The growing interest in novel functional foods and naturally derived bioactive ingredients has led to intensified research on plant-based compounds with potential health-promoting effects. Oxidative stress, defined as an imbalance between antioxidant capacity and reactive oxygen species formation, plays a significant role in the development of chronic diseases. Since synthetic antioxidants have limitations and may cause side effects, consumers are increasingly demanding clean label or natural foods that are minimally processed. In addition, the need to reduce the environmental impact of the food industry through sustainable production and circular economy strategies has increased interest in the valorisation of agro-industrial by-products, such as olive leaves and green walnut husks, as sources of high-value bioactive compounds (1-3).

Among such natural sources, extracts of olive leaf, thyme, and green walnut husk are particularly rich in polyphenolic components. These raw materials are especially relevant from a sustainability perspective, as they largely originate from by-products of the food industry and therefore represent an underutilised resource with high potential for value-added applications. In addition to being investigated in both in vitro and in vivo systems, their incorporation into food products, especially those with added functional value, is increasingly being explored. Dairy products such as yogurt, cheese and fermented beverages are considered suitable carriers for such bioactive ingredients due to their widespread consumption, favourable technological compatibility, and the fact that they are naturally low in polyphenolic compounds, which makes their enrichment particularly meaningful. Furthermore, functional dairy products belong to the most frequently consumed category of functional foods, highlighting their relevance for the delivery of plant-derived antioxidants and other beneficial constituents. Incorporating such extracts into dairy products may enhance their stability, improve nutritional and functional properties, and potentially contribute to health-promoting effects (4). Although several reviews have addressed plant-derived polyphenols, natural antioxidants or functional dairy products in general, there is still a lack of focused synthesis comparing olive leaf, thyme and green walnut husk extracts specifically in the context of dairy matrices. In particular, limited attention has been given to the relationship between their phytochemical composition, extraction approaches, technological performance, sensory limitations, regulatory aspects and the level of evidence supporting their functionality in real dairy products. Therefore, this review provides an overview of the polyphenolic profiles and antioxidant properties of olive leaf, thyme and green walnut husk extracts, as well as their technological performance, sensory implications and potential applications in functional dairy products.

This narrative review was prepared based on a literature search conducted in scientific databases including Web of Science, Scopus, PubMed, ScienceDirect and Google Scholar. The search focused primarily on publications from 2000 to 2026, with particular emphasis on recent studies published during the last ten years. The following keywords and their combinations were used: “olive leaf extract”, “thyme extract”, “green walnut husk extract”, “polyphenols”, “phenolic compounds”, “antioxidant activity”, “antimicrobial activity”, “dairy products”, “yogurt/yoghurt”, “cheese”, “fermented milk”, “milk matrix”, “functional foods”, “green extraction” and “sustainable extraction”. Relevant original research articles, reviews, book chapters and regulatory documents were considered. Priority was given to studies directly addressing the phytochemical composition, extraction methods, biological activity and application of olive leaf, thyme and green walnut husk extracts in dairy or food matrices. Studies not directly related to plant polyphenols, dairy applications or the selected plant materials were excluded from the main discussion.

Efficient extraction of bioactive compounds such as polyphenols is a critical step in the valorisation of plant-based materials. Conventional extraction methods are widely used due to their simplicity and reproducibility. These include solid–liquid extraction, Soxhlet extraction, maceration and percolation, most often performed with organic solvents such as ethanol, methanol or acetone. Maceration, based on prolonged soaking of plant material at ambient temperature, offers a low-cost and accessible option but often requires long extraction times and may result in lower yields. Percolation, on the other hand, allows continuous solvent flow through the sample, improving mass transfer and recovery efficiency. Soxhlet extraction, although widely recognised for its effectiveness, involves elevated temperatures and large solvent volumes, which raises safety and environmental concerns. Additionally, these methods are generally time-consuming, energy-intensive and associated with high solvent consumption, which can compromise both sustainability and the stability of heat-sensitive compounds (12-14). To address the limitations of conventional techniques, increasing attention has been directed towards green and innovative extraction methods. These modern approaches aim to reduce environmental impact and improve efficiency by shortening extraction time, minimising solvent usage, and preserving the functional integrity of sensitive bioactive molecules. Among the most studied are ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), pressurised liquid extraction (PLE) and supercritical fluid extraction (SFE). These techniques offer advantages such as lower energy input, higher selectivity and improved extraction kinetics, particularly when optimised for solvent polarity, temperature and pressure (15). Importantly, the choice of extraction technique significantly influences the chemical profile of the extract, including the relative proportions of phenolic acids, flavonoids and other bioactive constituents. This compositional variability affects not only antioxidant and antimicrobial activity but also the functional behaviour of the extract in complex food matrices, determining solubility, stability and potential interactions with proteins and lipids (10).

Furthermore, the extraction method can be strategically selected to tailor the extract composition to its intended food application. For instance, when the extract is intended for incorporation into fermented products such as yogurt or kefir, techniques using aqueous or hydroalcoholic solvents, such as UAE or PLE, are preferred, as they provide extracts enriched in water-soluble polyphenols (e.g. hydroxytyrosol from olive leaves). These compounds ensure homogeneous distribution and functional stability during fermentation.

Conversely, in the case of cheeses, extraction techniques and solvent systems that yield less polar phenolic fractions may be considered as a potential formulation approach for lipid-rich dairy matrices. However, direct empirical evidence confirming their preferential incorporation and functional performance in the cheese fat matrix remains limited. Such extracts, for example those containing flavonoid aglycones or juglone derivatives from green walnut husks, integrate more effectively into the milk fat matrix and contribute to lipid protection and extended shelf life. Moreover, in the formulation of functional beverages, extracts with high water solubility are preferred, which can be achieved through UAE or maceration in aqueous solvents, while in dietary supplements (capsules or powders), techniques such as MAE or PLE are more suitable due to their ability to yield concentrated and stable extracts with longer shelf life and easier incorporation. Thus, the choice of extraction method determines not only the quantitative yield but also the functional suitability of bioactive compounds for specific food applications.

Although modern extraction methods offer numerous advantages in terms of efficiency, selectivity and sustainability, the choice of an appropriate technique largely depends on the physicochemical properties of the plant matrix and the targeted bioactive compounds. Parameters such as particle size, moisture content, structural complexity, and the polarity and thermal sensitivity of the compounds significantly influence the effectiveness of a given extraction method (16). When extracting polyphenolic compounds from olive leaves, UAE has been identified as one of the most efficient techniques. This method is characterised by relatively short extraction times, moderate temperatures that preserve thermolabile components and environmental compatibility, making it one of the most frequently employed techniques for polyphenol recovery from olive leaves. Giacometti et al. (17) demonstrated that the total polyphenol and flavonoid contents extracted from olive leaves using UAE were 14.31 and 19.50 % higher, respectively, than those obtained with conventional extraction techniques. In addition to UAE, other frequently used methods with higher efficiency than conventional techniques include MAE and SFE (18).

As with other aromatic herbs, the efficiency of extracting bioactive compounds from thyme largely depends on the morphological characteristics of the plant. Leaves and stems differ significantly in their phenolic content, with the leaves containing much higher concentrations of polyphenols and essential oils, particularly monoterpenoid phenols such as thymol and carvacrol, while the stems are poorer in these compounds and thus less suitable for the extraction of high-value components (19).

The choice of extraction method has a significant impact on the yield and antioxidant capacity of the extract. According to a study by Palmieri et al. (20), Soxhlet extraction was the most effective method for recovering polyphenols from thyme, achieving a yield of (9.25±0.02) % after two hours of extraction. This indicates that 9.25 g polyphenols were extracted from 100 g dry plant material. However, the main drawbacks of this technique are the long extraction time and high temperature, which may contribute to partial degradation of heat-sensitive compounds. As a faster and more energy-efficient alternative, UAE is frequently used. It operates at lower temperatures, requires less solvent and shortens processing time. Although UAE helps preserve thermolabile compounds, the same study reported a significantly lower polyphenol yield of (1.62±0.02) %, more than five times less than Soxhlet extraction. Despite this, UAE is still considered a suitable “green” technology due to its reduced energy use, lower environmental impact and potential for industrial application.

The efficiency of bioactive compound extraction from green walnut husk greatly depends on the selected method, type of solvent and processing conditions. As with other plant matrices, the morphological features and chemical composition of the raw material are crucial for determining the most suitable extraction technique. Given the high content of phenolics and thermolabile antioxidants, there is a growing shift towards “green” extraction technologies that preserve bioactivity while minimising energy and solvent consumption. For the efficient recovery of polyphenols from green walnut husk, UAE and MAE have proven particularly effective. According to Momen and Satari (21), combining microwave pre-treatment (MWP) with UAE (MWP–UAE) significantly enhances total antioxidant content, expressed as gallic acid equivalents (GAE) on dry mass basis, achieving 3.64 g/100 g. This represents a notable increase compared to standard UAE (3.32 g/100 g) and maceration (1.96 g/100 g, 24 h). Extraction performance is significantly influenced by factors such as solvent choice (typically ethanol, water, or their mixtures), pre-treatment techniques (e.g. ultrasound or microwave) and operational parameters including temperature, time, solid-to-liquid ratio, and energy input. Proper optimisation of these conditions is essential for maximising yield and preserving the functional integrity of the extracts. As previously demonstrated, the application of innovative and green extraction techniques significantly reduces the environmental footprint of the process, thereby contributing to the sustainable valorisation of agro-industrial waste such as olive leaves and green walnut husks. Beyond improving extraction efficiency, these technologies also foster circular economy principles, reduce waste generation and create high-value ingredients for the food and pharmaceutical industries (22). Therefore, green extraction methods not only enhance selectivity and efficiency but also represent an environmentally friendly and socially responsible approach to the industrial processing of by-products.

Phenolic compounds from olive leaf, thyme and green walnut husk extracts, particularly oleuropein, rosmarinic acid and juglone, exhibit pronounced antioxidant potential that, in dairy systems as well as in food matrices in general, results in the inhibition of oxidative reactions and limitation of microbial growth, thereby directly contributing to shelf-life extension. Their chemical structures, characterised by ortho-diphenolic and quinone groups, enable them to act through transition metal chelation, neutralisation of reactive oxygen species, and inhibition of oxidative enzymes. This reduces hydroxyl radical formation and interrupts lipid peroxidation chain reactions, resulting in the preservation of lipids, proteins and other sensitive food constituents.

The antimicrobial effects of these compounds are closely related to their antioxidant activity. By neutralising reactive species and altering redox balance, polyphenols interfere with microbial metabolic processes, while metal chelation reduces the availability of essential cations required for microbial proliferation. Juglone, due to its quinone structure, can induce redox perturbations in microbial cells and compromise membrane integrity and protein function, as demonstrated for Listeria monocytogenes ATCC 19115, thereby contributing to the antimicrobial potential of green walnut husk extracts (23). Oleuropein and rosmarinic acid inhibit enzymes such as lipoxygenase, which participate in oxidative reactions within food matrices, thereby preventing the formation of undesirable oxidation products responsible for off-flavours and deterioration of sensory quality (24, 25). The structural formulae of oleuropein, rosmarinic acid and juglone, which illustrate their key functional groups responsible for these mechanisms, are shown in Fig. 1, Fig. 2 and Fig. 3.

The combined effects of radical scavenging, metal chelation, and enzyme inhibition provide multilayered protection against both oxidative and microbiological degradation. As a result, polyphenolic extracts not only minimise the risk of oxidative rancidity, discolouration and nutrient loss but also function as natural preservatives that slow down the growth of spoilage microorganisms (23). These properties highlight oleuropein, rosmarinic acid and juglone as particularly valuable for application in food technology, with strong potential to replace or complement synthetic antioxidants and preservatives, thereby improving product stability, safety and shelf-life. Radical scavenging mechanisms of polyphenols from olive leaf, thyme and walnut husk share core antioxidant actions, but have distinctive features based on their molecular structures (Table 1 (26-28)). In essence, all polyphenols scavenge radicals chiefly by donating hydrogen atoms or electrons, but olive leaf polyphenols have outstanding radical-trapping efficiency due to their chemical structure, thyme compounds add metal chelation effects that enhance antioxidant protection, and walnut husk polyphenols combine radical scavenging with redox cycling typically associated with quinones (26-28). In milk matrices these antioxidant mechanisms can be further modulated by interactions with milk proteins and lipids, which may alter extract efficacy and biostability. Polyphenols bind non-covalently to milk proteins via hydrogen bonds, hydrophobic interactions and sometimes covalent bonds, stabilising polyphenols and protecting them from degradation during storage and digestion (29, 30). These interactions can affect the release and absorption of polyphenols in the gastrointestinal tract, sometimes reducing bioavailability but often improving stability through digestion (29, 31). In addition, the complexes may influence the texture and sensory properties of food products and affect their functional and health-promoting qualities (30, 32).

The use of plant-derived extracts in foods is governed by strict regulatory frameworks designed to ensure consumer safety, product quality and consistency. These regulations differ between jurisdictions and depend largely on how the extract is classified, whether as a food ingredient, food additive, dietary supplement, novel food or medicinal preparation. Because botanical extracts often contain complex mixtures of bioactive compounds, their approval requires detailed characterisation, toxicological evaluation and evidence of safe use. These requirements are particularly relevant for functional dairy products, where plant extracts are incorporated not only for technological improvements but also for their antioxidant, antimicrobial and health-promoting potential.

The regulatory status of plant extracts differs considerably across jurisdictions and hinges on product classification. In the European Union, extracts intended for food are subject to the Novel Foods Regulation when they lack a history of significant consumption prior to 15 May 1997; such applications must include compositional, toxicological and exposure data (60). EFSA guidance for botanicals and botanical preparations used in supplements further emphasises identity verification, contaminant control and standardised marker content. Nutrition and health claims require separate EFSA evaluation under EU health-claims rules. In addition to novel food and safety requirements, the use of extraction solvents must comply with EU legislation on extraction solvents used in the production of foodstuffs and food ingredients. Directive 2009/32/EC (61) defines permitted extraction solvents and, where applicable, maximum residue limits in food products. This is particularly relevant for plant extracts obtained using organic solvents such as ethanol, methanol, acetone or other solvent systems, as residual solvent content must be controlled to ensure consumer safety and regulatory compliance. Therefore, solvent selection for plant extracts intended for dairy applications should consider not only extraction efficiency and selectivity, but also food-grade status, residue limits and suitability for incorporation into food matrices.

In the United States, botanical ingredients used in conventional foods fall under the food additive or GRAS frameworks (62). For dietary supplements, a New Dietary Ingredient (NDI) notification is required for extracts not marketed before 15 October 1994. FDA also provides dedicated guidance for botanical drugs when extracts are intended for medicinal use.

Regulatory expectations include robust characterisation (botanical identity, plant part used, extraction solvent), contaminant testing (pesticides, heavy metals, PAHs, mycotoxins), stability data and human exposure assessment. Toxicity and allergenicity remain key considerations: olive leaf extracts and oleuropein generally show high acute LD₅₀ values (>1000–2000 mg/kg in animal studies), while thymol exhibits moderate acute toxicity (LD₅₀≈1000 mg/kg) and may cause irritation. Juglone, a naphthoquinone characteristic of green walnut husk, is a known irritant capable of causing contact dermatitis, and at high concentrations botanical extracts may show cytotoxicity in model systems (63).

Herb–drug interactions and allergic reactions also require consideration. Certain botanical constituents may modify pharmacokinetics or pharmacodynamics, e.g. thyme components may influence coagulation and potentiate anticoagulants, while olive leaf compounds may affect blood pressure regulation (64). For these reasons, regulatory dossiers and product labels must address contraindications, allergenic potential and interaction risks.

In Croatia, national regulations align with EU legislation, particularly Regulation (EU) 2015/2283 on Novel Foods (65) and Regulation (EC) 1333/2008 on food additives (66). Plant extracts without a documented history of consumption in the EU prior to 15 May 1997 require full safety assessment, including characterisation, contaminant analysis and exposure evaluation. The Croatian Ministry of Health oversees notifications for extracts used in foods and supplements, requiring data on standardised markers, contaminants and stability. Particular attention is paid to heavy metals, pesticides, mycotoxins and PAHs. Olive leaf and thyme extracts are commonly used in supplements, while green walnut husk extract requires additional caution due to the presence of juglone. Product labelling must comply with consumer information requirements, including allergenicity and potential interactions (67, 68).

Although regulatory frameworks permit the use of plant extracts in functional foods, strict requirements for identity, purity and safety testing may slow innovation and market entry, underscoring the need for clearer, harmonised guidelines for the safe application of botanicals in food systems.

In summary, while regulatory systems provide essential safeguards for consumer protection, their complexity and variability pose challenges for the development of plant-enriched functional dairy products. Ensuring compliance with compositional, toxicological and labelling requirements is particularly important for extracts containing potent bioactive compounds such as oleuropein, thymol or juglone. Future progress in this field will benefit from more harmonised regulatory approaches and clearer criteria for assessing botanical extracts intended for incorporation into food matrices, thereby facilitating safe and efficient innovation within the functional dairy sector.

This review highlights the strong potential of olive leaf and thyme extracts as natural, polyphenol-rich ingredients for functional dairy applications, while green walnut husk extract shows emerging potential that requires further validation in real dairy matrices. Their incorporation into fermented and non-fermented dairy products may improve antioxidant capacity, microbial stability and technological performance, while sustainable extraction techniques support the valorisation of agro-industrial by-products. However, challenges remain, including sensory limitations at higher contents, variability in extract composition, limited data on green walnut husk application in real dairy systems, and the need for a deeper understanding of polyphenol–protein interactions and in vivo bioavailability. A limitation of this review is the heterogeneity of the available literature, including differences in extract composition, extraction methods, dose expression, dairy matrices and analytical approaches. These differences limit direct comparison among studies and make it difficult to draw firm conclusions regarding efficacy across product types.

Future research should focus on standardised extract characterisation, dose optimisation in specific dairy matrices, sensory threshold determination, evaluation of polyphenol stability and bioavailability during digestion, and well-designed in vivo or human intervention studies to confirm potential physiological effects. Particular attention should be given to green walnut husk extract, for which validation in real dairy matrices remains limited. Addressing these gaps, alongside navigating complex regulatory requirements for botanical ingredients, will be essential to enable wider industrial adoption of plant extract-enriched dairy products that align with clean-label and sustainability trends.