Enzymes-Assisted Extraction of Plants: History
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Subjects: Food Science & Technology
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Paulina Streimikyte

Enzymes were used for enhancing production yield with mild and not hazardous applications. However, enzyme specificity, activity, plant origin and characteristics, ratio, and extraction conditions differ depending on the goal. The latest studies show that oligosaccharides released and formed by enzymes have a high potential to be slowly digestible starches (SDS) and possibly be labeled as prebiotics. Additionally, they excel in new technological, organoleptic, and physicochemical properties. Released novel derivatives and phenolic compounds have a significant role in human and animal health and gut-microbiota interactions, affecting many metabolic pathways. 

  • enzyme-assisted extraction
  • plant material
  • phenolic compounds
  • prebiotic
  • nanocellulose
  • nanofibers
  • fermentation

1. Introduction

The demand for new and natural compounds, ‘clean label’ trend, rising drug resistance, holistic wellbeing approach of the post-pandemic period, and sustainable living has intensified the development of plant-derived compounds called biologically active components [1][2][3]. Biologically active substances bind by interaction or binding to specific receptors in stem cells, improving a particular physiological function of the body. Unfortunately, many such compounds are present in cytosolic cell spaces and plant cell walls [4]. Many extraction methods cannot achieve these compounds and thus obtain the highest components yields. That is why enzymes incorporation in various extractions is currently one of the few methods to provide this result. Enzymes with specific hydrolytic properties are used to degrade this matrix to gain access to biologically active components from cytosolic spaces and cell walls [5]. One of the advantages of the usage of enzymes is that they can be added to hydrophilic and multi-step lipophilic extractions, especially for by-product valorization [6]. For example, in Europe, grain, fruit, and vegetable food loss from post-harvest to distribution varies from 20, 41, and 46%, respectively [6][7]. However, enzyme-assisted incorporation increases phenolic content in lipophilic extracts, which is potently applicable for nutraceuticals or pharmaceuticals. However, limitations in the safety of by-products have risen, and greater attention is given to this topic [7]. In comparison, for hydrophilic extracts, enzymes efficiently increase the water-soluble content of novel derivatives applicable in food industries [8].
Gut microbiota modulates lipogenesis and cholesterol synthesis. Dysbiosis initiates higher absorption of sugars in the small intestines by modulating membrane transport [9][10]. Moreover, acetate, the metabolite made by the gut microbiome in the proper amount, can boost immune responses by promoting B10 cells, and in higher amounts can lead to adiposity [11][12][13]. These challenges invite scientists to search for sustainable and functional food development worldwide. One scope is enzymes, usually used for plant-based drinks production and syrups for saccharification, decreased viscosity, higher yield, and low toxicity in the food industry. However, lately, studies suggest that controlled enzyme-assisted extraction could lead to a higher and broader density of nutrients [14][15]. For example, dietary fibers with three or more monomeric units, phenolic compounds, and complexes can be suggested prebiotics and used for functional food development [16]. 
Because the plant material is complex, with varied compositions and matrices, enzymes are used in mixtures or cocktails. Besides releasing secondary metabolites and small peptides, they cleave long-chain molecules into shorter ones. Likewise, these substances are soluble in the solvent and can enhance organoleptic, technological, and functional properties. Moreover, enzymatic extraction methods are characterized by mild reaction conditions, substrate specificity, industrial applicability, and many other advantages [17]. These extracts may be used continuously in many fields and, surprisingly, in green synthesis development [14][18][19].
Green nanoparticle synthesis in aqueous plant extracts has increased over the last decade. Scientific discussion and research indicate the appropriate size of nanoparticles with high potential antimicrobial properties, involving the most common pathogenic bacteria like Escherichia coli, Staphylococcus aureus, and widely spread, highly resistant Candida albicans [20][21]. Studies identify that phenolic compounds and sugars play an essential capping and stabilizing role in green nanoparticles synthesis, and enzymes incorporation could increase the synthesized media yield with economically friendly conditions [22][23].

2. Carbohydrases and Phenolic Compounds in Plants

Carbohydrases and hydrolases get a more profound overview due to the plant cell wall mainly consisting of various carbohydrates, trapping active biological components. The cell contains various linear heterogeneous polymeric carbohydrates homologous to cellulose, such as xyloglucans and mannan, and hemicellulose is covalently linked to cellulose microfibrils and lignin to form complex structural branches. This multi-component structure in the plant cell wall is called lignocellulose. However, plant polymeric substances are usually categorized to waste. 
Enzymes are derived from bacteria, fungi, yeasts, archaea, animal organs, or plant extracts. However, microbial enzymes are more stable compared to ones having a plant or animal origin. Moreover, the production of the enzyme during microbial fermentation is cost-effective and easily adapted to modifications and high purity [24]. Carbohydrases can be categorized in starch-degrading enzymes: amylases and glucoamylases; and non-starch polysaccharides (NSP) catalyzing enzymes with cellulolytic, pectinolytic xylanolytic activities [24][25][26]. 
NSP enzymes are preferred as a part of commercial enzyme mixture, thus ensuring complete lysis of cell walls while contributing to a cost-effective means [5][27]. Various fungi, including Trichoderma sp. and Aspergillus sp., produce carbohydrate-hydrolyzing enzymes. For many years, Trichoderma sp. has been extensively studied for high cellulase production [28]. However, most strains of Trichoderma are known to have low β-glucosidase activity, which causes cellobiose accumulation. Although much effort has been made to obtain T. reesei mutants by classical mutagenicity, such as RUT-C30, the relatively low activity of β-glucosidase remains one of the significant barriers to efficient cellulose hydrolysis [29]. Aspergillus sp. is important in xylanase production, and the latest studies showed UV-irradiated Aspergillus mutants for a higher yield of enzymes [30][31]. 
In general, biological raw material systems range from 5000 to 25,000 individual phytochemicals that can have biological activity. Biologically active substances are metabolites synthesized in plants that perform plant protection and other functions. There is growing evidence that biologically active substances can help maintain optimal health and reduce the risk of chronic diseases such as cancer, cardiovascular disease, stroke, and Alzheimer’s disease (AD) [32][33]. 

3. Enzymatically Treated Polysaccharides as Possible Functional Components

Lignocellulose and starch content differs depending on plant material. Grain materials usually consist of more starches because of the endosperm in the seed, whereas other plant parts, like brans, leaves, and others, consist of non-starch polysaccharides. Shah et al. [34] described three categories of starches: rapidly digestible starch (RDS), resistant starch (RS), and slowly digestible starch (SDS), which relate glucose release during digestion. Mainly, starches are found in cereals and pseudocereals where β-glucans are the most widely investigated non-starch polysaccharide and have enormous health-promoting properties [35]. However, β-glucans properties may vary depending on molar mass, which can be from 209 up to 2500 (kg mol−1) depending on the cultivar, variety, and the location of growth [36][37]. 

4. Enzymes-Assisted Processes for Plant Materials

4.1. Bioactives Extraction from By-Products

In order to use the enzyme or their mixtures efficiently in extraction methods, it is essential to understand their catalytic mechanism of action and the optimal activity conditions for the recovery of individual biological raw materials and substances: e.g., a mixture of cellulose, pectin, and hemicellulose enzymes in a grapefruit peel during hydrolysis releases sugar into monomeric compounds that microorganisms can use later to produce ethanol and other fermentation products [38]. Another example is in tomatoes: lycopene is found mainly in the peel, giving it a red color. Carotenoids, especially lycopene, are one of the most potent antioxidants of plant origin, with a role in more than twenty different induced-signaling pathways and cell cycles described by Qi et al. [39]. According to scientific knowledge, lycopene is better absorbed from processed products than fresh tomatoes [40][41]. The digestive enzyme pancreatin is recommended before the solvent extraction of lycopene. Its use increased the yield of lycopene 2.5-fold compared to that obtained using the traditional extraction method [42]. 
Enzyme-assisted extraction (EAE) presents applicability to extract pectins from wastes and by–products by increasing plant cell wall permeability [43][44]. Enzymes are applicable to extract many phenolic compounds, including flavonoids and anthocyanidins [45]. Enzyme activity, treatment time, substrate ratio, and particle size are essential to get the highest efficiency during enzymatic treatment.

4.2. Plant-Based Drinks from Grains and Fermented Drinks Production

There is also no surprise that dairy milk substitute from grains production often requires enzymatic assistance for increasing extraction yields, proteins, and total solids content [19]. Annually, global plant-based dairy substitutes were marked to be grown by 10% and by 2019 had reached US 1.8 billion dollars [46]. Moreover, created derivatives and released sugars create sensory-acceptant organoleptic properties [14]. Amylolytic enzymes are required due to the amylose and amylolytic ratio of the starches, resulting in different rheological and textural properties. However, disrupting amylose and amylopectin molecules increases liquefying properties of grain beverages.

4.3. Nanocrystals, Nanofibers, and Nanocellulose

The latest studies indicate that phenolic content increased in fermented products [47][48][49]. As a sidestream nanocellulosic material, it is usually produced by Komagataeibacater, Acetobacter, Gluconacetobacter strains which might be used as, e.g., wound healing biofilm [50]. Specific enzymes release, cleave, transport, and form derivatives from different plant origins by opening the ability to discover green synthesis applications for nanofibers, nanocrystals, and nanoparticles. Aqueous different plant extracts are the new scientific approach for synthesizing nanoparticles by changing environmentally disruptive chemical and physical methods. Enzyme-assistance by disrupting plant cell wall microfibrils and amorphous zones is visible through Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), or Scanning Electron Microscopy (SEM), which also implies an increase of extract yield [51][52]. Plant extracts contain high phenolic content and reducing sugars and reducing or stabilizing agents [23][53]. 

5. Conclusions

The use of enzymes in extracting biological raw material compounds is an up-and-coming area from small-scale, laboratory optimization studies to large-scale, industrial applications. It implies food processing, functional components, and medical devices development for high antioxidant, anti-inflammatory, and antimicrobial characteristics. However, success in this area requires interdisciplinary research from various life sciences disciplines. An important area of research is investigating the stability of enzymes and their interaction with other food and plant ingredients during processing and storage; repeatability is also questionable because the plant material differs from the origin, cultivars, and growing, harvesting, and storage conditions. Additionally, limitations occur in the form of worldwide regulations of enzymes usage and dosages due to the novel components that are produced during these processes. However, enzyme-assisted processes are reaching for more sustainable development of innovations in a broad spectrum of industries.

This entry is adapted from the peer-reviewed paper 10.3390/ijms23042359

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