Combined Enzyme- and Ultrasound-Assisted Extraction of Botanical Bioactives for Nutraceutical Ingredients: Mechanisms, Process Design, Scale-Up and Safety

1. Introduction

Botanical extracts are the core raw materials of the nutraceutical industry, and the extraction step largely determines their composition, yield, cost and safety. Conventional solvent extraction by maceration, percolation or reflux is slow, uses large solvent volumes and may degrade heat-sensitive constituents [1,2]. Ultrasound-assisted extraction has become the most studied alternative. Acoustic cavitation fragments and erodes plant tissue, opens cell walls and accelerates mass transfer, so that higher yields can be obtained in less time, at lower temperature and often with water or ethanol in place of more hazardous solvents [1,3].

Two recent publications, which form the starting point of this review, qualify this picture. Lucas-González and colleagues separated date palm seed polyphenols into free, soluble-conjugated and insoluble-bound fractions and found that probe sonication extracted the first two efficiently but was not efficient for the insoluble-bound fraction; they concluded that cavitation did not affect the covalent bonds between polyphenols and the cell wall [4]. Because the three fractions in date seed were of similar size, this limitation is not marginal: the best ultrasound run recovered 189 milligrams per 100 grams of the summed polyphenols, against about 260 milligrams per 100 grams for the three conventional fraction-specific extractions combined [4]. Demesa and colleagues reviewed the potential for toxic side reactions in ultrasound-assisted extraction and found no direct evidence of toxic compound formation from plant materials under optimised conditions, but noted that ultrasound can co-extract natural toxins, can promote oxidation and Maillard-type reactions under extreme conditions, has rarely been reported at industrial scale, and needs experimental safety studies [3].

Enzyme-assisted extraction addresses a different barrier. Cellulases, hemicellulases, pectinases and proteases hydrolyse the cell wall polymers that physically enclose intracellular constituents and the ester and glycosidic bonds that hold bound phenolics, under mild aqueous conditions [5,6]. Its drawbacks are slow reaction rates and the cost of enzyme preparations [7]. The combination of the two technologies, often called ultrasound-assisted enzymatic extraction, is therefore attractive: ultrasound may speed up and extend enzymatic action, and enzymes may reach the bound fraction that ultrasound alone leaves behind [8,9].

The aim of this review is to evaluate this combination for botanical ingredients intended for nutraceutical use. It explains the mechanisms by which the two technologies interact, compares treatment sequences and operating windows, reviews representative applications, discusses scale-up, and sets out the safety and quality considerations that follow from adding a biological catalyst to a cavitation process. The perspective is that of a manufacturer of botanical extracts, and particular attention is given to what must be specified, controlled and reported.

2. Methods of Literature Selection

This is a narrative review. The two source publications provided by the author [3,4] were read in full. Further literature was identified through structured searches of the Europe PubMed Central database and publisher websites, using combinations of the terms ultrasound-assisted extraction, enzyme-assisted extraction, ultrasound-assisted enzymatic extraction, bound phenolics, cell wall, sonication, enzyme activity, scale-up and degradation, with priority given to highly cited reviews and to primary studies that reported quantitative comparisons between single and combined treatments. Bibliographic details of all cited works were checked against the Crossref registry. Because extraction conditions, matrices and outcome measures differ widely, no quantitative synthesis was attempted.

3. Where Phenolics and Other Actives Reside in Plant Tissue

Plant phenolics occur in three operational forms. Free phenolics are dissolved in the vacuole or cytoplasm and are extracted readily by aqueous–organic solvents. Soluble-conjugated phenolics are esterified or glycosylated to small soluble molecules such as sugars and organic acids. Insoluble-bound phenolics are covalently linked through ester, ether or glycosidic bonds to cell wall polymers such as cellulose, hemicellulose, pectin, lignin and structural proteins [4,10]. The bound fraction is often large, particularly in seeds, brans, hulls and fibrous tissues, and it is usually omitted when extraction yields are reported, so the efficiency of a method can be overstated [4,10].

The bound fraction is released analytically by alkaline or acid hydrolysis, which are harsh and not compatible with food-grade ingredient manufacture. Fermentation, malting, extrusion and enzymatic hydrolysis can release it under food-compatible conditions, sometimes assisted by microwave or ultrasound [10]. The distinction matters for nutraceutical ingredients for two reasons. First, the bound fraction represents unrecovered value. Second, bound phenolics that reach the colon are released by microbial enzymes and contribute to biological effects there, so an extract that excludes them has a different composition, and potentially a different activity, from the whole plant material [10].

Non-phenolic actives face analogous barriers. Lipophilic constituents such as the gingerols of ginger and the curcuminoids of turmeric are enclosed in cells embedded in a starch- and fibre-rich matrix, and polysaccharides are themselves part of the wall [8,11,12].

4. Mechanisms of the Two Technologies and Their Interaction

4.1. Ultrasound

Ultrasound at 20 to 40 kilohertz propagating through a liquid produces cycles of compression and rarefaction that nucleate, grow and violently collapse cavitation bubbles. Collapse generates local temperatures of thousands of kelvin, high pressures, microjets and shock waves [1,3]. On plant material this produces fragmentation, surface erosion, capillary penetration of solvent, detexturation and sonoporation of membranes, which together increase the contact area and mass transfer [1]. These are physical effects. They explain why ultrasound accelerates the release of free and soluble-conjugated compounds, and why it does not by itself cleave covalent ester and glycosidic bonds to the cell wall under mild conditions [4].

Cavitation in water also generates hydroxyl radicals. Under excessive power, long duration or poor temperature control, these radicals and local heating can oxidise lipids and phenolics, alter proteins and polysaccharides and produce off-flavours [3,13].

4.2. Enzymes

Enzyme preparations used for extraction are usually mixtures. Cellulases and hemicellulases (including xylanases) depolymerise the structural polysaccharides of the wall; pectinases loosen the middle lamella; amylases remove starch that traps lipophilic constituents; proteases hydrolyse wall proteins and protein–phenolic complexes; and esterases, such as feruloyl esterases, cleave the ester bonds by which hydroxycinnamic acids are attached to wall polysaccharides [5,6,10]. Enzymes act under mild conditions, are selective, and leave no hazardous solvent residue. Their limitations are reaction times measured in hours, dependence on pH and temperature, substrate accessibility, inhibition by phenolics and other plant constituents, and cost [6,7].

4.3. How Ultrasound Changes Enzymatic Action

Ultrasound affects enzymatic extraction in three ways. First, it increases access: fragmentation and erosion expose new substrate surface, and cavitation-driven mixing reduces the diffusion layer around particles, so that enzyme molecules reach the substrate and products leave it faster [8,9]. Second, it can change enzyme activity directly. At mild intensities and appropriate duty cycles, sonication has increased the activity of several enzymes, attributed to favourable conformational changes and dispersion of aggregates; at high intensity, long duration or elevated temperature, it inactivates enzymes through structural disruption and radical attack [14]. Third, in complex matrices ultrasound may release cofactors from the biomass. In a study comparing purified pectin with grapefruit peel and apple pomace, ultrasound applied during enzymatic hydrolysis increased hydrolysis only in the complex matrices, accompanied by greater release of calcium and potassium ions, which the authors interpreted as release of enzyme cofactors [9].

The same principles apply to protein-rich materials. Ultrasound-assisted hydrolysis of goat milk casein increased the reaction rate by up to 120 per cent and the final degree of hydrolysis by 23 to 48 per cent with three commercial proteases, and reduced the time to reach maximum solubility up to tenfold [15].

4.4. Synergy Depends on Sequence

Whether the combination is synergistic, additive or neither depends on when ultrasound is applied. In the study by Kabawa and colleagues, ultrasound applied before or after the enzymatic step had no significant effect on pectin hydrolysis, whereas ultrasound applied during the reaction increased hydrolysis in complex matrices, indicating synergy [9]. This finding has practical consequences: a brief sonication pretreatment followed by conventional incubation, a common industrial shortcut, may not deliver the benefit expected from the combined process. Table 1 summarises the interactions.

5. Process Design

5.1. Treatment Sequences

Four sequences are used (Table 2). In ultrasound pretreatment, the material is sonicated and then incubated with enzymes; the plant tissue is opened, but enzyme activity is not affected and, as noted above, synergy may be absent [9]. In enzyme pretreatment followed by ultrasound-assisted extraction, the wall is hydrolysed first and the loosened material is then extracted by sonication, often in a solvent unsuitable for the enzyme, such as aqueous ethanol; this sequence suits lipophilic actives and was used, for example, with ginger before solvent extraction [11,16]. In simultaneous treatment, ultrasound is applied, continuously or pulsed, during enzymatic incubation in aqueous medium; this is the sequence in which synergy has been observed and in which conditions must satisfy both the enzyme and the cavitation process [8,9]. Finally, after any of these, a heat step is needed to inactivate the enzymes before concentration and drying.

5.2. Operating Window

Simultaneous treatment must be operated within a window that satisfies both technologies. Temperature should be near the enzyme optimum, typically 40 to 55 degrees Celsius for commercial carbohydrases, and must be controlled because sonication heats the medium; the date seed study used an ice bath to prevent overheating [4]. pH is set by the enzyme, usually between 4 and 6 for carbohydrases. Acoustic intensity and duty cycle should be high enough to disrupt tissue and mix the slurry but low enough to avoid inactivating the enzyme; pulsed operation helps both temperature control and enzyme stability [14]. The liquid-to-solid ratio affects both the acoustic field and the concentration gradient; in the date seed study it was the most important factor for polyphenol recovery, with higher ratios giving better results [4]. Probe geometry also matters: a smaller-diameter sonotrode with higher displacement amplitude gave the best recovery and antioxidant activity [4]. Particle size, enzyme dose expressed in activity units per gram of substrate, incubation time and agitation complete the list of critical parameters.

Optimisation should begin with a screening design such as a Plackett–Burman design, which identified the critical variables among seven factors in the date seed study [4] and in a recent combined extraction of mulberry bark flavonoids [17], followed by response surface or machine-learning optimisation of the few factors that matter.

5.3. Solvent Choice

Enzymes require an aqueous medium, which restricts simultaneous treatment to water or dilute buffers. This suits polysaccharides, flavan-3-ols and other hydrophilic phenolics, which were extracted efficiently by water alone from date seed [4], but not lipophilic actives such as curcuminoids, gingerols or methoxyflavones. For these, a two-stage process is appropriate: an aqueous enzyme–ultrasound stage to open the matrix and release bound and hydrophilic constituents, followed by an ethanol or other food-grade solvent stage to recover lipophilic ones. Enzyme pretreatment of ginger with amylase or a multi-enzyme preparation followed by acetone extraction raised oleoresin yield from 15 to 20 per cent and gingerol content of the oleoresin from 6.4 to 12.2 per cent [11], and amylase–amyloglucosidase pretreatment of turmeric raised curcumin extraction from 3.58 to 5.73 per cent [12].

6. Applications

Table 3 summarises representative studies. In sesame bran, ultrasound-assisted enzymatic extraction gave the highest protein yield, total phenolic content and antioxidant capacity among enzyme-only, ultrasound-only, combined and alkaline methods [18]. In mulberry bark, combined extraction optimised by statistical screening and machine learning was used to recover flavonoids, with mechanistic support from infrared spectroscopy, microscopy and molecular simulation [17]. Polysaccharides from agricultural by-products are the largest application area, and a 2026 review concluded that the combination increases extraction efficiency, shortens processing and preserves structural integrity, but requires optimised multi-enzyme mixtures to avoid antagonism between enzymes [8]. For the major lipophilic rhizome actives used in nutraceuticals, most published work has tested enzyme pretreatment followed by solvent extraction rather than simultaneous enzyme–ultrasound treatment [11,12,16], and systematic comparisons of the four sequences for these materials are lacking.

Two limitations run through this literature. Most studies measure total phenolic content by the Folin–Ciocalteu assay and antioxidant capacity by chemical assays, rather than identified compounds by chromatography, so changes in composition, including deglycosylation and degradation, are not visible. And few studies analyse the bound fraction remaining in the residue, so it is rarely possible to tell whether a combined process releases bound compounds or simply extracts free ones faster.

7. Scale-Up

Laboratory studies use ultrasonic baths or probes of a few hundred watts treating millilitres to litres. Industrial use requires continuous flow cells or large probe arrays in which every volume element receives a defined acoustic dose. High-power ultrasound has been commercialised for extraction, emulsification and other operations, but it is not an off-the-shelf technology and must be scaled up for each application [19]. Industrial and pilot-scale reports of ultrasound-assisted extraction remain few, and they are concentrated in olive oil, beverages and a small number of botanical and fungal extracts [3].

For combined processes, scale-up adds the constraints of enzymatic reactors: residence times of tens of minutes to hours, temperature and pH control, and a subsequent inactivation step. A practical configuration is a stirred, temperature-controlled enzymatic tank with an external recirculation loop through an ultrasonic flow cell, which allows the acoustic dose to be set independently of the tank volume. The specific energy delivered, in kilojoules per kilogram of slurry, should be the basis for scale-up rather than nominal generator power, because the fraction of electrical power converted to acoustic power varies widely between devices; calorimetric measurement provides a simple estimate of absorbed acoustic power [20]. Probe-type systems are considered easily scalable, which supports this approach [4].

Economic assessment should weigh enzyme cost, which is often the largest operating cost of enzymatic extraction [7], against gains in yield, shorter time, lower temperature and reduced solvent use. Integration with other technologies, such as microwave heating, is also possible but adds complexity [21].

8. Safety and Quality Considerations

Demesa and colleagues concluded that ultrasound-assisted extraction does not increase toxicity relative to conventional processing when parameters are optimised, while calling for experimental studies [3]. Combining ultrasound with enzymes raises additional considerations (Table 4).

8.1. Radical Formation and Degradation

Cavitation-generated radicals and local heating can degrade phenolics and oxidise lipids, particularly with prolonged sonication at high power [3,13]. Simultaneous enzyme–ultrasound treatment typically involves longer sonication than ultrasound extraction alone, because enzymatic reactions are slow; pulsed operation, temperature control and minimising total acoustic energy reduce this risk. Marker compounds should be quantified, not only total phenolic content, so that degradation is detected.

8.2. Enzyme Preparations and Residues

Commercial enzyme preparations are produced by fermentation and contain, in addition to the declared activity, side activities, residual production-organism material and formulation agents. In regulated markets food enzymes are subject to safety evaluation, which covers the production strain, toxicological data, allergenicity and, where relevant, the presence of production-strain DNA and antimicrobial resistance genes; such issues have led to negative conclusions for specific preparations [22]. Manufacturers should use food-grade enzymes with documented safety evaluations in their target markets, verify inactivation by measuring residual activity in the extract, and consider the allergenic potential of residual enzyme protein.

8.3. Compositional Change

Enzyme preparations with glycosidase side activities can convert glycosides to aglycones during extraction. This can be desirable, since deglycosylated forms such as ginsenoside compound K are often better absorbed [23], but it changes the identity of the extract: a specification based on glycoside markers may be failed by an extract that is in other respects of higher quality, and the safety profile of the transformed extract should not be assumed to be identical to that of the traditional preparation. Changes in composition should be documented and reflected in the specification.

8.4. Co-Extraction of Natural Toxicants and Contaminants

By opening the matrix more completely, combined treatments may increase the extraction of natural toxicants as well as actives, as reported for ultrasound with cyanogenic glycosides and glycoalkaloids [3]. Hydrolysis of cyanogenic glycosides by glycosidases releases hydrogen cyanide, so materials containing them require specific evaluation. More complete extraction may also increase the transfer of pesticide residues, heavy metals and mycotoxins from the raw material; these should be tested in the extract, not only in the raw material.

8.5. Microbiological Control

Enzymatic incubation at 40 to 55 degrees Celsius in aqueous slurry for extended periods favours microbial growth. Raw material bioburden, incubation time and hygiene of the recirculation loop must be controlled, and the enzyme inactivation step should also be validated as a microbial control point.

9. A Reporting Standard for Combined Extraction Studies

The following minimum information is proposed for studies and process dossiers.

• Botanical identity, plant part, pretreatment, moisture and particle size distribution of the raw material.
• Enzyme preparation by trade name, source organism and declared activities, with the dose expressed in activity units per gram of dry substrate, and the pH, temperature and time of incubation.
• Ultrasound system (bath, probe or flow cell), frequency, sonotrode geometry and amplitude, duty cycle, and the acoustic power absorbed, measured calorimetrically, with the specific energy delivered per kilogram of slurry.
• The treatment sequence (pretreatment, simultaneous or post-treatment) and the temperature profile during sonication.
• Controls with enzyme alone, ultrasound alone and neither, under otherwise identical conditions, so that synergy can be assessed.
• Fraction-resolved analysis of free, soluble-conjugated and insoluble-bound compounds, including the bound fraction remaining in the residue, with identified compounds quantified by validated chromatography.
• Enzyme inactivation conditions and residual enzyme activity in the extract.
• Safety-relevant analyses of the extract, including natural toxicants relevant to the species, contaminants and microbiological quality.
• Specific energy and enzyme consumption per kilogram of extract, to allow economic and environmental comparison.

10. Conclusions

Ultrasound-assisted extraction efficiently releases free and soluble-conjugated constituents of botanicals but does not cleave the covalent bonds that retain insoluble-bound phenolics, and its industrial use and safety have been less thoroughly documented than its laboratory performance. Enzyme-assisted extraction attacks precisely those bonds and wall polymers but is slow and costly. The combination addresses both limitations: enzymes release what cavitation cannot, and ultrasound improves enzyme access, mass transfer and, within a mild operating window, enzyme activity. Synergy is most likely when ultrasound is applied during enzymatic hydrolysis of complex matrices, and less likely when it is used only as a brief pretreatment. For nutraceutical manufacturers, the combination offers higher and more complete recovery under food-compatible conditions, but it also changes extract composition, introduces enzyme-related safety obligations and requires scale-up based on delivered acoustic energy. Studies that include single-treatment controls, measure the bound fraction and report energy and enzyme activity in standard units would allow the technology to be compared and adopted on evidence rather than claims.