Impact of Fermentation on Phenolic Compounds and Antioxidant Capacity of Quinoa

: Quinoa ( Chenopodium quinoa Willd.) is increasingly singled out as a healthy food with an excellent nutritional profile. Besides being suitable for gluten ‐ free diets, it is rich in proteins of excellent quality and is a good source of minerals and vitamins, as well as of natural antioxidants, such as phenolic compounds. The aim of this work is to present how fermentation can affect phenolic compound content and antioxidant capacity of quinoa. It emerged that fermentation can be used to increase phenolic compound content and antioxidant capacity in both quinoa seeds and flours. The use of fermented quinoa flours allowed obtaining bread and pasta richer in phenolic compounds and with a greater antioxidant capacity. Fungi are the main starters used in quinoa seed fermentation, while Lactobacillus strains have been applied to produce sourdoughs. Quinoa has been also fermented to obtain yogurt ‐ like beverages with a higher content in phenolic compounds and a greater antioxidant activity. Strains of Lactobacillus sp. and Bifidobacterium sp. have been used as starters.


Introduction
Quinoa (Chenopodium quinoa Willd.) is a pseudocereal crop, indigenous to the Andean region, increasingly singled out as a healthy food with an excellent nutritional profile. It has recently attracted a lot of attention, as it is rich in proteins of excellent quality, with a balanced essential amino acid profile similar to milk and close to the ideal balance recommended by the Food and Agriculture Organization of the United Nations (FAO); it is a good source of minerals (e.g., calcium, iron, and zinc), vitamins, and also natural antioxidants. In addition, it is a gluten-free (GF) crop, and its seeds have been milled and widely used to overcome the technological challenges of GF breadmaking [1].
Quinoa is also a crop with resilience traits. It can withstand extreme environmental conditions, in terms of soils, rainfall, temperature, and altitude. It can thus grow from sea level up to 4500 m above sea level and can adapt to frost, drought and salinity [2].
The inclusion of pseudocereals in the diet of populations other than those from Southern America has been long limited because of traditional dietary habits, disadvantages of longer cooking times, a limited variety of products made from them, and the presence of anti-nutritional factors, such as phytates [3]. However, in recent years, the global demand for quinoa has exploded, because of its claimed "superfood" quality.
A great deal of evidence has shown that consumption of fermented foods can have a preventive effect towards non-communicable diseases, due to the presence of bioactive molecules, which can have antioxidant capacity, cholesterol-lowering effect, or antidiabetic activity [4]. Fermentation of grains and derivatives thereof specifically determines the production of health-promoting components, such as γ-aminobutyric acid,

Phenolic Compounds in Quinoa
Phenolic compounds (PCs) are a diverse group of phytochemicals ranging from simple phenols to complex polyphenols. These chemical structures share the presence of one or more hydroxyl groups on aromatic ring(s).
PCs are produced by plants in normal and stress conditions; hence, they are ubiquitous in plant-derived foods. In cereals and pseudocereals, they are commonly found in the outer layers of the grains. An appropriate dietary intake of PCs contributes to maintaining health and well-being while lowering the risk for the onset of noncommunicable diseases thanks to their antioxidant, anti-inflammatory, and antiproliferative activity [12].
Phenolic acids and flavonoids are the most abundant dietary PCs [12]. The former include hydroxybenzoic and hydroxycinnamic acids derivatives (Figure 1), which occur, respectively, as components of hydrolysable tannins and lignin, and hydroxyacid esters covalently conjugated to the plant cell wall components [13]. Flavonoids show a threering structure in the C6-C3-C6 form. They are further classified into flavonols, flavan-3ols, flavones, isoflavones, flavanones, anthocyanidins, and chalcones, based on the connection of an aromatic ring to the heterocyclic ring ( Figure 1). So far, phenolic compounds have been the most studied non-nutrient phytochemicals in quinoa [14]. They have been mainly quantified by spectrophotometric methods as Total Phenolic Content (TPC). A great variability in TPC has been observed among studies investigating quinoa samples originating from different regions worldwide.
In raw seeds of Titicaca and Puno quinoa, grown in Morocco, TPC was 31.67 and 105.85 mg Gallic Acid Equivalents (GAE) 100 g −1 dry matter (dm), respectively [15]. TPC values ranging from 66 to 202 mg GAE 100 g −1 dm were observed in five quinoa genotypes grown in South Sinai (Egypt) [16]. In the raw seeds of the Chinese quinoa cultivar Jinli-1, TPC was 200.40 mg GAE 100 g −1 dm [17], while in quinoa seeds grown in Korea it was 14.37 mg GAE 100 g −1 dm [18]. Recently, quinoa cultivation has spread also in Europe, and phenolic compounds were determined also in European samples. In raw quinoa seeds grown in Finland, Mattila et al. found that TPC was 181 mg GAE 100 g −1 dm [19], while lower values were reported by Multari et al. [20]. As regards quinoa from Southern America, total free and bound phenolic compounds ranged from 123 to 341 mg GAE 100 g −1 fw (fresh weight) and between 128 and 452 mg GAE 100 g −1 fw, respectively [21]. A lower content of PCs in free form was observed by Vega-Gálvez et al. in samples from Chile [22].
Variability in TPC is due to several factors, such as genetic traits, growing conditions, and post-harvest processing of quinoa seeds. However, differences in analytical procedures for phenolic extraction also contribute to data variability.
Quinoa processing, such as milling and cooking, can modulate the dietary intake of PCs. For instance, upon milling (degree of milling of 27.23%), TPC of the Chinese quinoa cultivar Jinli-1 decreased to 137.36 mg GAE 100 g −1 dm, compared to raw seeds (200.40 mg GAE 100 g −1 dm) [17]. The effect of different types of processing on TPC in quinoa from Brazil has been also recently reported [23]. Compared to the control, toasting significantly decreased TPC, while cooking increased TPC up to 110.65 and 127.54 mg GAE 100 g −1 dm, when it was performed at atmospheric pressure and under pressure, respectively. A higher TPC upon cooking was also observed in cereals [24] and was possibly due to a softening or disintegrating effect on grain tissues that promotes the release of phenolics from the food matrix.

Impact of Fermentation on Phenolic Compounds in Quinoa
Fermentation can have multiple effects on phenolic compounds, such as modifications of their content and/or profile, as well as formation of metabolites.

Fermentation and Phenolic Compound Content
The relationship between fermentation and phenolic compound content has been studied in quinoa seeds and flours.
Microorganisms naturally present in quinoa seeds or previously isolated from them, as well as fungi and Saccharomyces cerevisiae strains (generally applied to the baking and brewing process), were used as fermentation starters.
Strains of Lactobacillus were used to ferment quinoa seeds by Li et al. [25] and Rocchetti et al. [26]. Lactobacillus casei CICC 20995 was used to ferment quinoa seeds from Jiaqi-1, which is an improved variety from the Peruvian altiplano and cultivated in the Shanxi region (China) [25]. In unfermented quinoa seeds, phenolic compounds were found mainly in bound form, which accounted for 85.20%. The level of bound phenolics was 1180 mg GAE 100 g −1 dm, while free phenolic content was 205 mg GAE 100 g −1 dm. In fermented seeds, a 4-fold increase in free phenolics was observed, while the bound phenolic content decreased by approximately 1.5 folds (Table 1). It was supposed that the ability of the enzymatic system of L. casei to catalyze combined polyphenol-protein compounds into a free state was responsible for the decrease in bound phenolic content. Autochthonous lactic acid bacteria (LAB)-Lactobacillus paracasei A1 2.6 and Pediococcus pentosaceus GS Bwere used as starters to ferment commercial quinoa seeds native to South America [26]. Quinoa seeds were cooked and acidified before fermentation according to common industrial practices. It was found that fermentation with P. pentosaceus GS B strain was more efficient than with L. paracasei A1 2.6 strain in increasing TPC of quinoa seeds (70.9 vs. 59.6 mg GAE 100 g −1 , respectively). However, the most effective process was fermentation of cooked and acidified quinoa seeds by both P. pentosaceus GS B strain and L. paracasei A1 2.6 strain (Table 1). Compared to raw quinoa, TPC increased by 13.8%. The chromatographic analysis of phenolic extracts showed that quinoa was abundant in flavonoids and phenolic acids; however, alkylphenols and lignans were also identified.
Fungi were successfully applied to quinoa seed fermentation, and the effect on PCs was studied. Xu et al. investigated the effect of solid-state fermentation (SSF) by three filamentous fungi (Agaricus bisporus AS2796, Fomitiporia yanbeiensis G1 and Helvella lacunosa X1) on phenolic compound content and antioxidant capacity of quinoa seeds [27]. A. bisporus sp. is also known as the white button mushroom and has anticancer activity, and Fomitiporia (Hymenochaetales, Basidiomycota) has been greatly applied for preventing chronic diseases, while H. lacunosa sp. has been used for enhancing immunity [27]. The application of the above-mentioned strains showed that TPC after SSF was influenced by the starter strains applied and mainly by the fermentation time. When H. lacunosa X1 and F. yanbeiensis G1 were used, after a fermentation time of 14 days, the longer the fermentation time was, the higher TPC was (Table 1). In contrast, the highest TPC value in quinoa fermented by A. bisporus AS2796 was obtained at 35-day fermentation. Rhizopus microspores var. oligosporus was also used to ferment white quinoa seeds [28]. An increase in TPC from 4.1 to 7.4 mg GAE 100 g −1 dm was observed after a 3-day fermentation. A higher TPC was found after a 5-day fermentation (Table 1). Total Flavonoid Content (TFC) was also determined and a significant increase upon fermentation was observed (Table 1) [28].
The effect of fermentation with three strains of fungi-Rhizopus oligosporus, Aspergillus oryzae and Neurospora intermedia-which are commonly used for the preparation of tempe, koji, and oncom molds, respectively, was explored [29]. The highest value of total phenolics was found when a 6-day fermentation with R. oligosporus was applied. It increased from 0.47 (raw) to 1.96 μg 100 g −1 dm in black quinoa, and up to 0.89 μg 100 g −1 dm in red quinoa ( Table 1).
The effect of standard (30 h) and prolonged (40 h) tempe-type fermentation with R. oligosporus ATCC 64063 strain was studied in white, red, and black quinoa [30]. It emerged that both fermentative processes increased TPC compared to raw seeds (Table 1). Moreover, in white and black quinoa, a 40 h fermentation determined a greater increase in TPC than the 30 h treatment.
Baker's yeast (Saccharomyces cerevisiae NBRC 2375) or brewer's yeast (S. cerevisiae NBRC 1951) were used to ferment quinoa seeds from Argentina [31]. It was found that TPC in raw quinoa seeds was 39.3 mg GAE 100 g −1 dm and increased by approximately 46% after fermentation with S. cerevisiae NBRC 2375 and NBRC 1951. No statistical differences were found between the two microorganisms (Table 1).
Several Lactobacillus strains were applied also to quinoa flour fermentation. Ayyash et al. studied the effect of solid-state fermentation with two strains of Lactobacillus plantarum-L. plantarum DSM2648 and L. plantarum KX881779-and one strain of Lactobacillus reuteri, KX88177 [32]. TPC in quinoa flour was monitored over 72 h (with 24 h intervals). In non-inoculated quinoa, TPC ranged from approximately 0.11 to 0.18 mg GAE 100 g −1 dm. During 72 h of fermentation, TPC increased significantly (p < 0.05). The highest TPC was found after 72 h fermentation with L. reuteri KX88177 (Table 1).
Quinoa-based ingredients to be used in pasta-making and formulation of bakery products have been obtained from fermentation of quinoa seeds and flours. Lorusso et al. used fermented quinoa flour in pasta-making and investigated the effect on phenolic compound content [33]. Fermentation of raw materials is, in fact, among the possible approaches to obtain functional ingredients for pasta fortification [5]. The autochthonous L. plantarum T6B10 and Lactobacillus rossiae T0A16, isolated from quinoa flour, were used as starters. It was found that the replacement of semolina with raw quinoa flour increased TPC by approximately 37%, and a greater increase (approx. 84%) was observed when fermented quinoa flour was used (Table 1). It was supposed that acidification during fermentation promotes the hydrolysis of complex phenolic compounds and their glycosylated forms into the corresponding phenolic acids which results in a higher content of free total phenolics.
The two LAB strains were also used by Rizzello et al. to obtain a quinoa sourdough for bread-making [34]. TPC in quinoa sourdough was 16.19 mmol kg −1 . In the dough obtained by using quinoa sourdough, TPC was 8.78 mmol kg −1 , while in the quinoa-based control dough it was 3.65 mmol kg −1 (Table 1).
A quinoa sourdough, prepared by fermenting quinoa wholemeal flour with L. plantarum ATCC 8014, was used to make muffins [36]. TFC was 997 mg QE 100 g −1 fw in the wholemeal flour, it decreased in quinoa sourdough and increased up to 1317 mg QE 100 g −1 fw in muffins (Table 1).
Quinoa flour has been fermented to obtain yogurt-like beverages, in order to meet the increasing interest in and demand for non-dairy beverages made with vegetables, fruits, and cereals. Pseudocereals, as well as minor cereals like oat or spelt, and legumes have been, in fact, increasingly investigated as raw ingredients for making functional beverages [39].
Lorusso et al. prepared three yogurt-like beverages by fermenting quinoa with L. rhamnosus SP1, L. plantarum T6B10, and Weissella confusa DSM 20194 [37]. The concentration of total phenols in the fermented beverages was significantly higher than in the non-fermented products. Fermentation with L. rhamnosus SP1 enabled the highest TPC value to be obtained. After 20-day storage, an additional TPC increase was observed (Table 1).
Karovičová et al. prepared fermented quinoa beverages by mixing quinoa flour with water and by fermenting the obtained solution for 6 h with a probiotic culture, including Bifidobacterium sp., Lactobacillus acidophilus, and Streptococcus thermophilus [38]. After fermentation, TPC increased by 27% (Table 1).

Fermentation and Phenolic Compound Profile
The effect of fermentation on the profile of phenolic compounds has been also investigated. Hydroxybenzoic and hydroxycinnamic acids derivatives, as well as flavonoids, were identified in fermented quinoa and quinoa-based products.
Among hydroxybenzoic acid derivatives, gallic, protocatechuic, and vanillic acids were mainly identified. Gallic acid was detected in quinoa seeds fermented with Rhizopus microspores var. oligosporus (Table 2). It was observed that after 3-day fermentation, gallic acid content increased from 0.01 to 2.37 mg kg −1 but decreased upon longer fermentation ( Table 2). Protocathecuic acid was identified in white, red, and black quinoa seeds, and its variation in content was studied upon fermentation with Rhizopus oligosporus ATCC 64063 strain [30]. Protocathecuic acid content was the highest in cooked black quinoa seeds (20.4 μg/g dm) and lowest in white seeds (0.60 μg/g dm) ( Table 2). Upon fermentation, protocatechuic acid content ranged from 28.40 to 34.45 μg/g dm in red and black quinoa seeds, while its content was the lowest in white quinoa (1.94-3.69 μg/g dm) ( Table 2). Vanillic acid has been determined upon fermentation of quinoa seeds with strains of the Rhizopus sp. and S.cerevisiae. Starzyńska-Janiszewska et al. investigated the content of vanillic acid in bound and free form, in both red and black varieties fermented with a strain of R. oligosporus [29]. As regards the free form, fermentation determined an increase in vanillic acid content in black seeds and a decrease in red seeds, while in the bound form, a 1.13-and 6.23-fold increase occurred upon fermentation of both black and red varieties, respectively ( Table 2). The R. oligosporus ATCC 64063 strain was also used to ferment cooked white, red, and black quinoa [30]. The highest increase in vanillic acid content occurred by fermenting for 40 h red quinoa, where it increased from 10.07 to 46.21 μg g −1 dm ( Table 2). Vanillic acid content was also monitored in quinoa seeds after a 3-and 5day fermentation with Rhizopus microspores var. oligosporus [28]. A 1.2-1.4 fold increase occurred ( Table 2). A comparable increase was observed by fermenting quinoa seeds with a strain of S. cerevisiae generally used in baking, while a 3.43 fold increase was observed upon fermentation with brewer's yeast (Table 2) [31]. Alongside vanillic acid, phydroxybenzoic acid content also increased when baker's and brewer's yeast were applied (Table 2) [31]. On the other hand, a decrease in hydroxybenzoic acid was observed when red and black quinoa seeds were fermented with a strain of R. oligosporus (Table 2) [29]. Regarding hydroxycinnamic acid derivatives, caffeic, chlorogenic, ferulic, and pcoumaric acids were found in quinoa samples. Caffeic acid was identified only among the bound phenolics of red and black quinoa seeds, pre-cooked and fermented with R. oligosporus for 4 days [29]. Its content was 0.034 mg 100 g −1 in fermented seeds of the red variety and 0.160 mg 100 g −1 in fermented black quinoa (Table 2). Chlorogenic acid content increased upon 3-day fermentation with Rhizopus microspores var. oligosporus, while its content did not vary by fermenting quinoa seeds for 5 days (Table 2) [28]. Ferulic acid content decreased upon fermentation with both a strain of R. oligosporus and a strain of S. cerevisiae generally used in brewing (Table 2) [29,31]. On the other hand, when quinoa was fermented with a strain of S. cerevisiae generally used in baking, a 2.35-fold increase in ferulic acid content was observed ( Table 2) [31]. Fermentation showed to impact greatly on the content of p-coumaric in quinoa, which increased by 10 and 8.1 fold when the strains of S. cerevisiae generally applied in baking and brewing were used, respectively ( Table 2) [31]. On the other hand, when red and black quinoa seeds were fermented with a strain of R. oligosporus sp. [29], p-coumaric decreased ( Table 2). In the same sample, a significant decrease in hydroxycinnamic acid was also observed upon fermentation (Table 2) [29].
Regarding flavonoids, catechin was found only in bound form in black and red varieties, and fermentation with R. oligosporus determined a decrease from 0.205 to 0.165 mg 100 g −1 in black seeds and an increase from 0.055 to 0.130 mg 100 g −1 in red seeds ( Table  2) [29]. The flavonol kaempferol was found only in free form in red and black quinoa seeds ( Table 2) [29]. Upon fermentation with a strain of R. oligosporus sp., its content increased from 0.047 to 0.102 mg 100 g −1 in red quinoa seeds, while in the fermented black seeds it was under the limit of quantitation. After fermentation with the two strains of S. cerevisiae, kaempferol was detected in neither red nor black varieties (Table 2) [31]. Quercetin was found in black and red quinoa in both free and bound form ( Table 2). Bound quercetin content increased after fermentation with a strain of R. oligosporus sp., while the free form decreased [29] (Table 2). After fermentation with S. cerevisiae strains, quercetin was also not detected [31]. Free rutin content decreased in red and black quinoa seeds fermented with a strain of R. oligosporus sp. (Table 2) [29], while it increased when it was fermented with the strain R. oligosporus ATCC 64063 for 30 h (Table 2) [30]. Longer fermentation (40 h) negatively affected rutin content ( Table 2).
Changes in the phenolic compound profile of quinoa upon fermentation can be due to the activity of microbial enzymes that degrade the grain cell wall structure and thus promote the release of bound phenolic compounds. In addition, biotransformation of phenolic acids by the metabolic pathways of the applied strain can occur. For instance, a 130-fold increase in vanillin content was observed in black quinoa seeds fermented with a strain of R. oligosporus sp. [29]. It is likely that the increase in vanillin was due to microbial biotransformation of ferulic acid to vanillin [40].
The relationship between fermentation and the bioconversion of phenolic compounds in quinoa has been, however, poorly explored and should be further investigated.

Impact of Fermentation on Antioxidant Capacity in Quinoa-Based Foods
The antioxidant capacity of a food of plant origin is determined by the concentration and chemical form of components acting as antioxidants. Hence, any processing able to modulate the content of antioxidants in foods impacts their antioxidant capacity.
The antioxidant capacity of quinoa seeds of Jiaqi-1 variety increased after fermentation with L. casei CICC 20995 [25]. It was observed that the half-inhibition concentration (IC50) values of quinoa seeds were 6.65 and 3.43 mg mL −1 before and after fermentation, respectively [25].
Antioxidant capacity of commercial quinoa seeds, native to South America, fermented by L. paracasei A1 2.6 and P. pentosaceus GS B was measured by FRAP and ORAC assays [26]. As far as ORAC assay is concerned, the radical scavenging of fermented quinoa seeds was greater than in raw seeds, and P. pentosaceus enabled the highest value to be obtained (Table 3). When the FRAP assay was applied, a not-detectable reducing power activity was found in all quinoa samples, except for raw quinoa (Table 3).
Antioxidant capacity of quinoa seeds fermented with different fungi was measured by different assays.
Xu et al. measured the antioxidant capacity of quinoa seeds fermented with the three filamentous fungi A. bisporus AS2796, F. yanbeiensis G1, and H. lacunosa X1 by DPPH assay [27]. They found that upon 21-and 35-day fermentation EC50 values were lower than the control, regardless of which fungus was used ( Table 3).
The antioxidant capacity of quinoa seeds fermented with R. microspores var. oligosporus was significantly different from the control (raw seeds) only after 5 days of fermentation [28] (Table 3). Antioxidant capacity of colored quinoa fermented with another strain of the Rhyzopus genus, that is, R. oligosporus, and strains of A. oryzae and N. intermedia was measured by ABTS [29]. It emerged that R. oligosporus was the most efficient of the studied strains in enhancing the antioxidant capacity of colored quinoa. In detail, upon a 6-day fermentation, ABTS increased by 276% in black quinoa and by 148% in red quinoa (Table 3).
When quinoa seeds from Argentina were fermented with baker's yeast (S. cerevisiae NBRC 2375) or brewer's yeast (S. cerevisiae NBRC 1951), an increase in the antioxidant capacity after fermentation with both strains was observed [31]. Compared to raw quinoa, DPPH values increased by 43 and 33% when baker's and brewer's yeast were used, respectively. ABTS values raised by 22 and 27%, and FRAP content increased by 51 and 50% (Table 3).
Antioxidant capacity of quinoa pasta and sourdough started with LAB was measured by DPPH. When two Lactobacillus strains, L. plantarum T6B10 and L. rossiae T0A16, previously isolated from quinoa, were used to produce fermented quinoa pasta [33] and quinoa sourdough bread [34], it was observed that the antioxidant capacity increased. In the study by Lorusso et al. [33], DPPH values increased from 26% in quinoa pasta to 35% in fermented quinoa pasta (Table 3). In quinoa sourdough obtained by fermenting quinoa flour with L. plantarum T6B10 and L. rossiae T0A16, the antioxidant capacity increased up to 71.8% (Table 3) [34].
Montemurro et al. determined the radical scavenging activity in sourdough obtained by raw and sprouted quinoa flour, started with three LAB strains: L. rossiae LB5, L. plantarum 1A7 and L. sanfranciscensis DE9 [35]. Fermentation of raw flour increased DPPH values from 81 to 90%, while a slighter increase was observed when sprouted flour was fermented (from 94 to 97%) ( Table 3).
Antioxidant capacity of yogurt-like beverages produced by fermenting quinoa with the Lactobacillus strains L. rhamnosus SP1, L. plantarum T6B10, and W. confusa DSM 20194 was significantly different depending on the strain used. The fermented beverage showing the highest antioxidant capacity after fermentation was the one started with L. plantarum T6B10 (Table 3) [37]. Before fermentation, all three beverages showed comparable DPPH values.
The antioxidant capacity of the quinoa-based beverage produced with quinoa flour and water, and fermented with a commercial probiotic culture including Bifidobacterium sp., Lactobacillus acidophilus, and Streptococcus thermophilus increased from 60.31 to 69.20% (Table 3) [38]. Hur et al. [28]

Conclusions
Fermentation can be used to increase phenolic compound content and antioxidant capacity in both quinoa seeds and flours. Fermented quinoa flours were applied to breadand pasta-making, and food products richer in phenolic compounds and with greater antioxidant activity were obtained. Fungi were mainly used as starters to ferment quinoa seeds, while Lactobacillus strains were applied to produce sourdoughs. Quinoa has been also fermented to obtain yogurt-like beverages with a higher content in phenolic compounds and a greater antioxidant activity.
In order to maximize the increase in phenolic content, attention should be paid not only to fermentation conditions, but also to quinoa variety used. Studies investigating the effect of fermentation on phenolic compound profile should be encouraged, as well as on the bio-conversion of phenolic compounds.
Author Contributions: Authors equally contributed to the conceptualization, investigation, writing and editing of the paper. All authors have read and agreed to the published version of the manuscript.