Fermentative and Enzyme-Assisted Production of Phenolic Antioxidants from Plant Residues

Miklos Tako, Carolina Zambrano, Alexandra Kotogan, Erika Beata Kerekes, Tamas Papp, Judit Krisch and Csaba Vagvolgyi


Phenolic antioxidants are secondary metabolites participating in defence mechanisms in plants. They also have significant roles in other processes, such as pigmentation, adaptation to adverse environmental conditions and incorporation of attractive substances to accelerate pollination. Phenolic compounds could affect beneficially the human health and have a great potential in the prevention and treatment of certain chronic diseases. Thus, there is a growing interest in using them as additives in functional foods. Accordingly, the bioprocesses able to produce bioactive phenolics from natural sources have significant attention in the food industry. Phenolics in plant residues are mainly in glycosidic form, which results in reduced bioavailability. However, solid-state fermentation (SSF) of the substrate using cellulolytic filamentous fungi and carbohydrase-assisted extraction are well applicable methods to liberate the phenolic aglycons from their glycosides. The extracted phytochemicals are then potentially applicable as natural food additives and antimicrobials.

The present chapter summarizes some general properties of plant phenolics, the current trends on their mobilization through fungal fermentation and enzyme treatment, the most important plant waste sources, as well as fungal species and enzymes, which have been utilized for this purpose.

Main Groups of Plant Phenolics

Phenolic Acids

Phenolic acids are found in most foods of vegetable origin, generally in conjugated form. They are divided into two main groups, namely, hydroxybenzoic and hydroxycinnamic acids, according to the pattern of hydroxylation and methoxylation of the aromatic rings. The most common hydroxybenzoic acids in plants are gallic acid, /^-hydroxybenzoic acid, vinyl acid and protocatechuic acid. Hydroxycinnamic acids are generally found in glycosidic forms or esters of shikimic, quinic and tartaric acids. The most common hydroxycinnamic acids in plants are ferulic,/>-coumaric, sinapic and caffeic acids. Being antioxidants, several phenolic acids are outstanding in reducing power and free radical scavenging activities. Many caffeic acid derivatives can inhibit enzyme activities such as lipoxygenase and protein kinase (Kang et al. 2008; Doiron et al. 2017).


Flavonoids are common phenolics in plants. Their structure, composition and biological activities are summarized in the study of Panche et al. (2016). Most flavonoids are soluble according to the polarity and chemical structure, for instance, degree of glycosylation, hydroxylation and acylation. They have a common structure consisting of two benzene rings joined by three carbon atoms, which form an oxygenated heterocycle with various levels of hydroxylation and methoxylation.


They are non-flavonoid polyphenols that are characterized structurally by the presence of a 1,2-diphenylethylene nucleus. The most studied representative of this group is the resveratrol that has gained a global attention due to its beneficial health effects including cardiovascular, antiobesity, antidiabetic and neuroprotective properties. It can also be used to minimize or prevent the oxidation of lipids in pharmaceutical products, prolonging their shelf life and maintaining the nutritional quality (Gtikjin 2010).


Tannins are highly hydroxylated water-soluble compounds. They can interact with carbohydrates and proteins that result in different insoluble complexes. Tannins can be divided into two groups according to the chemical structure: (1) hydrolyzable tannins that are esterified derivatives of gallic acid and hexahydroxydiphenic acid; (2) condensed tannins known as proanthocyanidins, which are polymers of high molecular weight catechins formed by oxidative condensation of the phenolic ring and the adjacent units or by enzymatic polymerization of flavan-3-ol and flavan-3,4-diol units. Both the condensed and hydrolyzable tannins have high bioactive potential. The antimicrobial activity of hydrolyzable tannins has been summarized recently by Ekambaram et al. (2016). Condensed tannins can inhibit the growth of many microorganisms as well (Assefa et al. 2017).

Fruit and Vegetable Processing By-products

Processing technologies in the agro- and food industry generate large amounts of by-products annually. Treatment and disposal of these wastes are important both economically and environmentally. There are many applications for their utilization, for instance, as animal feed, natural fertilizer or biomass for bioethanol production (Kruczek et al. 2016). However, peels, pulps and seeds wasted from fruits and vegetables could be good sources of bioactive phytochemicals such as dietary fibers, carotenoids and phenolic compounds. In most cases, the bioactive phenolic content of these by-products is comparable to what can be found in the final product. Most of these phenolics are bounded in glycosides that need to be hydrolyzed to improve the bioavailability. Fermentation with microorganisms and the different enzymatic treatment methods can be effective tools for this purpose.

In the last few years, there has been an increasing interest of using agro-industrial by-products for production of natural additives and supplements rich in valuable compounds. The recycling and recovery of these substances could be economically attractive in the development of functional food ingredients (Galanakis 2012). Globally, grape, citrus fruit and apple processing technologies generate large amounts of by-products having bioactive constituents (De Ancos et al. 2015). As seen in Table 12.1, these plant materials store several extractable phenolic compounds. In addition, residues of other food crops such as blueberry, watermelon, avocado, banana, guava, mango, litchi, pitahaya, tomato, potato and wheat bran also contain valuable phytochemicals (Deng et al. 2012; De Ancos et al. 2015). It is worth mentioning, however, that the composition and concentration of the phytochemicals could highly differ between fruit cultivars. Environmental factors and agronomic conditions during the cultivation, and postharvest storage parameters can also affect the crop’s phenolic composition.

TABLE 12.1

Extractable Phenolic Compounds in Apple, Grape and Citrus Fruit Residues



Phenolic Compounds



Peel, pomace

Phenolic acids (gallic, syringic, chlorogenic, caffeic, p-coumaric, ferulic, protocatechuic), quercetin in glycosylated forms, catechin, epicatechin, anthocyanins, phloridzin, rutin

Kalinowska et al. (2014)



Hydroxybenzoic acids (gallic, protocatechuic, syringic, p-hydroxybenzoic), hydroxycinnamic acids (caffeic, p-coumaric, ferulic, sinapic, caftaric, coutaric, fertaric), flavanols (catechin, epicatechin, epicatechin gallate), stilbenes (resveratrol), flavonols (kaempferol, quercetin, and their derivatives, rutin), anthocyanins, procyanidins

Fontana et al. (2013)

Citrus fruits

Peel, seed

Naringin, naringenin, hesperedin, neohesperedin, eriocitrin, neoeriocitrin, and caffeic, p-coumaric, ferulic and sinapinic acids

Sharma et al. (2017)

Substrate Pretreatment

Prior to fermentation and enzymatic treatment, the by-product residues are subjected to drying and subsequent grinding in most studies. Hot air drying (known as oven-drying) and freeze-drying (known as lyophilization) are the frequently used techniques to completely dry the substrates. This preparation step has great importance since it can influence the fungal growth and the stability of the phenolics during the treatments. Accordingly, there are several studies aimed to evaluate the effects of different drying methods on the phenolics composition of plant waste materials (Tseng and Zhao 2012; Orphanides et al. 2013).

Heating has an impact on the action of some enzymes (e.g. esterases, oxidases), which determines the final amount and quality of bioactive compounds in the residues (Galanakis 2012). In addition, some phenolic substances are also sensitive to heat, especially to the temperatures above 50°C, which could cause their chemical degradation, isomerization or polymerization (Yu and Ahmedna 2013). The common oven-drying techniques use temperatures of around 60°C and the time could be a few hours, which therefore implies a low operating cost. The study of Khanal et al. (2010) showed that the heating above 60°C significantly reduced the procyanidin and anthocyanin content in blueberries and grape pomace. Drying at low temperature (40°C) for 3 days proved to be a more gentle process compared to a short-time (8 hours) treatment at higher temperatures (more than 60°C) that caused a considerable loss in phenolics concentration. Alternatively, ohmic heating, also called electro-conductive heating, could be applied to avoid the thermal damage of heat-sensitive substances (Aggarwal and Jain 2019).

Lyophilization is a more costly and slower technique compared to oven-drying because it needs a vacuum and it could take several days to reduce the water content in the pomace to the desired level (Galanakis 2012). Due to the low temperature conditions, however, this technique could protect the phenolic compounds from thermal degradation, retaining the high-degree antioxidant activity in the substrate (Michalczyk et al. 2009). For instance, mango peel and kernel residues subjected to freeze-drying showed superior phenolic content and antioxidant activity to samples treated with oven, cabinet, vacuum and infrared-drying techniques (Sogi et al. 2013).

Chopped samples can support both the fermentation and the enzymatic treatment for effective phytochemical recovery. Grinding of the by-product before and/or after the drying process promotes the degradation of the lignin-cellulose network by reducing the crystallinity and increasing the surface contact area for the mycelial growth and enzymatic actions. When solvent extraction is also applied, grinding provides higher diffusion to extractants into the by-product matrix (Galanakis 2012).

Advantages of SSF in the Production and Recovery of Biomolecules

Several studies have shown that SSF is a well applicable method for bioactive compound production and enrichment from fruit and vegetable processing by-products (Lizat'di-Jimenez and Hernandez-Martmez 2017). SSF could be defined as a process in which the microbial growth occurs on solid particles in the absence or with very' low amount of water. It is also known that SSF can provide conditions for the cultivated microorganisms similar to those in their natural environment. SSF has several biotechnological advantages (e.g. high fermentation capacity and end-product stability, high- yield enzyme production, and reduced substrate inhibition, stirring, and downstream processing). In addition, various agro-industrial by-products and wastes can be utilized as cultivation substrates in SSF systems. By this approach, different industrially relevant molecules, such as enzymes, organic acids, pigments and bioactive phenolic acids have been produced in recent innovations (Soccol et al. 2017; Sadh et al. 2018). Therefore, SSF is a useful technology not only in the waste management but in the generation of value-added products from plant residues. The most common by-product materials used are sugarcane bagasse, cassava bagasse, oil cakes, orange peel, apple pomace, grape pomace, coffee husk and wheat bran. Many factors (i.e. substrate pretreatment, moisture level, water activity, particle size, growth medium supplementation, temperature, pH, agitation and aeration) can influence the bioconversion (Nigam and Pandey 2009).

SSF processes have several advantages over the conventional submerged fermentation (SmF) approaches. For example, the final yield of various extracellular enzymes of filamentous fungi is significantly higher in SSF than in SmF (Barrios-Gonzalez 2012). In addition, some extracellular enzymes (mainly carbohydrases) are formed in a cell-wall bounded form under SmF conditions (Oda et al. 2006). The major difficulties in SSF systems can be associated with the scale-up, because of the variation of several parameters (e.g. moisture content, temperature and oxygen transfer), which affect the fungal growth and the enzyme activities. Fungi are the preferred fermenting organism in SSF because of their ability to grow well under low moisture content surroundings. Among them, filamentous fungi are frequently used to produce enzymes, organic acids and bioactive compounds. Concerning bioactive phenolics, some important studies are discussed below.

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