How food wastes can be converted into new products: European legislation and analysis of enzymatic hydrolysis

How food wastes can be converted into new products: European legislation and analysis of enzymatic hydrolysis Laura Esposito, Francesca Accardo * , Barbara Prandi , Tullia Tedeschi Department of Food and Drug, University of Parma, Parco Area delle Scienze, 27/A, Parma 43124, Italy ARTICLE INFO Keywords: enzymatic hydrolysis food residues sustainability food waste management valorisation pathway legal framework nutrient recovery ABSTRACT The generated food waste has a significant economic and environmental impact. Since most of this is considered nutrient-rich substrate, it can be reduced or converted to avoid negative effects. In recent years, new technologies have increasingly focused on this aim by extracting and recovering valuable components for the formulation of new products. Herein, this review analyses food waste management strategies and emerging scientific advancements. Among these, enzymatic hydrolysis represents a promising sustainable alternative to traditional chemical extraction. Unlike chemical methods, it operates under mild conditions, reducing energy consumption and harmful by-products, while efficiently recovering fibres, proteins, phenolic compounds, and other biomolecules of interest from food waste. Furthermore, its effectiveness can be significantly enhanced when combined with other techniques. However, most of these applications are currently at the laboratory scale, and a thorough assessment of the potential benefits and the feasibility at an industrial level is required. In this context, integrating enzymatic hydrolysis within circular economy models can further improve resource efficiency by promoting waste valorisation in industrial applications. This approach aligns with sustainable development goals, fostering the creation of bio-based products and reducing dependence on non-renewable resources. Despite facing challenges such as regulatory constraints and the need for scalable, cost-effective processes, the development of innovative and sustainable practices can bring significant economic, social, and environmental benefits. 1. Introduction By 2050, the world population is expected to reach approximately 10 billion. As a result, demand for food, feed, and energy is also expected to grow up [1]. Despite this growing demand, a significant amount of food is wasted: in 2022, an estimated 1.05 billion tonnes, or 19 % of all food, was wasted in retail, foodservice, and households, and approximately 13 % was lost in the supply chain [2], accounting for 8–10 % of global greenhouse gas emissions. Therefore, reducing avoidable waste could reduce the amount of resources used and their overall impact [2]. For this reason, the United Nations 2030 Agenda for Sustainable Development includes a specific goal to address the problem of food waste: Ensuring Sustainable Patterns of Production and Consumption. By 2030, global per capita food waste must be halved at the retail and consumer levels, and food losses must be reduced along production and supply chains, including post-harvest losses [3]. In recent years, many researchers have focused on the recovery and the valorisation of food-related waste, applying different processes to extract several compounds. Among them, the use of chemical and supercritical CO 2 extraction, ultrafiltration, ultrasound, and microwave-assisted extraction, and finally mild hydrolysis are currently applied as processes. Different from traditional chemical hydrolysis, biochemical approaches involve the use of hydrolytic enzymes, in immobilized form or in solution. This technique can be applied as a main process, used as a preliminary step, or combined with other technologies, for different purposes: i) to obtain multiple compounds of interest; ii) to extract a Abbreviations: EAHPE, Enzyme-Assisted High Pressure Extraction; EASCFE, Enzyme-Assisted Supercritical Fluid Extraction; EU, European Union; FAO, Food and Agriculture Organization; FLW, EU Platform on Food Losses and Food Waste; FOGs, Fats Oils and Greases; ILAEE, Ionic Liquid-Assisted Enzyme Extraction; LCA, Life Cycle Assessment; LCC, Life Cycle Costing; MAEE, Microwave-Assisted Enzyme Extraction; PEF, Pulsed Electric Fields; UAEE, Ultrasound Assisted Enzyme Extraction.. * Corresponding author. E-mail addresses: [email protected] (L. Esposito), [email protected] (F. Accardo), [email protected] (B. Prandi), [email protected] (T. Tedeschi). Contents lists available at ScienceDirect New BIOTECHNOLOGY journal homepage: www.elsevier.com/locate/nbt https://doi.org/10.1016/j.nbt.2025.09.005 Received 21 March 2025; Received in revised form 31 July 2025; Accepted 14 September 2025 New BIOTECHNOLOGY 90 (2025) 122–133 Available online 20 September 2025 1871-6784/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). target compound within the matrix and increase its availability; iii) to promote the formation of new compounds during hydrolysis. For this purpose, different works highlighted the advantages of the use of enzymatic hydrolysis to convert food waste efficiently [4,5]. According to the different nature of the enzyme used, enzymatic hydrolysis is currently employed for the extraction of essential oils from fruit processing residues [6], proteins from legumes, meat, fish residues [7–10], and fibres from lignocellulosic biomass [11,12]. Furthermore, it is important to consider that the economic feasibility depends on the availability of food waste, process costs, technological solutions, and the scalability of the process [13,14], given that many solutions are still at laboratory level [15]. Herein, the conversion of food waste represents a global sustainability challenge that encompasses technological, economic, and regulatory limitations. However, only a limited number of publications provide a detailed overview of these issues. Consequently, this work aims to provide an in-depth analysis by linking scientific evidence, technical aspects, regulatory considerations, and management practices that could be employed as a useful tool to support researchers and food industries when the development of new products from waste is the target. Using a top-down approach, a critical discussion on food waste management strategies and the current European regulatory framework about food wastage valorisation is given. Furthermore, this work provides an in-depth insight into enzymatic hydrolysis, which is now considered a relevant and sustainable valorisation pathway to convert food-related waste. Finally, the applications of this valorisation pathway are analysed, with particular focus on protein hydrolysis. 2. Exploring food waste, loss, processing residues and byproducts: definitions, management and legal framework 2.1. Definitions and associated data Over the last 20 years, the term “food waste”, referring to all kinds of wasted food, has become prevalent in the scientific literature, as demonstrated by the increasing presence of this term in scientific papers: from 188 publications in 2004, to 3225 in 2024 (annual publications, as reported on PubMed) [16] (Fig. 1 A). Interestingly, before 2003 there were no scientific articles that included the term “valorisation” in the topic of food waste. While from 2003 to 2024 the total number of publications on this topic has increased exponentially: from 1 publication in 2003 to 1947 in 2024 (as reported on PubMed) [17] (Fig. 1B). This increasing trend can be attributed to several reasons, including: i) the growing environmental awareness; ii) the evolution of policies aimed at reducing and recovering wasted food; iii) scientific progress regarding the development of new methods for the valorisation of wasted food; iv) the resulting economic interest. The proposed methodology for the classification of wasted food is based on when it occurs along the food chain and the main causes. Food loss refers to the decrease of food intended for human consumption that is removed from post-harvest or post-slaughter production, but excluding the retail level [2]. Generally, roots, tubers, and oilseed crops record the highest level of losses (25 %), followed by fruits and vegetables due to their perishable nature (over 20 %). Meat and animal products, together with cereals and legumes, show a lower percentage of loss: 10 % and approximately 12 %, respectively [1]. Among the most significant causes of loss are extreme events in the pre-harvest stages, inadequate conditions in post-harvest practices, lack of adequate transportation and storage facilities, poor management of storage temperature, and inadequate process capacity [1]. Food waste is defined as food and its inedible parts removed from the food supply chain for human consumption [2]. The increase in food waste is linked to the high standards required by the retail market, poor storage or stock management, and the uncertainty between expiry and preferred consumption date labels at home [1]. Food processing residues are residues coming from the production and transformation of food, such as peels, seeds, bones, plant fibres, and other materials. These are not intended as final products of the process, but they can have great potential for recycling since they are considered nutrient-rich substrates. Once food processing residues are given an alternative destination through a secondary transformation, they can be defined as food by-products. These products originate from a primary production process and can be further used through direct reuse or reprocessing. Their applications must strictly comply with the legal framework to ensure public health safety [18]. 2.2. Food waste legislation: European regulatory framework Over the past few years, different countries around the world implemented regulatory actions on food waste management, highlighting an increasing interest of public governments in this topic. For instance, European countries, Uruguay, Argentina, Chile, Japan, and China implemented several guidelines and policies to promote the adoption of circular economy model systems, aiming to reduce food loss and/or reuse food waste [19]. Although some countries promote green practices on food waste management, there are no common regulations that specifically control the conversion and utilization of food waste, Fig. 1. (A) Number of annual publications on PubMed that include the terms "food waste" from 2004 to 2024; (B) Number of total publications on PubMed that include the terms "food waste valorisation" from 2003 to 2024. L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 123 emphasizing the heterogeneity of the system at the international level. Furthermore, even at the regional level, as in the case of the U.S., policy supports ambitious national goals and initiatives aiming to valorise waste and reduce food losses. Nevertheless, a lack of a homogeneous regulatory framework and federal standardization among the different member states is an issue for the U.S. when it comes to the circular economy in the food system [20]. In addition, there are some regions located around the world that have no regulation in place (e.g., Africa) [19]. For this purpose, given the fragmentation of policies in place at international level, our work focus on common guidelines that are built thought the years in Europe. Recently, reducing food loss and waste has been a priority for the European Commission, which has established a common guideline defining the waste hierarchy [18]. This strategy considers prevention and reuse of food intended for human consumption as the best action, followed by recycling (including composting), energy recovery, and reuse for animal feed and by-products. Therefore, landfill disposal is considered the last option to be applied for wasted food [18]. To prevent the latter, the European Platform on Food Losses and Waste (FLW) was created in 2016 to help different stakeholders to identify critical steps to reduce wasted food, exchange best practices, and monitor progress over time. The platform connects international organizations, stakeholders, specialists from EU member states, and EU authorities [21]. Furthermore, the FLW theme is a component of the 2019 EU Farm to Fork Strategy: a programme that seeks to prevent FLW while achieving sustainable production, processing, distribution, and consumption [22]. To determine the extent of wasted food, the Commission will examine food losses at each stage of production and seek solutions to stop them [23]. At the European level, several regulations and directives provide procedural guidelines about the recovery of food by-products. Regulation (EC) No. 1069/2009 laying down health rules as regards animal byproducts and derived products not intended for human consumption classifies animal by-products according to their health risk, specifies disposal methods, and possibilities for reuse. The main objective of this regulation is to manage and reduce the risk associated with animal byproducts, which also ensures that animal by-products are not misused in the feed chain or do not compromise human or animal health. A category 1 material can be used as a fuel after processing or without prior processing or used to produce derived products. A material belonging to category 2 can be recovered to produce organic fertilizers, soil improvers, composted, or transformed into biogas (used as fuel). Finally, category 3 material can be recovered to produce feed, organic fertilizers, or soil improvers, composted, or transformed into biogas or used as fuel. The handling, transportation, storage, processing, and disposal of all categories of animal by-products are regulated by strict measures. Potential derivative products must ensure high safety standards. Each application is regulated by specific rules and standards [24]. Furthermore, Directive 2009/28/EC promotes the use of energy from renewable resources, including food by-products, by setting mandatory national targets for the overall share of energy from renewable sources and sustainability criteria for biofuels and bioliquids [25]. Finally, Directive (EU) 2018/851 on waste promotes waste reduction and recycling, introduces the concept of circular economy, establishes measures to avoid or reduce waste generation, improve sustainable waste management, reduce the overall effects of resource use, and improve efficiency [26]. At the national level, each state in the EU adopts the European legal framework and specific strategies in accordance with the laws of its own state. A schematic representation of the current legal framework at the European level is presented in Fig. 2. 2.3. Food waste management Given the persistent nature of waste streams and their potential, developing strategies for the recovery of wasted food categories could have a positive impact on the economy and the environment. In this context, the bioeconomy is based on the use of renewable and biological resources from land and sea, such as crops, fish, forests, and microorganisms, to produce food, energy, and materials that can help to create new value chains, green, and more sustainable industrial processes [27]. In addition to these latest definitions, according to FAO [28], the concept of bioeconomy includes the production, use, and conservation of biological resources, involving related knowledge, science, technology, and innovation, to create new products and services, while promoting sustainable economic growth. A sustainable circular bioeconomy supports the objectives of the European Green Deal by eliminating pollution, promoting clean energy, investing in smarter and more sustainable transport, financing green projects, protecting nature, and fighting wasted food by valorising it in a wide range of value-added products [29]. Consistent with these purposes, biorefineries transform biomass sustainably into products such as food, feed, chemicals, biomaterials, and bioenergy [30]. The growing interest of the scientific community in the industrial use of wasted food as a secondary resource for biorefineries suggests that this represents a substantial potential for material recycling [32]. The achievement of these initiatives depends on the standardization and the safety of food-related waste recovery processes. Standardization measures of collecting, transporting, and processing enable to provide an efficient recovery of food-related waste, to avoid contamination risk, and to preserve the quality of the new added-value products. The pathway from wastage to new product must be supported by processes that ensure the safety and quality of the final product, especially if it is intended for human consumption. Although advantageous, considering the environmental aspect, biorefineries are not completely pollution-free due to factors such as the diversity of raw materials, local conditions, and process design. Therefore, a comprehensive environmental assessment is needed to evaluate and compare their performance with fossil fuel-based systems [15]. A useful tool to assess the environmental impact of biorefineries in the valorisation of wasted food is the Life Cycle Assessment (LCA). The latter is defined by ISO 14040:2006 as the compilation and evaluation of the inputs, outputs, and potential environmental impacts of a product system throughout its life cycle. This allows to study the environmental aspects and potential impacts throughout the life cycle of a product from raw material acquisition to production, considering the used materials and the related disposal [31]. Laboratory scale assessment allows for Fig. 2. Flowchart depicting the European regulatory framework about food waste and by-products. In light grey the reference law [23–26], in dark grey the main content. L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 124 evaluating the environmental impact of the chosen technology and selecting the most environmentally friendly to scale the process [15]. Wasted food can be recovered and valorised through different valorisation processes to extract and recover several components from it for industrial uses such as cosmetics, nutraceuticals, pharmaceuticals, and food packaging. Wasted food can also be converted into biomaterials such as bio ceramics and biopolymers. Recycling can produce lowadded-value bioenergy (biogas, bioethanol, etc.) and high-added-value products. The technologies applied are different and combine biochemical (i.e., hydrolysis, fermentation, extraction) and thermophysical (i.e., supercritical CO 2 , ultrafiltration, ultrasound) techniques. However, many of these technologies are still at the laboratory level and need to demonstrate their industrial feasibility [14], prioritizing economic and environmental benefits, ensuring lower impacts than disposal methods. Accurate cost-benefit assessments and the adoption of sustainable technologies are essential to achieve significant improvements [32]. Regarding the economic framework, a Life Cycle Costing (LCC) analysis is necessary before implementing food wastage valorisation processes. Cristobal et al. [33] presented a technical-economic study to evaluate the profitability of valorisation processes in which the amount of wasted raw materials, the resources needed for the valorisation of wasted food, the distance from the valorisation plants, the costs of transportation, handling, and storage costs were analysed. The results showed that each waste could have a different profitability potential, but it is necessary to conduct a market analysis before implementing the biorefinery. As expected, the most profitable options were obtained when the final product had a higher market price. However, the authors highlighted the importance of several aspects during production that can have a positive impact on the result, namely concentrating production and exploiting economies of scale, optimizing logistics and transport management, to not compromising the environmental impact. Furthermore, Ghinea et al., 2025 [13] provided a concrete example of a cost-benefit study of the employment of enzymatic hydrolysis to produce three different alternatives for food waste management, demonstrating that this technique seems to provide significant economic, environmental, and social opportunities even if further developments are required. Despite the need to reduce waste and promote a circular economy, the valorisation of residues in the agri-food sector encounters several obstacles, such as complex processes for their purification and handling, restrictive regulations aimed at ensuring food safety, and a reduced or non-competitive economic value compared to primary products. Regulations and innovation must coexist to maximize the value of food byproducts. 3. Valorisation of food waste and food processing residues based on biopolymers hydrolysis Food waste and food processing residues can be recovered and extracted to create new products using various technologies, including biopolymers hydrolysis. It is a chemical reaction in which the bonds of a complex compound are broken, forming smaller molecules [34]. The main substrates of organic biomass intended for hydrolysis are carbohydrates, lipids, and proteins, from which monosaccharides, fatty acids, amino acids, and small peptides are obtained. Chemical hydrolysis is traditionally performed in solution in presence of inorganic acids or bases to thereby increase the solubility of complex substrates. Chemical hydrolysis has been employed as the conventional technique for years because it is considered very efficient and fast, but it requires rough conditions and a large amount of energy, it is not selective and leads to the formation of unwanted and dangerous compounds. For this reason, in recent years, chemical hydrolysis has been partially replaced by enzymatic hydrolysis, which involves the use of enzymes instead of rough chemical reagents, such as strong acids and bases, requiring mild conditions and less energy. Furthermore, the use of environmentally friendly catalysts, which are chemoselective, regioselective, enantioselective, and do not require purification steps, is also promoted. However, it is considered an expensive and slow method, and careful considerations during the optimization phase, including a thorough study of the matrix, enzyme, and working parameters (i.e., pH, temperature, and reaction time) are needed [34]. A schematic representation of chemical and enzymatic hydrolysis, with their advantages and limitations (panel A), and the mechanism of the hydrolysis of carbohydrates, proteins and lipids (panel B, C, D) is presented in Fig. 3. Despite the specific disadvantages of both techniques, according to the objective to achieve, many investigations highlight the relevance of these valorisation routes, which will be detailed in the paragraphs below. 3.1. Principles of chemical hydrolysis Chemical hydrolysis is extensively used to obtain hydrolysates consisting of smaller compounds of interest, which enhances the commercial value of the final product [35]. However, it requires extreme conditions in terms of pH and/or high temperatures. Chemical hydrolysis is commonly applied to various substrates, such as lignocellulosic biomass, to obtain cellulose nanocrystals and nanocellulose fibres. Hydrolysis into monosaccharides also enables the fermentation process, aimed at the production of biofuels, and represents an effective method to recover glucose and other sugars from lignocellulosic biomass. Access to cell walls allows the release of polysaccharides from biomass, breaking the structural bonds between lignin, cellulose and carbohydrates. Commonly used acid solutions are sulfuric, hydrochloric and phosphoric acid. The most used alkaline solution is sodium hydroxide. The acid or alkaline solution can be used either concentrated or diluted. Concentrated solutions require a low hydrolysis temperature. However, the risk of corrosion and pollution is increased, and an additional step for final neutralisation is also needed. Reactions with diluted solutions are less polluting but require more energy [36]. Chemical hydrolysis has also been studied using lipid-rich residues to produce biofuels. In the study of Quijote et al. [37], the simultaneous extraction and conversion of fatty acids from spent coffee grounds into methyl esters was carried out by in situ transesterification without the addition of a catalyst for biodiesel production. Hydrolysis of protein is influenced by various factors such as temperature, reaction time, type of reagents, and concentration; therefore, 100 % protein yield has never been obtained [38]. In the study by Rojo et al. [39], alkaline hydrolysis of microalgae biomass proved to be efficient for the recovery of small peptides, which increased with temperature. In contrast, acid hydrolysis, even at mild temperatures, degraded proteins but allowed a greater recovery of carbohydrates. This highlights the importance of establishing process parameters based on the starting substrate and the final target. Izydorczyk et al. [40] demonstrated that chemical hydrolysis of slaughterhouse waste for fertiliser production is an alternative to incineration, achieving a reduction in CO 2 emissions. This method allowed the recovery of nitrogen and the release of free amino acids, with a positive impact on plant growth. Bhavsar et al. [41] studied alkaline hydrolysis of wool to produce keratin hydrolysates as biofertilizers, comparing it with superheated water hydrolysis. Although both methods efficiently hydrolyse keratin, hydrolysis with superheated water has proven to be the most sustainable and economic option, due to the reduced chemical input. Chemical hydrolysis of organic waste has also shown promise as an alternative to commercial synthetic fungicides. Jindˇ richov´ a et al. [42] emphasised their potential to inhibit disease development and activate plant defence mechanisms. The efficiency of chemical hydrolysis can be further improved when combined with advanced extraction techniques, such as ultrasonic and microwave extraction, as reported by Yuan et al. [43] and by Karanicola et al. [44]. L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 125 In many cases, chemical hydrolysis can also be applied as a pretreatment step for enzymatic hydrolysis, especially for difficult substrates such as lignocellulose. This preliminary reaction promotes depolymerisation, facilitating the sequential saccharification of the lignocellulosic biomass and ensuring the complete release of sugars for the subsequent hydrolytic steps [45,46]. These cases illustrate how chemical hydrolysis can be effectively applied in various contexts. 3.2. Valorisation pathways through enzymatic hydrolysis The use of enzymes on food residues is widely studied because it is considered an effective and sustainable method to extract and obtain biomolecules of interest. In the literature, enzymatic hydrolysis and enzyme-assisted extraction are sometimes referred to as the same technique, but they differ in their ultimate purpose. Indeed, as the name suggests, enzyme-assisted extraction is used to break the cell walls of biomass to improve the extraction of compounds of interest inside the cells (i.e., phenols, carotenoids, essential oils) [47]. Enzymatic hydrolysis, on the other hand, represents the process of catalytic decomposition of polymers to obtain smaller molecules using specific enzymes, which is the final goal of using this method. In both cases, the reaction efficiency is influenced by the pretreatment of the substrate and the activity of the selected enzyme, the concentration of the enzyme and substrate, the temperature, the pH, the reaction time and the presence of inhibitors or activators. Each enzyme has a pH and temperature range in which it shows high efficiency, otherwise, the enzyme activity may be compromised or inactivated. Inhibitors decrease the affinity of the enzyme for the substrate, while activators enhance the enzyme activity. Enzymatic hydrolysis is preferred over chemical hydrolysis due to its higher sustainability, specificity, tolerance for mild conditions, better Fig. 3. (A) Schematic comparison between chemical and enzymatic hydrolysis, highlighting the advantages and limitations of each technique; Hydrolysis mechanisms using chemicals (acid or alkaline solutions) or enzymes (Amylase, Cellulase, Hemicellulase, Laccase, Protease, Lipase): (B) Hydrolysis of Carbohydrates; (C) Hydrolysis of Proteins; (D) Hydrolysis of Lipids. L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 126 conversion efficiency, and fewer secondary products [48]. However, unlike chemical hydrolysis, it is costly and limited at scale due to enzyme expenses, reaction time, substrate properties (e.g., porosity and surface area), and the need for specific enzyme stability conditions such as pH and temperature [49]. It can also produce bitter and undesirable flavours depending on the enzyme used, making products intended for human consumption difficult to market [48]. Proteases such as Alcalase®, Protamex®, Flavourzyme®, trypsin, pepsin, chymotrypsin and papain are the most widely used for protein extraction and production of protein hydrolysates [50–52]. For the extraction of oils and fatty acids, lipases or lipase mixtures are commonly used [53], but some proteases have also been tested for this purpose [54]. Cellulolytic enzymes such as Viscozyme®, pectinase, cellulase, and β-glucosidase are studied for the extraction of bioactive compounds [55–57] and as a saccharification agent before alcoholic or lactic fermentation [58,59]. Hydrolysis can occur using free enzymes in solution or immobilised enzymes (Fig. 4). Hydrolysis with free enzymes faces some problems, such as the stability of the enzyme and the difficulty in recovering the enzyme, which is inactivated at the end of the reaction. A single enzyme or a mixture of multiple enzymes can be used; tandem or one-pot reactions can also be considered. Enzyme hydrolysis by immobilisation allows the recovery of continuously usable enzymes and can improve enzyme selectivity. However, it requires the enzyme to be recoverable and maintain its activity during multiple application cycles, and it is more expensive due to the costs of immobilisation techniques [60]. Enzyme immobilisation can be achieved by adsorption, encapsulation, entrapment, or cross-linking [61]. Any immobilisation technique can be implemented either suspended or fixed to the walls of the batch. Suspension is generally applied to maximise enzyme-substrate contact, and when it is expected that the carrier will be easily separated from the solution. The attachment to the walls of the batch avoids the separation step, it is an easier system to manage, especially for long operations, but it can avoid the access of the substrate to the enzyme. The applications of immobilised enzymatic hydrolysis are manifold, for example, the conversion of lactose by β-galactosidase into sugars with reduced caloric content and high sweetness [62]; the production of biofuel from waste cooking oil [63]; the extraction of bioactive peptides from whey by protease [64]. As mentioned above, the choice of enzyme used to conduct hydrolysis depends on the nature of the substrate and the intended final product. The purpose of enzymatic hydrolysis varies depending on the goal of the study: it may serve as a pretreatment, or it can function as the primary step of the process to extract or enhance the release of compounds already present in the starting material. 3.2.1. Enzymatic hydrolysis using carbohydrate sources Enzymatic hydrolysis has been commonly studied as a pretreatment for the fermentation of plant-based residues. Enzymatic hydrolysis is widely used as a saccharification method of biomass rich in polysaccharides such as starch and lignocellulose, allowing their conversion into many valuable products [59]. Using enzymatic hydrolysis with a mixture of pectinolytic and cellulolytic enzymes as a saccharification step, lactic acid [58], probiotics [65], 2,3-butanediol [66], acetic acid, bacteriocins, and biosurfactants [67] have been obtained during different types of fermentations with considerable importance for the pharmaceutical, food, and chemical industries. The amylase activity of enzymes is exploited for the hydrolysis of starch to release reducing sugars as a pretreatment and produce pure glucose for chemical application [68] and alcoholic fermentation [69]. In their study, Fagundes et al. [69] conducted an LCA analysis of bioethanol production from banana, papaya, and potato residues through a pretreatment of the starting material, enzymatic hydrolysis and fermentation, highlighting the need to optimise the process to enhance its sustainability. The use of enzymatic hydrolysis as a pretreatment is one of the most effective techniques for processing lignocellulosic biomass, a major source of energy and chemicals for the planet. Lignocellulose is a biomass that is very difficult to hydrolyse enzymatically, as it is resistant to the action of cellulase. Therefore, the raw material must undergo pretreatment before enzymatic hydrolysis with cellulase, followed by fermentation to produce bioethanol [59]. The use of ultrasound coupled with enzymatic hydrolysis showed an increase in glucose release yield to produce bioethanol from lignocellulosic biomass [70]. An integrated biorefinery model was created to chemically and enzymatically hydrolyse a flour industry residue into sugars, which are then converted into high value triacylglycerols and carotenoids by the biocatalytic activity of yeasts [49]. Sugars present in abundance in food and beverage waste can be recovered as such, through enzymatic hydrolysis as a saccharification step. Kwan et al. [71] studied the amylase-inhibitory effects of preservatives and additives in beverages: as a result, benzoic acid, sorbic acid, and caffeine in food and beverage wastes did not inhibit the enzymatic activity when glucoamylase and sucrase were used. Dietary fibre-rich residues can be transformed through enzymatic Fig. 4. Schematic representation of possible enzymatic hydrolysis techniques. (A) Free enzymatic hydrolysis using a single enzyme; (B) Free enzymatic hydrolysis using a mixture of enzymes in a single vessel; (C) Free enzymatic tandem hydrolysis; (D) Enzymatic hydrolysis by immobilization using the adsorption technique; (E) Enzymatic hydrolysis by immobilization using the entrapment technique; (F) Enzymatic hydrolysis by immobilization using the encapsulation technique; (G) Enzymatic hydrolysis by immobilization using a cross-linked carrier. L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 127 hydrolysis, which depolymerises lignocellulose into prebiotic ingredients that promote the growth of Bifidobacterium and Lactobacillus species in the host’s gastrointestinal tract. To this end, the combined cellulase and xylanase treatment was tested to convert guava puree byproducts into potential prebiotic sources [72], while the prebiotic potential of hydrolysed pectic oligosaccharides from citrus peel was investigated [73]. Enzyme-assisted methods for the recovery of multiple macronutrients from food wastage have also been implemented: soluble fibre, lipid, and protein fractions were extracted from fruit by-products using a protease in the study by Fuso et al. [12] as biorefinery flow optimisation. The improvement of the technological and sensorial characteristics of food by-products has been studied by applying enzymatic hydrolysis. Viscozyme® on wholemeal okara flour allowed the reduction of insoluble fibre and emulsification, increasing soluble fibre and foaming capacity [55]; hydrolysis with xylanase and cellulase on cassava peel allowed to obtain cellulose nanofibers that act as emulsion stabilizers [74]; on oil palm leaves, it has been studied how to formulate emulsifiers, comparable to those found in plant foods, using Alcalase® [75]. Alcalase® and ficin were tested on sweet potato waste for the formulation of plant-based foaming additives [76]. 3.2.2. Enzymatic hydrolysis using oil sources FOGs (fats, oils and greases) residues that can be further exploited include used cooking oils, trap greases, mill effluents, rendering fats, spent earths from filtration and bleaching, deodorisation distillates, soap stocks, and sewage sludges. The hydrolysis products of interest are mainly free fatty acids, which are widely used in oleochemical applications. However, their heterogeneity, the presence of water and impurities make their management difficult. Hydrolysis of these byproducts by lipase allows for milder conditions. The most effective catalysts turned out to be non-specific and highly resistant enzymes [77]. Souza et al. [78] tested a mixture of lipases on cooking oil waste for the extraction of fatty acids and glycerol to produce an oleochemical bioproduct. Enzymatic hydrolysis offers a more energy-efficient and environmentally friendly alternative to conventional fish oil extraction, which typically requires high temperatures and the use of organic solvents. During the process, proteases break peptide bonds, releasing oil from the protein matrix. However, it remains costly due to the inactivation and discard of the enzyme after its use. The enzymatic hydrolysis process was optimised in the study by Liu & Dave [79]: Alcalase® was immobilised, and the oil extraction yield from salmon by-products was comparable to hydrolysis using free Alcalase®, with the advantage of being able to recover the enzyme up to three hydrolysis cycles. 3.2.3. Enzymatic hydrolysis using protein sources Enzymatic hydrolysis is applied to different protein-rich food residues to extract proteins and hydrolyse them to obtain their main constituents (i.e., amino acids and peptides). This technique is employed to improve the techno-functional, organoleptic and nutritional properties, including digestibility of the selected substrates [80–83]. These target characteristics are directly enhanced by the used enzyme and substrates. For this reason, an in-depth analysis of the process parameters and the final protein hydrolysate is always required. These products, upon hydrolysis, can be used in agriculture, food, feed, cosmetics, pharmaceuticals, or biotechnological applications. For this purpose, several matrices were tested, both animal and plant sources, including residues derived from cattle, poultry, pigs, fish, cheese, and legumes. These exhibit a different hydrolysis rate: ranging from very high to very low, depending on the protein nature. The different structures of animal and plant proteins result in distinct technofunctional properties, digestibility, accessibility to enzymes, and peptide fragmentation. Animal proteins are usually considered more accessible due to their structure, while plant proteins show a low accessibility to the enzyme, due to their cross-linked structure and the presence of complexes with fibres and polyphenols in the matrix [84]. Table 1 details several studies using protein hydrolysis on different substrates, including the parameters employed. Moreover, legumes and cereals can contain high amounts of antinutritional factors that reduce the digestibility of proteins [99]. Their content can be reduced by cooking, roasting, hulling, fermenting, and enzymatic hydrolysis. The latter appeared efficient in degrading wheat proteins into low molecular weight compounds [100], leading to nutrient enrichment and a reduction of antinutritional compounds in canola oil residues [87]. Enzymatic hydrolysis on legumes is also considered a valid tool to reduce the allergenicity [101,102], and it can also be applied to different residues [10]. In recent years, enzymatic hydrolysis of proteins has been increasingly explored, primarily for its efficiency in protein breakdown. While the focus of this technique is on the valorisation of protein-rich substrates, by increasing the protein recovery and digestibility, an additional aspect involves evaluating the release of peptides that may have potentially bioactive properties (i.e., antioxidant, antimicrobial, antihypertensive). This possibility adds further interest to enzymatic hydrolysis, particularly in food, health, nutraceutical, and packaging purposes [103, 104]. Fish and meat residues, as well as other protein-rich sources such as brewer’s yeast, have been investigated for their potential to release bioactive peptides using trypsin and alkaline protease [104,105]. Additionally, peptides derived from collagen have shown antioxidant, anti-aging, and collagenase inhibition properties, which may be used in nutritional, cosmetic, and pharmaceutical applications [7,106, 107]. While the extraction of potentially bioactive peptides is not the primary goal of enzymatic hydrolysis, it remains a relevant and promising pathway of research. Beyond health-related applications, enzymatic hydrolysis of proteinrich waste can also have a role in agriculture. Plant and animal protein hydrolysates can act as organic soil improvers and plant biostimulants, essential agronomic tools to promote sustainable agriculture without chemical fertilizers [108]. Organic bio-stimulants obtained from protein-rich waste through enzymatic hydrolysis are composed of a mixture of peptides and amino acids, which are beneficial for soil, foliar system, growth, yield, and quality of crops, while also improving stress tolerance [52,109,110]. Therefore, protein hydrolysis has received significant interest in recent years for its effectiveness across a variety of residues. 3.2.4. Enzymatic hydrolysis combined with other techniques Enzymatic hydrolysis is often combined with other pretreatment or extraction techniques to enhance overall efficiency and improve extraction yield. This integrated approach addresses some inherent limitations of the enzymatic process, such as enzyme sensitivity to high temperature or inhibitory compounds present in complex and heterogeneous substrates. For instance, physical methods like ultrasound or microwave pretreatments can disrupt the structure of lignocellulosic biomass, increasing enzyme accessibility, and thus accelerating hydrolysis. Consequently, these synergic combinations reduce enzyme loading requirements, lower energy consumption, and minimise the generation of harmful by-products compared to the application of each technique individually. However, such multi-technique approaches demand careful optimization and control of operational parameters, as well as thorough evaluation of the compatibility and potential interaction between enzymes and pretreatment methods. This ensures process stability, maximizes efficiency, and preserves enzyme activity [111]. The technologies used in combination can be applied before, after, or simultaneously with the enzymatic hydrolysis. Several studies have used coupled technologies, as detailed in Table 2. The combination of other L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 128 Table 1 Overview of processing conditions applied to different substrates to produce protein hydrolysates using the free enzyme technique. Substrate Solution Hydromodule Enzyme Enzyme concentration pH Temperature Reaction time Inactivation Objective of the study Reference Chickpeas and peas residues 10 mM phosphate buffer (for Alcalase®, Papain, Trypsin and mix of Alcalase® and Papain) 1:5 Alcalase®, Trypsin, Pepsin, Papain, mix of Alcalase® and Papain 1 % (w/w for pepsin, papain, and trypsin, v/w for Alcalase®) Alcalase®: 6.5–8.5, Trypsin: 7–9, Pepsin: 2–4, Papain 6–7, mix of Alcalase® and Papain 6.5–7 Alcalase®: 60 ◦C; Trypsin: 37 ◦C; Pepsin: 37 ◦C; Papain: 65 ◦C; Mix Alcalase® and Papain: 62.5 ◦C 2 h - Protein recovery/ digestibility increase [10] Soy (okara) Water 1:50 Alcalase® and Flavourzyme® From 1 % to 5 % (w/ w based on E:S ratio) 6.5–9 40–60 ◦C 70–500 min 90◦C for 10 min Optimization of enzymatic hydrolysis [85] Rice (rice bran) Water 1:5 Alcalase® Flavourzyme® Neutrase® 1 % (w/w based on E:S ratio) 8 8 7 50 ◦C 2–4–6 h 85 ◦C for 10 min Enzyme hydrolysis optimization/ new product development [86] Rapeseed (canola press cakes) Water 1:10 Alcalase® Viscozyme® Alcalase® Protamex® Viscozyme® +Phyzyme® 1 % 9 % 1 % 1 % 9 % +0.5 % (v/w) 9.5–7.5 ≈4.5 8 7.5 4.5 50 ◦C 45 ◦C 50 ◦C 50 ◦C 45 ◦C 4 h 20 h 4 h 4 h 20 h - Recovery of proteins and dietary fibres [87] Bovine (skin) 10 mM phosphate buffer 1:3 Alcalase®1 % (v/w) 7 40 ◦C 19 h 100 ◦C for 10 min Protein recovery [88] Bovine (liver) Phosphate buffer 1:1 Alcalase® and Protamex® 0.1 % (w/w) 8 45 ◦C 50 ◦C 55 ◦C 12 h 18 h 24 h 95 ◦C for 10 min Evaluation of antioxidant activity [89] Porcine, bovine, chicken, salmon Water and 100 mM phosphate buffer 1:1 Alcalase® Papain 0.02–0.5 % (w/w) 7 – 8 60 ◦C 2 h 100 ◦C for 30 min Enzymatic hydrolysis optimization /protein recovery [90] Porcine (meat and bones) Water 1:1 Alcalase®1 % (v/w) 8 50 ◦C 6 h - Extraction of different compounds/ protein recovery [91] Porcine (liver) Water 1:1 Papain Bromelain Alcalase® Flavourzyme® 1 % (w/w) 6 6 8 5.5 37 ◦C 40 ◦C 50 ◦C 50 ◦C 7 h 95 ◦C for 3 min Assessment of antioxidant capacity/ mapping of antioxidant peptides [92] Porcine (bones) Water 1:12 Neutrase®between 0.1 % and 2.5 % (w/w) 5 – 8 40–60 ◦C From 30 min to 90 min 90 ◦C for 20 min Optimization of enzymatic hydrolysis/ evaluation of functional properties [93] Poultry (chicken and turkey) Water 1:1, 1:2 and 1:3 Alcalase®0.5 % and 1.0 % (v/ w) ≈7 65 ◦C Serial hydrolysis (3 times) 2 h each 90 ◦C for 20 min Enzymatic hydrolysis optimization /protein recovery [8] Poultry (duck blood) Water 1:5 Neutrase® Papain 5 % (v/w) 7 7 50 ◦C 65 ◦C 4 h 95 ◦C for 30 min Production of protein hydrolysate/ evaluation of functional properties [94] Catfish Water 1:1 Alcalase®0–0.6 % (v/w) ≈7 45–70 ◦C 2–15 h Thermal treatment Recovery of bioactive compounds [95] Yellowfin tuna (viscera) Water 1:2 (w/v) Protamex®1.5 % (w/w) 6.5 41 ◦C 150 min 90 ◦C for 10 min Evaluation of antioxidant and antibacterial activity [96] Salmon (skin gelatin and trimmings) Water 1:5 skin gelatin 1:1.75 trimmings Alcalase®, Alcalase® and Flavourzyme® Promod 0.74 % (w/w or v/ w) 7 50 ◦C 4 h 90 ◦C for 20 min Optimization of enzymatic hydrolysis/ evaluation of biological properties [97] Cheese (rinds) 10 mM phosphate buffer 1:10 Alcalase®1 % (w/w) 6.5 60 ◦C 4 h and 16 h 90 ◦C for 10 min Lipid and protein recovery [98] L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 129 techniques with the use of enzymes has been widely studied to increase the extraction efficiency, especially of phenolic compounds and oils, and to optimize the hydrolytic process of polysaccharides and proteins. The common goal is to facilitate cell wall disruption, enzyme-substrate contact, and release of components of interest. The use of ultrasound in ultrasound-assisted enzymatic extraction (UAEE) increases the interaction surface and mass transfer rate between the matrix and the enzyme, due to the formation of cavitation and the high localized pressure and temperature produced. This reduces time, energy, and extraction solvent use [112–114]. Therefore, ultrasounds were tested in combination with enzymatic hydrolysis of pectin to improve the extraction yield of bioactive compounds from fruits: in the study by Niglio et al. [6], the sequential ultrasound step did not enhance the extraction yield of phenolic compounds, suggesting the need for process optimization. A similar operation is based on MAEE (Microwave Assisted Enzymatic Extraction), where the rapid localized heating, generated by microwaves, improves the penetration of the solvent into the cell walls and the interaction with the enzyme in a shorter time [115,116]. Similarly, EAHPE (Enzyme Assisted High Pressure Extraction) exploits the ability of high pressure to disrupt cellular structures and enhance enzyme-substrate interactions, resulting in higher extraction efficiency and faster processing times [117,118]. EASCFE (Enzyme Assisted Supercritical Fluid Extraction) combines enzymatic and supercritical fluid extraction to improve the extraction and processing of sensitive bioactive compounds. This technique uses a non-toxic extraction fluid and mild operating conditions, resulting in a selective extraction and the preservation of the enzyme used. When enzymatic extraction is performed first, the enzymes hydrolyse the structural components, and supercritical fluid extraction is used to extract the compounds selectively [119]. Supercritical fluid extraction combined with enzymatic hydrolysis can also be applied in a conjugated manner, selectively extracting compounds of interest in a single-step process. Sequential and simultaneous enzyme-assisted supercritical fluid extraction was compared in the study of Mikˇ sovsky et al. [120]. ILEAE (Ionic Liquids Enzymatic Assisted Extraction) offers a promising, efficient, and versatile method for the extraction of bioactive compounds, especially for difficult matrices such as lignocellulosic biomass. However, some ionic liquids can be toxic, and their biodegradability and recycling are limited. Enzymatic treatment combined with ionic liquid extraction was used simultaneously in the study by Liu et al. [121], resulting in shorter extraction times and higher yields compared to conventional techniques, due to the disruption of cellular structures. PEF (Pulsed Electric Fields) treatment combined with enzymatic hydrolysis is efficient in the hydrolytic process of proteins. Its application had the effect of promoting the enzyme penetration into cells, improving its efficiency [122–124]. Therefore, the combination of different techniques with the use of enzymes represents an innovative solution for several applications, thanks to their ability to optimize yields. 4. Conclusion and future perspectives Nowadays, recovery and valorisation of wasted food are fundamental in the face of economic and environmental challenges, which include food security, preservation of resources, and mitigation of environmental impacts. In the scientific community, increasingly innovative technologies for the recovery and valorisation of wasted food are spreading. Enzymatic hydrolysis represents a promising solution to convert residues into valuable products. This, as an environmentally friendly processing method, allows the recovery of macromolecules and Table 2 Applications of enzymatic hydrolysis combined with other extraction methods. Substrate Purpose of the study Enzyme Combined technique 1 Time of application 2 Main results Reference Tomato peels Lycopene extraction Cellulase and pectinase Ultrasound Simultaneously Extraction improvement up to 1.3 times compared to the single extraction process [114] Orange peels Phenolic compounds extraction Pectinex® Ultra SP-L Ultrasound After No improvement in extraction yield by applying an ultrasound step [6] Pumpkin seeds Pumpkin seed oil extraction Cellulase, hemicellulase, pectinase, β-glucosidase and neutral proteinase Microwave Simultaneously Physicochemical properties of the oil extracted by MAEE similar to those of the Soxhlet extraction [116] Rice hull Tricin Celluclast®, Rapidase®, Viscozyme® High pressure Simultaneously Tricin extraction yield higher than solvent extraction and HPP extraction alone [117] Tomato processing residues Carotenoids Cellulyve AN 3500, Pectinex® Ultra AF-P High pressure Simultaneously Higher extraction yield using HPAE compared to solvent extraction at ambient pressure [118] Moringa oleifera seeds kernel Kernel seeds oil Mixture of Neutrase® and Celluclast® High pressure Before Improved extraction yield using HPP prior to aqueous enzymatic extraction compared to solvent extraction [112] Pomegranate peels Phenolic compounds Acid Cellulase, Pectinex® Ultra SPL, Alcalase®, Viscozyme®, Kemzyme® (alone and in mixture) Supercritical fluid After EASCFE doubled the extraction yield of phenolic compounds [119] Apple pomace Phenolic compounds Snailase Supercritical fluid Simultaneously and after Improved enzyme efficiency in the simultaneous process [120] Eucommia ulmoides leaves Chlorogenic acid Cellulase, Dextranase, Pectinase, Xylanase Ionic liquids Simultaneously More efficient extraction using ILEAE than using conventional techniques [121] Microalgae biomass Protein hydrolysates Mixture of Alcalase® and Flavourzyme® Pulsed electric field Before Enhancement of enzymatic hydrolysis of proteins by application of PEF [124] Pomelo peels Pectins Cellulase Pulsed electric field Before, after and simultaneously Improved enzyme efficiency and financial benefits using PEF as pretreatment [123] Peanut shell Soluble dietary fibre Glucose amylase and thermostable α -amylase Pulsed electric field After Better technological properties using PEF [122] 1 Methodology applied in combination with enzymatic hydrolysis. 2 Time when the combined technique is applied (before enzymatic hydrolysis, after enzymatic hydrolysis or simultaneously). L. Esposito et al. New BIOTECHNOLOGY 90 (2025) 122–133 130