the microbiological profiles of individual cultivars. In detail, OMWW from Kalamon cv. was dominated by Klebsiella pneumoniae (49%) and Acinetobacter lwoffii (15%), with additional representatives of γ-Proteobacteria such as Stenotrophomonas maltophilia and Pseudomonas stutzeri [72]. In OMWW from Xondroelia cv., the prevalence of A. lwoffii (33%) and members of Enterobacteriaceae (K. pneumoniae, Serratia marcescens), followed by 13% of β-Proteobacteria (Oxalobacteraceae family), was detected. Finally, the OMWW from Ladoelia cv. showed a prevalence of Oxalobacteraceae, with Massilia timonae isolated exclusively from this sample, followed by Enterobacteriaceae and Moraxellaceae [72]. Although the OMWWs from the three cultivars share a similar phylotaxonomic core, this study underscores the pivotal role of olive variety as a key factor influencing the microbial composition of OMWW.

The Gammaproteobacteria group commonly includes members of the Enterobacteriaceae, Moraxellaceae, Xanthomonadaceae, and Pseudomonadaceae families, which together account for approximately 30% of OMWW’s total sequences. In parallel, Betaproteobacteria, comprising Oxalobacteraceae and other related genera, represent another relevant group. These two groups together represent about half of the bacterial 16S rRNA gene sequences available in the databases [17]. Alphaproteobacteria and Actinobacteria, represented by the Micrococcaceae, Microbacteriaceae, and Propionibacteriaceae families, account for approximately 20% of the identified microbial communities. Other phyla, such as Firmicutes, which include Bacillaceae, Clostridiaceae, Lactobacillaceae, and Paenibacillaceae, represent a minor fraction. Noteworthy, the presence of pathogenic bacteria has also been reported. In particular, fecal bacteria belonging to the Prevotellaceae, Lachnospiraceae, Peptococcaceae, and Peptostreptococcaceae families, as well as the Ruminococcus-Eubacterium-Clostridium group, have been frequently detected in OMWW [17,70]. It has also been reported that approximately 20% of the bacterial communities identified in OMWW are coliforms. Such communities include Acinetobacter, Enterobacter, Pseudomonas, Citrobacter, Escherichia, Klebsiella, and Serratia spp., as well as other enteric bacteria known for being opportunistic pathogens in humans. Moreover, their presence in OMWW even poses adverse environmental implications [70,73]. The presence of these harmful bacteria underscores the importance of the effective management of OMWW, particularly in terms of purification strategies [17].

Zooming in on yeast populations, the most frequently identified genera are Geotrichum, Candida, Pichia, Rhodotorula, and Saccharomyces [71]. The main species isolated by Sinigaglia and co-workers from OMWW obtained by a continuous process from two olive varieties (Peranzana and Ogliarola Garganica) of Southern Italy were Rhodotorula mucilaginosa, Pichia fermentans, Pichia holstii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida boidinii [74]. According to the authors, these microorganisms exhibited high enzymatic activity, particularly pectolytic and xylanolytic, which can be exploited to reduce the total phenolic content of OMWW, thus promoting its natural detoxification [74]. Bleve and co-workers [71] sampled OMWWs from five industrial oil mills in Salento (Puglia) and isolated 300 yeast strains belonging to the genera Geotrichum, Saccharomyces, Pichia, Rhodotorula, and Candida. Among them, Geotrichum candidum showed the ability to use OMWW as a carbon source, reducing COD and phenolic compounds. In another study, Ben Sassi and colleagues identified Pichia guilliermondii, Candida diddensiae, and Candida ernobii as the dominant yeasts in Moroccan OMWW [75].

Considering the fungal population, an analysis of GenBank sequences indicated that over 60% of fungal phylotypes isolated from OMWW belong to Basidiomycota, followed by Glomeromycota (19%) and unclassified fungi (17%), while Ascomycota were found as underrepresented (3%), probably due to the used primers [76,77]. Noteworthy, filamentous fungi, namely Aspergillus and Penicillium, have shown remarkable detoxification capabilities, strongly reducing OMWW’s phenol content. Lastly, despite being less commonly identified, Dothideomycetes (Alternaria) and Agaricomycetes (Phanerochaete, Trametes, and Pleurotus) exhibited high lignocellulolytic activity, which can be utilized for the removal of organic pollutants. Members of the genera Cerrena, Byssochlamys (syn. Paecilomyces), Lasiodiplodia, and Bionectria, identified through molecular techniques, are components of the OMWW indigenous microbiota and show the ability to degrade phenolic compounds [78]. In detail, Zaier and co-workers [79] collected 47 fungal isolates from OMWW sampled in different regions in Tunisia. The isolates, identified by ITS regions, belonged predominantly to the Aspergillus and Penicillium genera. Additional identified genera included Acremonium, Alternaria, Chalara, Fusarium, Lecythophora, Paecilomyces, Phoma, Phycomyces, Rhinocladiella, and Scopulariopsis. Screening for hydrolytic enzyme activities (lipase, protease, amylase, cellulase, invertase, phytase, tannase) revealed high metabolic potential under solid-state fermentation, indicating OMWW as a promising substrate for the biotechnological production of industrial enzymes [79].

Table 2. The main microbial communities in olive mill wastewater (OMWW) and their functional properties.

Microbial Group	Dominant Taxa	Key Functions/Roles	References
Bacteria	Gammaproteobacteria (Enterobacteriaceae, Moraxellaceae, Pseudomonadaceae, Xanthomonadaceae)	Dominant group (~30%), biodegradation, opportunistic pathogens	[70,72]
	Betaproteobacteria (Oxalobacteraceae and related genera)	Relevant in detoxification, co-dominant in microbial communities
	Actinobacteria (Micrococcaceae, Propionibacteriaceae, Microbacteriaceae)	Minor fraction, involved in nutrient cycling	[80]
	Bacteroidia (Prevotellaceae), Gammaproteobacteria (Acinetobacter, Enterobacter, Escherichia, Klebsiella), Clostridia (Clostridium)	Potential harmful pathogens with health/environmental risk	[73]
Yeasts	Microbotryomycetes (Rhodotorula)	Biodegradation of phenols and sugars, detoxification	[74]
	Saccharomycetes (Geotrichum candidum)	Ability to grow on OMWW, reducing COD, phenols, and antimicrobial compounds	[71]
	Saccharomycetes (Pichia, Candida, Saccharomyces)	Biodegradation, high enzymatic activity (pectolytic, xylanolytic, β-glucosidase, etc.)	[75]
Fungi (molds)	Eurotiomycetes (Penicillium, Aspergillus), Dothideomycetes (Alternaria), Agaricomycetes (Phanerochaete, Trametes, Pleurotus)	Strong biodegraders, phenol reduction, and detoxification	[17,75]
	Eurotiomycetes (Penicillium, Aspergillus), Dothideomycetes (Alternaria), Agaricomycetes (Phanerochaete, Trametes, Pleurotus)	Lignocellulolytic enzyme producers (low frequency)
	Others: Acremonium, Fusarium, Paecilomyces, Byssochlamys, etc.	Contribute to degradation, phenol reduction
	Aspergillus, Penicillium, Paecilomyces, Fusarium, Alternaria, Scopulariopsis	Multiple hydrolytic activities (lipase, protease, amylase, cellulase, pectinase, and tannase) are useful for biotechnological and environmental applications such as bioremediation and bioconversion	[79]
	Chalara, Lecythophora, Phoma, Rhinocladiella, Bionectria, Cerrena, Phycomyces	Ligninolytic and/or cellulolytic activity
	Acremonium, Scopulariopsis, Rhinocladiella	Selective proteolytic/lipolytic activity (i.e., specialized for specific protein or lipid substrates)

Table 2. The main microbial communities in olive mill wastewater (OMWW) and their functional properties.

Microbial Group	Dominant Taxa	Key Functions/Roles	References
Bacteria	Gammaproteobacteria (Enterobacteriaceae, Moraxellaceae, Pseudomonadaceae, Xanthomonadaceae)	Dominant group (~30%), biodegradation, opportunistic pathogens	[70,72]
	Betaproteobacteria (Oxalobacteraceae and related genera)	Relevant in detoxification, co-dominant in microbial communities
	Actinobacteria (Micrococcaceae, Propionibacteriaceae, Microbacteriaceae)	Minor fraction, involved in nutrient cycling	[80]
	Bacteroidia (Prevotellaceae), Gammaproteobacteria (Acinetobacter, Enterobacter, Escherichia, Klebsiella), Clostridia (Clostridium)	Potential harmful pathogens with health/environmental risk	[73]
Yeasts	Microbotryomycetes (Rhodotorula)	Biodegradation of phenols and sugars, detoxification	[74]
	Saccharomycetes (Geotrichum candidum)	Ability to grow on OMWW, reducing COD, phenols, and antimicrobial compounds	[71]
	Saccharomycetes (Pichia, Candida, Saccharomyces)	Biodegradation, high enzymatic activity (pectolytic, xylanolytic, β-glucosidase, etc.)	[75]
Fungi (molds)	Eurotiomycetes (Penicillium, Aspergillus), Dothideomycetes (Alternaria), Agaricomycetes (Phanerochaete, Trametes, Pleurotus)	Strong biodegraders, phenol reduction, and detoxification	[17,75]
	Eurotiomycetes (Penicillium, Aspergillus), Dothideomycetes (Alternaria), Agaricomycetes (Phanerochaete, Trametes, Pleurotus)	Lignocellulolytic enzyme producers (low frequency)
	Others: Acremonium, Fusarium, Paecilomyces, Byssochlamys, etc.	Contribute to degradation, phenol reduction
	Aspergillus, Penicillium, Paecilomyces, Fusarium, Alternaria, Scopulariopsis	Multiple hydrolytic activities (lipase, protease, amylase, cellulase, pectinase, and tannase) are useful for biotechnological and environmental applications such as bioremediation and bioconversion	[79]
	Chalara, Lecythophora, Phoma, Rhinocladiella, Bionectria, Cerrena, Phycomyces	Ligninolytic and/or cellulolytic activity
	Acremonium, Scopulariopsis, Rhinocladiella	Selective proteolytic/lipolytic activity (i.e., specialized for specific protein or lipid substrates)

4. Recovery Strategies and Commercial Relevance of Hydroxytyrosol (HT)

Among the various phenolic compounds found in OMWW, HT (3,4-dihydroxyphenylethanol) stands out due to its exceptional antioxidant capacity and wide range of health-promoting properties. Its presence in OMWW in relatively high concentrations (Table 1) has attracted growing interest in its recovery and application (Figure 2).

Compared to other natural antioxidants, such as ascorbic acid and vitamin E, HT exhibits superior capacity in protecting cells against oxidative stress [81]. This can be primarily attributed to its ortho-dihydroxy structure (Figure 2), which allows for efficient free radical scavenging and metal chelation [81]. In addition, HT also promotes the nuclear transcription of erythroid 2p45-related factor (Nrf2), which is implicated in the activation of several antioxidant/detoxifying pathways, including the synthesis of DNA repair proteins or phase II detoxifying enzymes [82].

Thanks to its antioxidant properties, HT is increasingly used as a natural preservative in the food industry. Qi and co-workers [83] reported that HT oleate can be effectively used as a safe antioxidant to prevent fatty acid oxidation and undesirable odor formation in olive oil [9]. Additionally, its excellent bioavailability and safety profile further boost its appeal for commercial applications, including in the nutraceutical sector. Today, HT is available in various products such as functional foods, beverages, and dietary supplements, often in capsule or tablet form [84]. Supplements may contain pure HT or standardized olive extracts with high HT levels, both marketed for cardiovascular health, metabolic support, and overall antioxidant benefits [84]. Numerous in vitro and in vivo studies have demonstrated HT’s anti-inflammatory, cardioprotective, neuroprotective, and antimicrobial effects, strengthening its role as a functional ingredient [81]. Importantly, HT’s health benefits gained regulatory recognition in 2011 when the European Food Safety Authority (EFSA) approved a health claim for HT and its derivatives related to protecting blood lipids from oxidative stress, with a daily intake of at least 5 mg per day [8].

The commercial significance of HT is largely influenced by improvements in its production methods. Efficient recovery and purification strategies are essential, requiring a combination of unit operations to achieve high HT yield and purity. The most effective processes integrate extraction, concentration, and chromatographic purification steps, often tailored to the source material. Specifically,

Table 3. Key unit operations in hydroxytyrosol (HT) recovery and purification from olive sources.

Unit Operations	Description	Typical Outcomes (Yield/Purity)	References
Liquid–liquid extraction	Counter-current or batch solvent extraction with organic (e.g., ethyl acetate, methanol, ethanol) or supramolecular solvents from olive waste and brines. Conditions (pH, solvent ratio, temperature) are optimized for maximum HT recovery.	Up to 88–90% recovery	[27,84,85,86]
Membrane filtration	Nanofiltration and reverse osmosis concentrate HT from aqueous extracts, separating it from sugars and other small molecules.	Concentration factors 7–9, stable solutions	[30,85]
Adsorption/desorption	Use of activated carbon or non-ionic resins to selectively adsorb HT, followed by elution (often with ethanol or water).	Up to 97% purity, 73–92% recovery	[24,87]
Chromatographic purification	Preparative liquid chromatography (C18, C8 columns), centrifugal partition chromatography, or HPLC to achieve high-purity HT.	>95–98% purity, not easily scalable	[16,88,89,90]

summarizes key operations applied for HT recovery and purification from olive sources, including table olives, leaves, pomace, and OMWW.

Traditional extraction from olive sources is being complemented and, in some cases, replaced by innovative chemical [91], enzymatic [92,93], and biotechnological methods, including microbial fermentation and metabolic engineering [94,95]. These approaches enable scalable, cost-effective, and sustainable production, making HT more accessible for industrial applications. Recent advancements in engineered microbial strains have greatly enhanced yields, further demonstrating their commercial viability [93,94,95]. The stability and effectiveness of HT have resulted in its integration into a wide range of commercial products. Its acknowledged health benefits and regulatory approval as a safe food supplement have contributed to a growing demand in the nutraceutical and functional food markets, a trend that is anticipated to persist in the coming years.

5. Hydrolytic Bioconversion of Olive Mill Wastewater (OMWW) for Hydroxytyrosol Production

The hydrolytic bioconversion of OMWW represents a promising approach to producing HT. Oligomeric olive polyphenols, including OLE, ligstroside, verbascoside, and HT glycosides, can undergo enzymatic hydrolysis, facilitating the release of HT from its conjugated forms. As shown in Figure 3, endogenous β-glucosidase (β-Glu) and esterase, which can be derived from olive fruit and microorganisms, are responsible for the hydrolytic cleavage of the glycosidic and ester bonds, respectively, present in olive secoiridoids.

Consequently, phenolic compounds abundantly present in undervalued OMWW can be effectively recovered through hydrolytic bioconversion. From a sustainable perspective, enzymatic and microbial bioconversion strategies provide environmentally friendly methods to valorize agro-industrial residues, simultaneously reducing pollution and producing high-value bioactive compounds. These processes closely align with the principles of the circular bioeconomy and the European Green Deal [21].

Enzymatic bioconversion is especially effective for producing HT from OLE-rich sources, such as OMWW. This method uses specific enzymes or engineered microbial systems to efficiently transform more complex compounds into HT, providing a scalable alternative to chemical synthesis. Additionally, it combines catalytic specificity, high production efficiency, and reduced environmental impact, aligning with green biotechnology principles [97]. For example, Hamza and Sayadi [98] demonstrated the successful biotransformation of rapeseed leaf extract and OMWW using fungal enzymes, reaching HT concentrations up to 1.1 g/L. Similarly, Macedo and colleagues [99] enhanced phenolic extraction and conversion from olive pomace using a combination of microwaves and pectinolytic, cellulolytic, and tannic enzymes, producing up to 59.29 mg of HT per kg of pomace.

Among the most efficient strategies, a two-stage enzymatic cascade for converting OLE into HT has been described. Firstly, hydrolysis is carried out by a thermo-halophilic β-glucosidase from Alicyclobacillus herbarius, allowing for the achievement of the complete conversion (>99%) of OLE into HT within 30 min. Then, an acylation step using an acyltransferase from Mycobacterium smegmatis is applied, yielding over 99% of HT in 24 h [100]. Dammak and co-workers evaluated the effectiveness of a local enzyme preparation from Aspergillus niger and six commercial fungal enzyme formulations for the hydrolysis of OMWW, highlighting a significant increase in HT release and reducing carbohydrates, with yields of up to 0.87% and 9.7%, respectively [101].

Another promising strategy is enzyme immobilization, where enzymes, like β-glucosidase and lipase, are fixed on supports such as chitosan-coated magnetic nanoparticles, mesoporous silica, or calcium alginate beads. This improves enzyme stability, reusability, and operational efficiency, making the process suitable for industrial applications [102,103].

In summary, the integration of microbial enzymes, non-genetically modified microorganisms, with OMWW exemplifies effective biotechnological models for HT production. Such approaches support the sustainable production of a high-value bioactive compound, contributing to waste reduction and resource recovery. Ongoing advances in biocatalyst optimization, microbial selection, and process integration are paving the way for scalable, eco-friendly HT production for food, nutraceutical, cosmetic, and pharmaceutical applications.

5.1. Spontaneous Fermentation in OMWW

Recently, a fourfold increase in HT levels has been observed by Papadaki and co-workers [104] in the spontaneous fermentation of OMWW of Chalkidiki cv processed in the Spanish style by indigenous lactic acid bacteria (LAB), namely Enterococcus casseliflavus and the plant-associated bacterium Bacillus amyloliquefaciens subsp. plantarum, for 120 or 150 days, underscoring the dynamic nature of HT recovery from OMWW. Analogously, Feki and colleagues [105] observed a simultaneous OMWW enrichment in HT and a notable decrease in other olive polyphenol content after spontaneous fermentation at 25 °C for 4–5 months. This evolution was attributed to hydrolysis reactions, likely performed by enzymes or microorganisms naturally occurring in OMWW. Accordingly, Ciafardini and co-workers attributed the disappearance of the bitter taste of a newly produced olive oil to the β-Glu hydrolysis of the bitter-tasting secoiridoid compounds during storage [106]. Similarly, Romero and colleagues reported changes in the phenolic profile of black olives during their natural fermentation, demonstrating that at the beginning of storage, the olive juice contained HT, hydroxytyrosol-4-β-glucoside, OLE, tyrosol, salidroside, and verbascoside. At the same time, after 12 months, the main phenol was HT [36]. In the context of biotechnological applications aimed at enhancing OMWW and recovering fractions rich in phenolic compounds such as HT, Nanis and co-workers [107] explored the valorization of OMWW solids through natural fermentation in saline solution (6% w/v NaCl) for 14 days at 20 °C, to obtain a product like olive paste. Microbiological analysis showed that fermentation was sustained exclusively by yeasts, in particular S. cerevisiae and P. membranifaciens, while no enterobacteria, pseudomonas, staphylococci, or LAB were detected. The molecular characterization of the isolates by the Restriction Fragment Length Polymorphism (RFLP) technique and sequencing of the 5.8S ITS region confirmed a high homology with the reference species. The main observed biochemical activity was that of β-glucosidase, which is involved in reducing the typical bitter taste of the matrix. From a sensory point of view, the pastas obtained showed no significant differences compared to conventional controls, suggesting that the spontaneous fermentation of OMWW solids represents a technologically valid and sustainable option for the recovery of agro-industrial by-products and the production of new added-value foods.

These findings provided a strong foundation for developing an integrated biorefinery that combines OMWW bioremediation with HT recovery. The fermentation of OMWW driven by microorganisms can effectively produce both bioaugmentation agents or starter cultures and a high-value compound, HT, in a cost-efficient manner.

5.2. Driven Microbial Fermentation

The enzymatic activity of selected microbial strains to convert metabolic precursors into HT offers a promising strategy for producing HT-rich matrices, from which the compound can be extracted or used directly as a functional ingredient. Fermentation-based strategies using selected yeast have also demonstrated potential, although HT yields remain limited (maximum 6.12 ng/mL) and are strongly influenced by precursor concentration and medium composition [108].

One key contributor to the enzymatic treatment of OMWW is β-glucosidase, specifically produced by yeasts such as A. niger, C. boidinii, and Wickerhamomyces anomalus [109,110], as well as by LAB, such as Lactiplantibacillus plantarum, isolated from naturally fermented table olives [111]. Research by Hamza and co-workers [112] highlights that when OMWW is treated with an enzyme mix rich in β-glucosidase, a significant release of high-value simple phenolics is found, thereby improving OMWW’s antioxidant properties. Subsequent studies by the same authors confirmed that the enzymatic approach yielded substantial amounts of HT, reaching a maximum of 1.53 g/L under optimized conditions [110]. Notably, the authors reported that enzyme treatment followed by ultrafiltration increased the recovery of HT to 7.2 g/L and significantly reduced the COD value, leading to a new natural healthy product [113]. These findings demonstrate the connection between using specific enzymes and decreasing common pollutants in OMWW.

In addition to β-glucosidase, esterase enzymes have also shown potential. For example, esterase from Aureobasidium pullulans has been effectively produced in culture broth supplemented with OMWW, indicating its possible use in synergistic enzymatic formulations [114]. Recently, strains belonging to Pseudomonas [17], Serratia, transformed Escherichia coli, or Halomonas have been found as able to convert tyrosol to HT [115]. Bouallagui and colleagues [115] used a strain of Pseudomonas, isolated from soil irrigated with OMWW, for the bioconversion of tyrosol into HT in laboratory fermenters. The optimization of operating conditions yielded 86.9% of 37.3 mM HT from 43 mM of tyrosol, with a specific productivity of 4.78 µM/min/g. The process was validated on a lab scale and allowed for the recovery of an HT-rich solution, with a final purity of 73.8% of the dry weight. The key differences between spontaneous and driven fermentation strategies for OMWW valorization are reported in Figure 4.

5.3. Microbiological and Regulatory Assessment of OMWW for Food Applications

The valorization of OMWW as a functional ingredient in food systems is currently hindered by some critical challenges, mainly technical and microbiological issues, as well as regulatory constraints. Under the current European legislative framework, OMWW is legally classified as an agro-industrial waste. According to Article 6 of Directive 2008/98/EC [116], a substance ceases to be considered waste only when it satisfies specific end-of-waste criteria, including the following: (i) the substance is intended for a specific use; (ii) there is an existing market or demand for it; (iii) it meets relevant technical and legal requirements for the intended use; and (iv) its use does not lead to overall adverse effects on human health or the environment. Despite the growing interest in the bioactive potential of OMWW, particularly its richness in phenolic compounds with antioxidant and antimicrobial activity, there are currently no harmonized EU-wide end-of-waste criteria applicable to OMWW for food-grade uses. As a result, its use in food production remains prohibited unless reclassification is granted through novel regulatory pathways or specific authorizations. Therefore, any future application of OMWW in the food chain must be supported by compliance with food hygiene regulations, such as Regulation (EC) No. 178/2002 [117] and Regulation (EC) No. 852/2004 [118], which require that all food ingredients derive from safe, approved, and traceable sources. Such evaluation is crucial before incorporating plant-derived products into food systems, as their natural origin often subjects them to microbial contamination from environmental sources such as soil, water, and air, thereby increasing the risk of transmitting undesirable microorganisms.

Typically, untreated fresh OMWW appears muddy due to a high concentration of suspended solid particles and undesirable pathogenic and opportunistic microorganisms, including E. coli, Pseudomonas spp., Klebsiella spp., and Clostridium spp. [17,70,73]. This poses critical technical and microbiological challenges that need to be addressed before the potential application of OMWW in the food sector. According to the European Regulation (EC) No. 1441/2007 [119], the absence of pathogens, such as Salmonella spp. and Listeria monocytogenes, is considered an essential criterion for the microbiological safety of vegetable products. While no mandatory microbiological criterion is set for the total aerobic mesophilic bacteria, some guidelines include E. coli and total aerobic mesophilic count as quality parameters. The following thresholds are generally applied: E. coli counts <10 CFU/g are considered satisfactory, counts 10 to ≤10² CFU/g are acceptable, and those >10² CFU/g are not acceptable. For total aerobic mesophilic count, ≤10⁴ CFU/g is satisfactory, from 10⁴ to 10⁶ CFU/g is acceptable, and >10⁶ CFU/g is not acceptable [120].

However, recent studies have provided encouraging data on the microbial profile of OMWW obtained from conventionally farmed olives. In detail, a specific screening for spoilage and pathogenic microorganisms (including Pseudomonas spp., yeasts, molds, aerobic spore-forming bacteria, coagulase-positive staphylococci, L. monocytogenes, members of the Enterobacteriaceae family, E. coli, and Salmonella spp.) performed by Sciurba and co-workers revealed no detectable levels (<1 log CFU/mL) in any of the OMWW samples tested [121]. Nevertheless, the same study also identified the presence of pesticide residues and micro-contaminants, underscoring an additional critical concern beyond microbiological safety. In a subsequent study, the same authors suggested using OMWW from organic olive farms as a safer alternative due to the lower risk of chemical contaminants compared to conventionally derived OMWW [122].

Purification processes are crucial for enhancing the microbiological safety of OMWW and preserving its phenolic content, ultimately rendering it a suitable substrate for fermentation and food production [30].

Numerous filtration treatments have been proposed, remarkably improving microbiological traits while maintaining the nutritional properties of its phenolic constituents [19]. OMWW is usually subjected to an initial purification step directly on the farm using cartridge filters with varying porosity. Foti and colleagues [30] applied successive filtration stages using specialized filters, namely Oenopad^® XF1, XF7, and XFSS. Such filters are designed to remove solids, clarify wastewater, and achieve complete sterilization with the XFSS filter (0.20–0.40 µm) employed at the final stage. This study highlighted microfiltration as an effective method for obtaining clarified and sterilized OMWW in a single step. Therefore, sterile filtration emerges as an effective strategy to overcome the challenges posed by the presence of opportunistic and pathogenic microorganisms.

6. OMWW as Functional Foods and Beverages

The growing demand for nutrient-rich and safe foods is accompanied by concerns about waste accumulation and a focus on the sustainable use of agro-industrial by-products [123]. In this context, the replacement of synthetic food preservatives with natural alternatives derived from plant matrices and agri-food processing residues is of considerable interest [124,125]. Some of these residues are rich in antioxidant compounds with promising effects in prolonging shelf life [126].

Given the diverse applications of phenolic extracts derived from OMWW in the food and beverage industry,

Table 4. Innovative uses of olive mill wastewater (OMWW) as a source of bioactive compounds with antimicrobial, antioxidant, and functional properties, applicable in both food preservation and the development of health-promoting products.

Product	OMWW Use	Main Characteristics	Applications	References
Natural preservatives	Phenolic extracts from OMWW	Antimicrobial, antifungal, antioxidant activity	Shelf life extension of food; alternative to synthetic preservatives	[127,128,129,130]
Encapsulated phenolics	OMWW bioactives encapsulated for food use	Stabilized, protected from degradation, controlled release	Functional ingredients in food matrices	[131]
Meat additives	OMWW phenols in raw meat products	Improved hygiene, oxidation prevention	Meat preservation and quality enhancement	[132]
Fermented sausages	OMWW extracts in fermented meat products	Antifungal activity against spoilage molds and spores	Clean-label antifungal agents	[133]
Functional beverages	Fermented OMWW with selected microorganisms	Increased HT and total phenolics; antioxidant and anti-inflammatory properties	Functional drinks with health benefits	[30,134]
Enriched fruit juice	Blood orange juice fortified with OMWW polyphenol concentrate	High HT content; EFSA-compatible levels after storage	Nutraceutical beverage; sensory acceptable	[8,24]
Acetic beverages	OMWW fermented into vinegar-like drinks (static and submerged)	Rich in acetic/gluconic acid; high antioxidant activity	Low-alcohol functional beverage	[134]
Olive wine	Spontaneous fermentation of OMWW	Contains HT, flavonoids, polyphenols; antioxidant activity in vitro and in vivo	Beverage with potential oxidative stress prevention abilities in animal models	[135]

provides an overview of the main products developed and their characteristics. As shown, OMWW can be effectively utilized to create various functional products, including natural preservatives, meat additives, fermented beverages, and enriched fruit juices. These applications leverage the high content of bioactive phenolic compounds, which possess antimicrobial, antioxidant, and antifungal properties. Additionally, several biotechnological strategies, such as fermentation, encapsulation, and membrane filtration, have been employed to enhance the stability and bioavailability of these compounds within food matrices. Overall, the data suggest that the potential industrial use of phenolic compounds from OMWW as clean-label functional ingredients is promising.

Several studies have explored the incorporation of OMWW into innovative food products and beverages [123,134]. Specifically, biotechnological advancements have enabled the recovery, stabilization, and biotransformation of OMWW phenolics into bioavailable forms. Additionally, encapsulation strategies offer the advantage of protecting the active compounds from degradation, improving their stability, and allowing for controlled release within food matrices [136].

If properly treated, the biophenols present in OMWW can be used in the food industry to prolong shelf life, given their high antimicrobial and antifungal activity. Obied and co-workers [127] documented that phenolic fractions derived from OMWW exhibit antibacterial activity against a wide range of spoilage and harmful bacterial species, such as Staphylococcus aureus, Bacillus subtilis, E. coli, and Pseudomonas aeruginosa. In the same line, Serra and co-workers [128] demonstrated that natural OMWW extracts were more effective in suppressing microbial growth than isolated biophenols such as quercetin, HT, and OLE when individually used. Tafesh and co-workers highlighted that individual isolated phenols were often less effective against major pathogens, including E. coli, Klebsiella pneumoniae, S. aureus, and Streptococcus pyogenes, while the entire phytocomplex obtained from OMWW maintained broad-spectrum antibacterial activity, against both Gram-positive and Gram-negative species [129]. Lastly, Fasolato and co-workers observed that the bactericidal effects of phenolic extracts purified from OMWW may also be useful in inhibiting the growth of technologically relevant starter cultures, including Staphylococcus xylosus and Lactobacillus curvatus, which were inhibited at relatively low concentrations (0.75 and 1.5 mg/mL, respectively) [130]. These studies highlight the remarkable antimicrobial potential of phenols extracted from OMWW against a wide spectrum of pathogenic and spoilage bacteria. The synergy between compounds is crucial; entire extracts are often more effective than isolated phenols, as already shown [128,129]. This aspect is significant for developing more potent natural preservatives.

However, Fasolato and co-workers highlight a crucial limitation: the poor selectivity of these extracts, which, at low concentrations, also inhibit useful microorganisms [130]. This side effect could compromise their application in fermented foods, where the vitality of starter cultures is essential.

Of particular interest is the use of OMWW extracts as functional ingredients for meat and meat-based preparations [132], where their addition as bioactive ingredients has been associated with improvements in both the hygienic status and rheological characteristics of the final products. In particular, olive phenolic compounds have shown higher efficacy in preventing lipid oxidation, especially in raw meat formulations, as evidenced by the TBARS test conducted after 72 h of storage at 4 °C, with dose-dependent results (100 mg/L for olive phenols) [132]. Even Lopez and colleagues successfully applied OMWW-derived polyphenols to ferment sausages. Specifically, the addition of 2.5% OMWW extract showed, both in vitro and in situ, the significant inhibition of Cladosporium cladosporioides, Penicillium aurantiogriseum, Penicillium commune, and Eurotium amstelodami, as well as their respective spores [133].

Lastly, OMWW can be processed and fermented with specific microbial strains to obtain beverages with high phenolic content and health benefits. In a recent study, fermented OMWW was proposed for developing an innovative functional beverage. After pre-treatment by filtration and microfiltration, OMWW was fermented using L. plantarum, C. boidinii, and W. anomalus. Fermentation led to increased levels of HT (up to 925 mg/L) and total phenolics, along with enhanced antioxidant, anti-inflammatory, and intestinal permeability properties. These results support the feasibility of recycling OMWW through microbial fermentation to produce health-promoting beverages [30]. Similarly, Foti and co-workers applied a tangential membrane filtration system to recover phenolic compounds from OMWW. Among the obtained fractions, the reverse osmosis concentrate showed the highest HT content (7203.7 mg/L), along with greater oxidant and antimicrobial properties [24]. This concentrate was then added to a commercial blood orange juice in different ratios (up to 4:250 v/v). In detail, formulations containing up to 2:250 v/v were sensorially acceptable. After two months of refrigerated storage, the enriched blood orange juice maintained HT levels compatible with the daily dose recommended by EFSA for health claims [8]. In the context of reusing OMWW in beverages, an interesting approach was provided by Signorello and co-workers, through selective fermentation aimed at producing a low-alcohol acetic beverage [134]. The OMWW, diluted to 100%, 75%, and 50%, was initially fermented with S. cerevisiae UMCC 855, and the product derived from the 75% matrix was further fermented by Acetobacter pasteurianus UMCC 1754 under static and submerged conditions. Static fermentation led to the depletion of residual ethanol and high concentrations of acetic acid (46.85 g/L) and gluconic acid (44.87 g/L), while the submerged process produced 31.63 g/L of acetic acid and 39.90 g/L of gluconic acid, maintaining a residual ethanol content (24.74 g/L). Furthermore, the results showed an enhancement in the antioxidant activity of the treated matrices, highlighting interesting prospects for the sustainable and functional recovery of this agro-industrial waste [134]. Among the various biotechnological approaches aimed at enhancing OMWW through fermentation, an interesting study recently described the production of an olive wine through spontaneous fermentation, evaluating its antioxidant activity in vitro and in vivo, demonstrating protective effects against oxidative stress in animal models [135]. In detail, to verify whether this process allowed for the original bioactivity of the matrix, the antioxidant potential of the obtained olive wine produced by the spontaneous fermentation of OMWW was evaluated. The obtained wine contained HT, total flavonoids, and total polyphenols equal to 0.14, 0.29, and 0.43 mg/mL, respectively, showing good free radical scavenging capacity with IC₅₀ values of 2.5% against DPPH and 3.2% against hydroxyl radicals. In in vivo tests, administration for 30 days to elderly mice significantly reduced malondialdehyde levels in the liver and carbonyl proteins in plasma and increased superoxide dismutase activity in both the plasma and liver, effects that were superior to those of γ-tocopherol. These results indicate that the spontaneous fermentation of OMWW not only mitigates the environmental impact of wastewater but also yields a functional product with antioxidant properties that are potentially useful in counteracting oxidative stress.

Overall, data on the potential industrial use of phenolic compounds from OMWW as clean-label functional ingredients for foods and beverages are promising.

6.1. Impact on Food Sensory Characteristics

While the incorporation of OMWW-derived compounds into food products offers numerous functional and health-related benefits, it also poses challenges from a sensory standpoint. HT and OLE are known for their intense bitterness and astringency, which can alter the organoleptic properties of the final product. The impact on sensory quality depends on multiple factors, including the type and concentration of phenolics, the food matrix, and the method of integration [36,120].

In dairy products, low HT levels have been shown to preserve sensory acceptability while enhancing oxidative stability. For example, soft cheeses enriched with an olive leaf extract exhibited improved antioxidant and antimicrobial activity. However, significant color changes were observed [136].

In bakery products, the inclusion of OMWW phenolics has been reported to improve crust color and delay staling, although excessive concentrations may impart off-flavors [122].

In beverages, particularly functional waters and plant-based drinks, the bitterness of OMWW phenolics often necessitates the use of flavor masking agents, natural sweeteners, or microencapsulation techniques to maintain palatability [25].

Encapsulation technologies such as loaded nanoparticles are emerging strategies used to incorporate both phenolics and microbial strains into food products while attenuating their negative sensory impact and increasing stability [25,136]. These systems can protect bioactive compounds from degradation, enhance solubility, and control release in the gastrointestinal tract while improving overall sensory appeal [136]. Therefore, the successful application of OMWW in functional foods depends on a careful balance between health benefits and consumer acceptability, which must be optimized through formulation and processing strategies [131,136].

6.2. Effect of Storage of Olive Mill Wastewater on Hydroxytyrosol Concentration

Storage conditions significantly influence the concentration and stability of HT in OMWW. This phenolic compound, which derives from the hydrolysis of OLE and tyrosol, can accumulate or be degraded depending on variables such as time, temperature, pH, oxygen exposure, and the presence of stabilizing agents like ethanol. Several studies have demonstrated that HT concentration generally increases over time in OMWW stored under anaerobic and ethanol-free conditions. For example, OMWW samples stored for five months without ethanol showed increased HT levels from 0.98 to 3.5 g/L and from 0.77 to 3.1 g/L in two separate trials [105]. This increase was attributed to the breakdown of more complex phenolic compounds, which diminish in parallel with the accumulation of HT [105]. Conversely, the addition of 10% ethanol was shown to stabilize HT levels during storage, preventing further increase, while lower or absent ethanol concentrations allowed for continued HT formation.

Stability is of crucial importance for OMWW extracts obtained through membrane-based purification methods, including reverse osmosis. These concentrated extracts demonstrated long-term stability, with minimal degradation of HT even after 24 months of refrigerated storage [24,137]. Interestingly, the pre-storage of OMWW before extraction also enhances yield. Studies have reported an increase in extraction efficiency from 85.5% to 96.8% when OMWW was stored before phenolic recovery [105]. This finding highlights the potential for strategic storage protocols to improve both the yield and cost-effectiveness of HT extraction, particularly for industrial applications where OMWW-derived concentrates may serve as natural antioxidants in food products.

Temperature is a key factor in phenolic stability. While the room-temperature storage of concentrated extracts results in minimal HT degradation, high temperatures are associated with a 20–24% reduction in total phenolic content [138]. Oxygen exposure, especially at lower temperatures, is another critical variable; samples stored under inert conditions, such as nitrogen, maintain greater phenolic integrity. The chemical stability of HT is also influenced by pH. Acidic conditions (such as pH 2.0) significantly accelerate the degradation of phenolics like OLE and HT, while neutral to mildly acidic environments offer much greater stability [138]. For instance, degradation rates can be up to 100 times slower at higher pH levels [139]. Other phenolics, such as verbascoside, display compound-specific degradation kinetics that are also pH-dependent. Similar trends are observed in thermal and oxidative degradation systems, where pH affects both rate constants and half-lives [139].

In summary, the storage of OMWW under optimized conditions, including the absence of ethanol, controlled temperature, limited oxygen exposure, and neutral pH, can significantly increase or preserve HT content. Concentrated extracts derived from OMWW demonstrate remarkable stability over extended periods, supporting their use as functional ingredients in food and nutraceutical applications.

6.3. Feasibility and Viability of OMWW Application in Food

The application of OMWW in food is receiving increasing attention due to its potential for recovering high-value bioactive compounds, particularly HT. However, despite promising laboratory and pilot-scale results, large-scale and economically viable implementation remains limited by technical, regulatory, and market barriers. Nonetheless, several laboratory and pre-industrial studies have demonstrated the feasibility of extracting and formulating phenolic compounds derived from OMWW into functional products, such as ophthalmic nutraceuticals, with proven in vitro safety and bioactivity [140,141].

Regarding the recovery of bioactive compounds from OMWW, several technologies have been investigated, including adsorption, membrane filtration, and the use of natural deep eutectic solvents (NADESs) [142]. Although these methods have demonstrated high extraction efficiency and selectivity at a small scale, their scale-up for industrial use still presents critical technoeconomic challenges. These include the need to optimize the process; manage the high variability in OMWW composition across years, which could pose economic losses for companies; and, most importantly, integrate these technologies into existing food production infrastructures [140,141]. The cost-effectiveness of the recovery process is also highly dependent on the technologies employed. Methods such as membrane filtration and adsorption have demonstrated high HT yields and product stability; however, their adoption by small and medium enterprises (SMEs) is often limited by the cost of equipment, maintenance (e.g., membrane fouling), and solvent use. Nevertheless, recent studies suggest that simple strategies, such as the pre-storage of OMWW under optimized conditions (e.g., anaerobic, ethanol-free, neutral pH), can significantly enhance HT formation and improve extraction efficiency, offering a low-cost solution to increase the economic sustainability of the process [105]. The economic success of OMWW valorization relies on three key factors: the value of the recovered compounds, the efficiency and scalability of the recovery process, and the demand in the target market. Although the market for OMWW-derived food ingredients is still emerging, its growth potential is supported by increasing consumer demand for clean-label, natural antioxidants and bioactive ingredients [143].

Another major limitation for incorporating OMWW into foods lies in the sensory characteristics. As discussed, major phenols such as HT and oleuropein (OLE) exhibit a marked bitter and astringent taste, which can compromise the sensory quality of food products. Although encapsulation and advanced formulation strategies can reduce these undesirable effects, they often lead to greater technological complexity and higher production costs. Therefore, further research is needed to develop products that preserve the functional benefits of OMWW compounds and meet consumer expectations in terms of taste, appearance, and overall acceptability. In this context, fermentation could represent a promising strategy for both improving the bioavailability of HT in OMWW and modulating flavor and aroma, enhancing the overall sensory appeal of OMWW-enriched products. For example, the enzymatic hydrolysis of plant proteins during fermentation can generate peptides with flavor-enhancing properties, helping to mask the unpleasant notes of by-products and improve their palatability [144]. Recent studies have demonstrated how the use of aroma-producing yeasts or the combination of yeast and LAB can play a key role in enhancing sensory quality. The use of controlled fermentation in the production of fermented beverages and sauces has enhanced aroma by generating volatile compounds, such as esters, alcohols, and organic acids [145,146,147,148]. These compounds contribute fruity, floral, and buttery notes. Such strategies could be adapted for formulations based on OMWW to improve both sensory properties and consumer acceptance. Furthermore, food safety and regulatory compliance remain key issues. Current European Union legislation does not comprehensively address the use of by-products such as OMWW in food applications. This regulatory gap often forces manufacturers to rely on private quality standards or apply for ad hoc authorizations, thus increasing costs and delaying time to market [149]. Overcoming these legal hurdles will be crucial for enabling the broader adoption of OMWW-derived ingredients in the food sector.

7. Conclusions and Future Perspectives

Currently, approximately one-third of food produced globally is lost or wasted along the food chain. This inefficiency underscores the urgent need to reconfigure food systems toward sustainability. In this context, the valorization and reuse of agro-industrial by-products represent strategic tools for reducing waste and promoting new circular economy-based economic systems. In the olive oil sector, despite the increasing adoption of two-phase extraction systems, which reduce water consumption compared to the traditional three-phase processes, the disposal of OMWW remains a critical environmental and economic issue, especially for small-scale olive oil producers in Italy and other Mediterranean countries. OMWW valorization is thus becoming an essential priority for the agri-food industry. By reconsidering OMWW not as waste but as a resource, it becomes possible to recover valuable bioactive compounds, especially HT, which have demonstrated strong antioxidant, anti-inflammatory, and antimicrobial properties. A summary of studies examining the potential reuse of OMWW in beverages and food suggests that fermentation, along with selective pre-treatment methods such as microfiltration and tangential filtration, can transform OMWW from a waste product into a valuable resource for producing functional foods.

The increase in HT and total polyphenol levels, combined with improved antioxidant and anti-inflammatory activity and intestinal barrier properties, shows that these processes are not limited to reducing environmental impact, but they generate matrices with real nutraceutical value. The multidimensional approach, which includes lactic and acetic fermentation, spontaneous fermentation, and integration with commercial juices, demonstrates its versatility and potential for industrial scalability. Particularly relevant is the possibility of obtaining products that retain HT concentrations compatible with EFSA health claim requirements [8], opening concrete prospects for the development of certified functional beverages. These strategies exemplify an effective model of a circular economy, yielding both environmental and economic benefits. Although significant research has been conducted on the chemical and biochemical properties of olive by-products, additional studies are necessary to improve extraction, processing, and application methods in order to fully realize their value. Recovering substantial quantities of HT from OMWW is technically feasible, but challenges persist. Seasonal and batch variability significantly affect HT content and OMWW composition, hindering reproducibility and complicating industrial scale-up. Future management strategies for OMWW should therefore involve a combination of physical and biotechnological processes, including membrane filtration, fermentation, and enzymatic treatments, followed by stabilization and formulation steps. The integration of advanced recovery technologies and biotechnological approaches for OMWW valorization, coupled with the broad health potential of HT, offers a compelling path toward sustainable innovation in the food system and beyond.

To move toward industrial scalability and standardization, future research should focus on several key areas. First, the development of robust and well-characterized microbial consortia tailored for OMWW fermentation could improve process stability, enhance phenolic biotransformation, and allow for better adaptation to variable matrices. Additionally, enzyme engineering, aimed at improving the specificity, catalytic efficiency, and stability of hydrolases, esterases, and oxidoreductases involved in HT release, represents a promising approach to optimizing extraction and detoxification steps. In parallel, efficient immobilization techniques for both microbial cells and enzymes could enable reuse across multiple fermentation cycles, lowering process costs and increasing yield consistency. Finally, applying comprehensive life cycle assessment (LCA) methods is crucial to quantify the true environmental and economic impacts of OMWW valorization strategies, pinpointing key issues and guiding the development of sustainable, circular processes. Despite these promising developments, the practical implementation of OMWW valorization in the food sector remains limited. Due to the constraints discussed above, OMWW reuse is currently mostly oriented toward biofuel production, as such processes are generally more scalable and face fewer regulatory barriers. However, as highlighted in this review, it is crucial to reconsider OMWW as a high-value by-product with significant potential in the food and pharmaceutical industries. Its high HT content makes it a valuable matrix for the development of innovative, bioactive, and sustainable products intended to promote human health. To enable the translation of scientific advances into viable industrial applications, it is essential to overcome current regulatory and economic obstacles.