Lentil and quinoa can be considered good providers of nutrients for microorganisms’ growth during fermentative processes. However, any modification (chemical or physical) of the starting substrate could impact the fermentative process even when the same microbial specie is used (Michael et al. 2011; Limón et al. 2015; Espinosa-Páez et al. 2017). Table 1 shows the nutritional composition (in dry basis) of lentil and quinoa, before (grain/seed) and after milling and sieving (flour). As it can be seen, quinoa is richer in lipids, minerals, phytates and phenols than lentils. Regarding protein content, all substrates presented higher than 30 g per 100 g of dry basis, excepting quinoa grain for which was lower. The remotion of some fibrous part of quinoa seeds during milling and after sieving could be responsible of the differences found between seed and flour in terms of protein, as well as in carbohydrates and lipids contents. The antioxidant activity values (mg trolox/ g dry basis) of the substrates based on radical-based scavenging assays 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and on non-radical redox potential-based one (FRAP) are also gathered in Table 1. According to the results, higher values were obtained in ABTS-assay than in DPPH and FRAP ones regardless the substrate. Moreover, lentils exhibited slightly higher capacity to quench the ABTS and DPPH radicals than quinoa, despite of the lower phenolic content of lentils. A positive relationship among total phenolic content and radical-based scavenging assays has been previously reported in vegetal foods (Marathe et al. 2011; Devi et al. 2019). In this study, it seems to be related to the phenolic profile rather than to the total content. Thus, phenolic compounds from lentils exhibited higher antioxidant activity than those from quinoa. Hence, chemical species with hydrogen atom or electron donating ability exert antioxidant properties. In the case of phenols, these capabilities seem to be related to the position and number of hydroxyl groups attached to the aromatic rings. Catechin and proanthocyanidin compounds represent 69% of the identified phenols in Pardina lentils (Aguilera et al. 2010); while phenolic acids together with flavanols comprised 60% of total compounds in white quinoa (Rocchetti et al. 2019).
Table 1
Proximate composition (g/ 100 g dry basis), phytic acid content (mg phytic acid/ g dry basis), total phenolic content (mg gallic acid/ g dry basis), and antioxidant activity by ABTS, DPPH and FRAP assays (mg trolox/ g dry basis) in lentil grain (LG) and flour (LF), and quinoa seed (QS) and flour (QF) before SSF.
| Lentil grain (LG) | Lentil flour (LF) | Quinoa seed (QS) | Quinoa flour (QF) |
Protein | 31.9 ± 0.5A | 32.3 ± 0.3A | 25.2 ± 0.2A | 31.1 ± 0.3B |
Lipids | 0.86 ± 0.08A | 1.19 ± 0.10B | 3.4 ± 0.3A | 8.6 ± 0.3B |
Ash | 2.76 ± 0.09B | 2.58 ± 0.05A | 3.60 ± 0.01A | 4.09 ± 0.02B |
Carbohydrates | 64.5 ± 0.7B | 63.9 ± 0.4A | 67.7 ± 0.6B | 56.2 ± 0.7A |
Moisture | 10.15 ± 0.05B | 9.35 ± 0.02A | 10.34 ± 0.09B | 8.97 ± 0.05A |
Phytic acid content | 4.5 ± 0.4A | 4.8 ± 0.4A | 15.2 ± 0.9A | 19.9 ± 0.2B |
Total phenolic content (TPC) | 1.28 ± 0.05A | 1.19 ± 0.07A | 1.57 ± 0.06A | 2.00 ± 0.08B |
Antioxidant activity (ABTS) | 3.8 ± 0.2A | 3.5 ± 0.2A | 2.4 ± 0.2A | 3.1 ± 0.2B |
Antioxidant activity (DPPH) | 1.26 ± 0.09A | 1.10 ± 0.06A | 0.82 ± 0.05A | 0.94 ± 0.04B |
Antioxidant activity (FRAP) | 2.09 ± 0.10A | 1.8 ± 0.2A | 1.8 ± 0.2A | 2.3 ± 0.2B |
The results represent the mean of three repetitions with their standard deviation. Different capital letters indicate significant differences between grain/seed and flour with a 95% (p < 0.05) significance level. |
On the other hand, no statistical significative differences were found among samples on their capacity to reduce the ferric 2,4,6-tripyridyl-s-triazine complex [Fe3+−(TPTZ)2]3+ from ferric iron (Fe3+) to ferrous iron (Fe2+) in acidic medium.
The evolution of the fermentation process was followed by estimating the unicellular biomass generation in the medium. Fungal biomass is difficult to assess, since fungal cells do not easily separate from the solid substrate. The measurement of glucosamine (chitin monomer and major constituent of the cell wall in fungi) is an acceptable indicator to the estimation of fungal mycelium development (Scotti et al. 2001). The biotransformation of the substrate by P. ostreatus will depend on its ability to grow and secrete certain enzymes (mainly oxidative and hydrolytic) able to metabolize substrates rich in lignocelluloses (Rodrigues Da Luz et al. 2012), which are not directly fermentable. White rot fungi such as P. ostreatus, present 2 types of extracellular enzyme systems: a hydrolytic system that produces hydrolases responsible for the degradation of polysaccharides and an extracellular and oxidative lignolytic system that degrades lignin (Ergun and Urek 2017). The growth of P. ostreatus (CECT 20311) observed by following the evolution of glucosamine content is not only affected by the amount of nutrients but also by the morphological characteristics, grain or flour, of the substrate (Fig. 1). The initial section of the curve, between day 0 and 4, corresponds to the latency phase of the fungus. On the fourth day of fermentation an exponential increase in the growth of the mycelium begins to occur without reaching a stationary phase during the observed period. Despite some differences observed in the growing rate between grains and flours, similar values were found after 14 days of incubation although higher biomass production was obtained over quinoa substrates as compared to lentils.
In order to evaluate the impact of biomass growing on the protein of the fermented samples, total protein content as well as the protein fraction soluble in TCA were evaluated at different times of the bioprocess (Table 2). Consistently with the biomass growth over the different substrates, a positive correlation between biomass and protein content was observed; the increase of total protein content was significantly higher in quinoa substrates (25.2% in seed after 14 days and 30.8% in flour after 6 days) than in lentils (25.5% in grain after 4 days and in 33.3% in flour after 14 days). However, no significant changes were observed in terms of soluble protein in TCA remaining constant along the process. The impact of the particle size of the substrate on the protein content is unclear and dependent on of the type of the substrate since the increase of protein was higher in lentil flour than in grain, in quinoa being the opposite. An increase of total protein content has been found in SSF with P. ostreatus in other pulses such as kidney beans (13%) and black beans (6%) (Phaselous vulgaris) (Espinosa-Páez et al. 2017) or in castellana lentils 18.5%), a different variety to the one used in this study. This protein increment could be explained as during fermentation, carbohydrates serve as an energy source for fungus growth and some of them may have been bioconverted into complex proteins, peptides or even free amino acids (Asensio-Grau et al. 2020). Furthermore, after fermentation with P. ostreatus of castellana variety lentils, an increase of soluble protein fraction was observed contributing to a higher digestibility in the resulting flour. Similar results were found by (Mora-Uzeta et al. 2019), in which protein content increased in tepary beans (Phaselous acutifolius) (+ 35%) fermented by Rhizopus oligosporus.
Table 2
Evolution of total protein (g protein/ 100 g dry basis) and soluble protein in TCA fraction* (g soluble protein/ 100 g protein), with respect to non-inoculated substrate (time 0), in lentil grain (LG) and flour (LF), and quinoa seed (QS) and flour (QF) at different SSF times.
Fermentation time (days) | Total protein content and soluble fraction in TCA |
Lentil grain (LG) | Lentil flour (LF) | | Quinoa seed (QS) | Quinoa flour (QF) |
0 | 24.4 ± 1.5abc (12.7 ± 0.7d) | 27.5 ± 0.9a (12.75 ± 0.15g) | | 20 ± 2a (14.30 ± 1.15c) | 25.5 ± 0.7a (18.5 ± 0.5c) |
2 | 24.5 ± 0.7abc (12.1 ± 0.7cd) | 29.0 ± 0.3b (11.0 ± 0.2f) | | 20.4 ± 1.0ab (13.1 ± 0.4b) | 27.7 ± 0.9b (20.0 ± 1.3d) |
4 | 25.5 ± 1.3c (11.3 ± 0.3abc) | 30.8 ± 0.4c (10.50 ± 0.04e) | | 21.7 ± 0.4bc (11.1 ± 0.4a) | 28.2 ± 0.6b (16.67 ± 0.15b) |
6 | 25.1 ± 0.6bc (11.3 ± 0.5abc) | 30.3 ± 0.3c (10.34 ± 0.12e) | | 22.5 ± 0.2cd (11.5 ± 0.2a) | 30.8 ± 0.3d (16.1 ± 0.3b) |
8 | 24.7 ± 0.4bc (10.6 ± 0.2a) | 30.48 ± 0.15c (10.04 ± 0.05d) | | 22.9 ± 0.3cd (12.6 ± 0.4b) | 29.36 ± 0.10c (14.9 ± 0.2a) |
10 | 24.5 ± 0.6abc (11.8 ± 0.4bc) | 30.4 ± 0.7c (8.86 ± 0.02b) | | 23.8 ± 0.8de (11.1 ± 0.2a) | 29.3 ± 0.7c (14.6 ± 0.4a) |
12 | 23.1 ± 1.0a (11.1 ± 0.6ab) | 31.9 ± 0.5d (8.26 ± 0.14a) | | 24.4 ± 0.4ef (11.5 ± 0.5a) | 29.36 ± 0.09c (14.8 ± 0.3a) |
14 | 23.8 ± 0.5ab (11.5 ± 0.5bc) | 33.3 ± 0.2e (9.37 ± 0.09c) | | 25.2 ± 0.5f (11.0 ± 0.3a) | 27.4 ± 0.7b (14.7 ± 0.3a) |
* Values in parenthesis corresponds to protein fraction soluble in TCA. The results represent the mean of three repetitions with their standard deviation. Different lowercase letters indicate significant differences between the different fermentation times with a 95% (p < 0.05) significance level. |
An important aspect when it comes the nutritional evaluation of a food or ingredient is the content of some antinutrient compounds. It is known the contribution of phytates to decrease the absorption of essential micronutrients such as calcium, iron (Hurrell et al. 2003), zinc (Guttieri et al. 2006) or magnesium (Bohn et al. 2004; Peng et al. 2010) and have a negative impact on protein digestibility since they can bond to dietary protein or digestive enzymes (proteases and amylases) inhibiting their hydrolytic activity (Espinosa-Páez et al. 2017; Muñoz-Llandes et al. 2019). Since solid-state fungal fermentation is presented as a strategy to reduce the antinutrient content of certain substrates (Garrido-Galand et al. 2021), the evolution of phytic acid content was followed along the fermentation process and results are shown in Fig. 2. According to literature quinoa seeds contain approximately between 1–2% of phytic acid (Hídvégi and Lásztity 2002; Febles et al. 2002) while lentils contain between 0.3–1.5% consistently with the initial values obtained in the raw materials used in this study (Table 1).
The degradation of phytate as consequence of fungal fermentation was observed, this being more pronounced in flours than in grains. A percentage of reduction of 27% and 89% was found in lentil grain and flour, respectively; while in quinoa, 45% in seed and 90% in flour was achieved. These changes begin to be significant (p < 0.05) from the tenth day of fermentation and depend on the substrate characteristics since the degradation of phytate after fermentation was greater in flours than in grains. These results are in accordance withCastro-Alba et al. (2019) who reported different level of phytate degradation in quinoa, canihua and amaranth according to their granulometry (seed or flour). The degradation rate of phytates seems to be also modulated by the pH reduction along fermentation because of organic acids production which depends on the inoculum employed at the same time. In this sense,Castro-Alba et al. (2019) reported differences between spontaneous fermentation and fermentation with a characterized specie such as L. plantarum. Furthermore, they suggest that the greater degradation of phytate in flour depends on both the exogenous phytase production of the microorganism and the activation of endogenous phytase. However, it must be considered that autoclaving prior to inoculation causes inactivation of endogenous phytase (Brejnholt et al. 2011). Therefore, the notable decrease in phytate in the last days of fermentation could be attributed mainly to the activity of the exogenous phytase from P. ostreatus, instead of the possible action of the endogenous phytase of the substrate.
On the other hand, P. ostreatus is known to be an excellent producer of hydrolytic enzymes, which contribute to the release of conjugated phenolic compounds chelated into the cell walls by hydrolysis during the fermentation. Phenolic compounds are the major contributors to antioxidant activity in fruit, vegetable, grain, or plant tissues. Thus, changes experimented by TPC along SSF time are shown in Fig. 3. Firstly, results evidenced a positive impact of thermal treatment on bound phenolic compounds release as TPC was much higher after autoclaving (time 0) than in the raw material (Table 1). Data are in agreement with those found by other authors (Bryngelsson et al. 2002; Madapathage Dona 2011). Hence, thermal treatment could promote cell walls disruption with the release of structural phenols and/ or the breakdown of insoluble polymeric phenols into smaller molecular weight compounds with enhanced extractability.
In contrast, a decreasing profile of the TPC was observed as long as fermentation progresses in lentil substrates, being higher in flour than in grain.Gebru & Sbhatu (2020) reported similar findings in white and brown teff solid-state fermented by P.ostreatus with a negligible, and even slightly decrease of TPC, after 6 days. Some authors, however, reported a significant increase of teff phenols when G. lucidum was used a starter and keeping the same condition of SSF. This fact evidences the relevance of each fungal mycelium metabolism, and enzymes synthesis, to conduct changes in the bioactive compounds.
In line, a negative correlation between TPC and fermentation time was also observed byXu et al. (2018) in 8 cereals and pseudocereals (wheat, corn, rice, millet, quinoa, oat, sorghum and buckwheat) and 2 legumes (soybean and pea) fermented with three different fungus and for 35 days. According to their results, only an increase of TPC were found at 14 days in oat. For the rest of the substrates, longer fermentation times were required to promote a significant increase of TPC content. In fact,Xu et al. (2018) reported a significant increase of TPC in fermented quinoa from 21 days of fermentation. This could be an explanation of the low TPC increase observed in the present study. It is well known that fermentation time is a kinetic parameter with importance for optimal enzyme production, thereby affecting transformation and production of particular compounds. The highest TPC value in quinoa seeds was detected after 8 days of fermentation (2.5 ± 0.3 mg GA/ g dry basis); while in flour, the highest value was reached after 10 days (3.36 ± 0.08 mg GA/ g dry basis).
The antioxidant activities of fermented lentil and quinoa are gathered in Table 3. Although SSF has been indicated with the aim of obtaining new ingredients with enhanced antioxidant properties, the capacity of the fermented substrates to scavenge free radicals such ABTS or DPPH, and to reduce ferric ions in FRAP assay decreased with fermentation time in this study. Besides, greater losses of antioxidant activities were achieved in lentils than quinoa. In addition to of total phenols, other metabolites formed, such as ergothionine, during the fermentation process may affect the antioxidant properties of the fermented products (Cai et al. 2011; Zhai et al. 2015; Bei et al. 2017). This fact could be responsible of the lack of correlation between TPC and extent of antioxidant activity (Torino et al. 2013; Alves Magro et al. 2019). Besides, competitive reactions between pro and antioxidant compounds can be done resulting in an increase or reduction of food’s antioxidant capacity. The ability of phenolic compounds to promote or inhibit oxidative damage depends on the different phenol concentration and pH, among other factors, Monohydroxylated phenols have been reported to exhibit a low radical scavenging activity (Briante et al. 2003; Villaño et al. 2005). Thus, (Fukumoto & Mazza (2000) found that benzoic and cinnamic acid derivatives, behave like prooxidants. According to this fact, a higher release of prooxidant phenols occurred in lentils than quinoa along fermentation time.
Table 3
Antioxidant activity (mg trolox/ g dry basis) and percentage of variation*, with respect to non-inoculated substrate (time 0), in lentil grain (LG) and flour (LF), and quinoa seed (QS) and flour (QF) at different SSF times.
Substrate | Fermentation time (days) | Antioxidant activity (mg trolox/ g dry basis) |
ABTS | DPPH | FRAP |
Lentil grain (LG) | 0 | 10.7 ± 0.5dA (0 ± 5) | 5.6 ± 0.5fB (0 ± 9) | 7.3 ± 0.3fA (0 ± 5) |
2 | 9.7 ± 0.4bcA (-9 ± 4) | 4.4 ± 0.2eB (-21 ± 3) | 4.8648 ± 0.0012eA (-33.30 ± 0.02) |
4 | 8.7 ± 0.2aA (-19 ± 2) | 3.92 ± 0.08dB (-30.1 ± 1.5) | 4.17 ± 0.06dB (-42.9 ± 0.9) |
6 | 9.3 ± 0.2abA (-13 ± 2) | 3.1 ± 0.2cB (-44 ± 3) | 3.03 ± 0.11cB (-58.5 ± 1.6) |
8 | 8.99 ± 0.12aA (-16.3 ± 1.2) | 2.5 ± 0.2abB (-55 ± 3) | 2.31 ± 0.08bB (-68.3 ± 1.1) |
10 | 10.2 ± 0.2cdB (-5 ± 2) | 2.79 ± 0.12bcB (-50 ± 2) | 2.54 ± 0.03bB (-65.2 ± 0.5) |
12 | 9.9 ± 0.9bcA (-7 ± 8) | 2.21 ± 0.11aB (-61 ± 2) | 1.8 ± 0.2aB (-75 ± 3) |
14 | 10.0 ± 0.3bcA (-7 ± 3) | 2.15 ± 0.03aB (-61.7 ± 0.6) | 1.72 ± 0.14aB (-76 ± 2) |
Lentil flour (LF) | 0 | 12.90 ± 0.02dB (0.00 ± 0.13) | 4.307 ± 0.006fA (0.00 ± 0.14) | 7.53 ± 0.06fA (0.0 ± 0.8) |
2 | 11.1 ± 0.8cA (-14 ± 6) | 3.1 ± 0.3eA (-29 ± 7) | 4.5 ± 0.6eA (-41 ± 8) |
4 | 10.90 ± 0.12cB (-15.5 ± 0.9) | 2.65 ± 0.08dA (-39 ± 2) | 3.68 ± 0.11dA (-51 ± 2) |
6 | 10.3 ± 0.2abB (-20.1 ± 1.5) | 2.06 ± 0.02cA (-52.2 ± 0.5) | 2.52 ± 0.15cA (-67 ± 2) |
8 | 10.06 ± 0.02aB (-22.1 ± 0.2) | 1.872 ± 0.014bA (-56.5 ± 0.3) | 1.92 ± 0.06bA (-74.5 ± 0.7) |
10 | 9.81 ± 0.08aA (-24.0 ± 0.6) | 1.54 ± 0.02aA (-64.3 ± 0.5) | 1.19 ± 0.04aA (-84.2 ± 0.6) |
12 | 9.99 ± 0.10aA (-22.6 ± 0.8) | 1.6087 ± 0.0008aA (-62.65 ± 0.02) | 1.43 ± 0.03aA (-81.0 ± 0.5) |
14 | 10.67 ± 0.13bcB (-17.3 ± 1.0) | 1.66 ± 0.05aA (-61.5 ± 1.1) | 1.36 ± 0.12aA (-82 ± 2) |
Quinoa seed (QS) | 0 | 11.54 ± 0.04bB (0.0 ± 0.3) | 3.08 ± 0.02eB (0.0 ± 0.7) | 2.74 ± 0.15dA (0 ± 5) |
2 | 10.84 ± 0.04aB (-6.1 ± 0.3) | 2.76 ± 0.09dB (-10 ± 3) | 1.75 ± 0.03bA (-36.2 ± 1.2) |
4 | 10.84 ± 0.04aB (-6.1 ± 0.3) | 2.576 ± 0.015cB (-16.3 ± 0.5) | 1.98 ± 0.05cA (-28 ± 2) |
6 | 10.84 ± 0.03aB (-6.1 ± 0.3) | 2.48 ± 0.04bcB (-19.3 ± 1.4) | 1.55 ± 0.03aA (-43.6 ± 1.0) |
8 | 12.83 ± 0.07cB (11.1 ± 0.6) | 2.30 ± 0.11aA (-25 ± 4) | 1.44 ± 0.07aA (-47 ± 3) |
10 | 11.40 ± 0.08bB (-1.3 ± 0.7) | 2.28 ± 0.10aA (-26 ± 3) | 1.5 ± 0.2aA (-45 ± 8) |
12 | 11.7 ± 0.4bB (2 ± 3) | 2.45 ± 0.02bB (-20.3 ± 0.7) | 1.42 ± 0.08aA (-48 ± 3) |
14 | 10.7 ± 0.4aB (-7 ± 4) | 2.555 ± 0.002bcB (-17.00 ± 0.06) | 1.458 ± 0.004aA (-46.7 ± 0.2) |
Quinoa flour (QF) | 0 | 9.37 ± 0.03cA (-0.0 ± 0.4) | 2.81 ± 0.04eA (0.0 ± 1.4) | 4.87 ± 0.06fB (0.0 ± 1.3) |
2 | 8.908 ± 0.007bA (-4.94 ± 0.07) | 2.55 ± 0.03dA (-9.2 ± 1.2) | 3.75 ± 0.08eB (-23 ± 2) |
4 | 8.5 ± 0.2aA (-10 ± 2) | 1.98 ± 0.06aA (-30 ± 2) | 2.4 ± 0.3cB (-50 ± 5) |
6 | 9.36 ± 0.04cA (-0.2 ± 0.5) | 2.291 ± 0.008cA (-18.4 ± 0.3) | 2.77 ± 0.02dB (-43.0 ± 0.3) |
8 | 9.60 ± 0.13deA (2.4 ± 1.4) | 2.39 ± 0.02cA (-15.0 ± 0.8) | 2.31 ± 0.08bcB (-53 ± 2) |
10 | 9.44 ± 0.09cdA (0.7 ± 1.0) | 2.12 ± 0.05bA (-24 ± 2) | 1.97 ± 0.11aB (-60 ± 2) |
12 | 9.516 ± 0.002cdeA (1.55 ± 0.02) | 2.09 ± 0.14bA (-26 ± 5) | 2.34 ± 0.02bcB (-51.8 ± 0.4) |
14 | 9.7 ± 0.2eA (3 ± 3) | 2.16 ± 0.06bA (-23 ± 2) | 2.21 ± 0.05bB (-54.5 ± 1.0) |
The results represent the mean of three repetitions with their standard deviation. Different lowercase letters indicate significant differences between the different fermentation times with a 95% (p < 0.05) significance level. Different capital letters indicate significant differences between grain and flour with a 95% (p < 0.05) significance level. * Values in parenthesis corresponds to percentage of variation of antioxidant activity. |
Nevertheless, and once more, autoclaving had a positive effect in terms of radical scavenging and reducing power activity of both substrates as a notable increase of mg trolox/ g dry basis at time 0 was found compared to the values found in raw materials (Table 1). These findings are in coherence with those of Rocchetti et al., (2019).