**4. Release and Production of Bioactive Compounds**

Improving the biological activity of pomace from food processing is one of the potential and emerging applications of fermentation. This strategy has been applied to obtain carotenoids, fatty acids, γ-linolenic acid, and polyphenols (Table 4). Polyphenols are an important class of bioactive compounds that are found in pomaces. From a broad perspective, polyphenols can be found either in free or bound forms. Polyphenols in free form are those present in the cytosol of vegetable cells, whereas the bound polyphenols are those bound to cell wall constituents [70]. For bound polyphenols in particular, their extraction is complex and conventional extraction methods have low efficiency to separate these compounds from structural components of food. In this context, the use of fermentation (by means of the action of microbial enzymes) has been indicated as a relevant strategy to recovery this compound [70,71].

**Table 4.** Bioactive compounds obtained from pomace fermentation.


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## **Table 4.** *Cont.*

DHA: Docosahexanoic acid, DPPH: (2,2-diphenyl-1-picrylhydrazyl) free radical, FRAP: ferric reducing antioxidant power, ORAC: oxygen radical absorbance capacity, TEAC: trolox equivalent antioxidant capacity, TFC: total flavonoid content, and TPC: total polyphenol content.

> For instance, studies carried out with grape pomace indicate that polyphenols [53,72], γ-linolenic acid and carotenoids [73] can be obtained from fermentation. In addition to the characterization of the content of these bioactive compounds, these studies also revealed aspects related to the preparation of samples, fermentation period, and the effect of the starter culture.

> Regarding the effects of sample preparation and fermentation period in the release of polyphenols, a recent experiment indicated that lyophilization is a better pre-treatment than oven-drying to improve the extraction of polyphenols from grape pomace [72]. Moreover, this study also indicated that long fermentation periods do not favor the accumulation of polyphenols. Additionally, this effect could be explained by the instability of free polyphenols during fermentation. The gradual decomposition of free polyphenols can occur, which may be compensated by the release of bound polyphenols from microbial activity. Another related study with pomace supports this consideration and the necessity to define the optimum fermentation period. The high polyphenol and bioactivity in the beginning of the fermentation period were followed by the reduction in both indicators (polyphenol content and biological activity) as fermentation progressed up to 15 days [74]. Additionally, Teles et al. [53] reported increasing polyphenol content and antioxidant activity during the fermentation of grape pomace with *Aspergillus niger* during a shorter period (96 h) in relation to these aforementioned studies. This study also indicated that polyphenol content was positively correlated with antioxidant potential.

> The production of γ-linolenic acid and carotenoids by solid-state fermentation also displayed the same dependency on fermentation time, wherein maximum yields were obtained after 6 days of fermentation [73]. In the case of carotenoids, the synthesis of lutein had a maximum yield after 8 days, whereas the production of carotene increased throughout the fermentation period (18 days).

> Apple pomace has also been explored as a relevant source of polyphenols and fatty acids. For instance, the effect of pre-treatment and fermentation on polyphenol accumulation during fermentation was studied by Zambrano et al. [72]. The maximum polyphenol content was not affected by the pretreatment (lyophilization vs. over-drying), but significant changes were reported during the fermentation period. The maximum polyphenol yield and antioxidant potential were obtained at day 10. Conversely, Lohani and Muthukumarappan [75] reported a gradual reduction in the polyphenol content of naturally fermented apple pomace. Madrera et al. [76] reported a slight reduction in the polyphenol content of fermented apple pomace with different yeasts. Additionally, this study also indicated that the production of fatty acids can be obtained from the fermentation of apple

pomace with yeasts. An interesting experiment with apple pomace explored the production of fatty acids in a 5 L bioreactor [77]. In this case, different concentrations of apple pomace were used as a carbon source for lipid biosynthesis. A concentration-dependent effect (40, 60 and 80 g substrate/L) in the production of fatty acids was reported. Moreover, the maximum yield for each tested apple pomace concentration was achieved in a short period (3 days).

In the case of olive pomace, the fermentation with *Kluyveromyces marxianus* led to a reduction in tannic acid content and an increase in the concentration of its depolymerized form, gallic acid [79]. Another interesting application of exhausted olive pomace (residue obtained after the removal of residual oil from olive pomace) is the production of microbial fatty acids, especially docosahexaenoic acid (DHA). A recent experiment indicated that the concentration of exhausted olive pomace had a concentration-dependent effect in the production of DHA by the microalgae *Crypthecodinium cohnii* [58]. Interestingly, another study with the same microalga revealed that detoxification with activated carbon reduced the production of fatty acids [80].

The simultaneous production of polyphenols and fatty acids from fruit pomace was also explored in a recent study with two *Sambucus* species [78]. In these fruits, optimum polyphenol production yield and antioxidant activity were obtained at day 3 and 4 (regardless of species), respectively. A similar effect was observed for the accumulation of linoleic and oleic fatty acids, which had maximum values at day 4. Similarly, the accumulation of polyphenols and antioxidant activity during the fermentation of chokeberry pomace were dependent on the time and starter culture [81]. Maximum values for total phenolic content were obtained between day 6 and 9 of fermentation for *Rhizopus oligosporus* and 9 days for *Aspergillus niger*.

Studies carried out with plum [82] and apricot [83] pomaces indicated that optimum fermentation periods for polyphenol accumulation and antioxidant activity from *Aspergillus niger* fermentation were 9 and 6 days, respectively. Another recent experiment indicated that the accumulation of polyphenols in pitahaya pomace from the activity of *Rhizomucor miehei* was improved by lyophilizing samples before fermentation [72]. A related experiment evaluated the accumulation of polyphenols and antioxidant activity in red bayberry pomace during 7 days during the sequential fermentation with *Saccharomyces cerevisiae* and a mix of lactic acid bacteria (*Lactobacillus bulgaricus*, *Bifidobacterium lactis*, and other lactic acid bacteria) [84]. A gradual increase in the polyphenol content was reported throughout the 7 days of fermentation. Moreover, the antioxidant activity of fermented pomace after this period was improved in relation to non-fermented pomace.

Since the fermentation of pomaces can lead to high polyphenol content and antioxidant activity (Table 4), the biological response to the consumption of fermented pomace was also explored in recent studies. Improvements in the antioxidant defense system and a reduction in the oxidative status of liver and ilium in mice fed with fermented blueberry pomace were reported [85]. The intestine inflammatory response (tumor necrosis factor-alpha and interleukin-10) was also improved in animals that consumed the diet supplemented with fermented blueberry pomace. Concentration-dependent effects were observed in the antioxidant and anti-inflammatory activities. Moreover, these effects ameliorated the modifications induced by a high-fat diet in terms of antioxidant and anti-inflammatory responses.

A further experiment carried out by the same research group explored the functional effect of fermented blueberry pomace in indicators of gut health of mice [86]. The consumption of supplemented diets improved the gut immunological response (secretory immunoglobulin A), affected the gut microbiota and also favored the production of butyric acid (a short fatty acid associated with health benefits). Again, the supplemented diet ameliorated the modifications induced by a high-fat diet in the gut immunological response and gut health. Another experiment in vivo that supports the health benefits associated with the fermentation of pomaces was carried out by Yan et al. [87]. In this case, the consumption of fermented blueberry pomace (rich in polyphenols) improved the resistance to fatigue in relation to control animals that ingested sterile water.

Along with the production of fermented pomaces with increased biological activity, it is also important to develop strategies to isolate active components from the bulk of fermented pomace. This aspect was recently explored by Espinosa-Pardo et al. [88] who optimized the extraction of polyphenols with super-critical CO<sup>2</sup> and co-solvents. The authors indicated that the extraction with CO<sup>2</sup> (25 MPa at 60 ◦C) and 90% ethanol as co-solvent was the most efficient extraction condition to obtain the highest polyphenol content and antioxidant activity. Another important aspect to consider is the effect of digestion in the stability of active compounds. Yan et al. [87] evaluated the impact of simulated digestion and indicated significant reduction in the polyphenol content and antioxidant activity of blueberry pomace fermented by *Lactobacillus rhamnosus* GG and *Lactobacillus plantarum*-1 (1:1).

The fermentation of pomaces can be seen as a relevant strategy to produce functional supplements with interesting biological effects, especially from berries. However, additional advances, especially in the application of extraction technologies and the characterization of biological effects in vivo, are still necessary.
