Solid‐state fermentation of lentil (Lens culinaris L.) with Aspergillus awamori: Effect on phenolic compounds, mineral content, and their bioavailability

Microorganisms have long been used in the production of a variety of foods, alcoholic beverages, additives, and supplements due to their cost effectiveness and environmental advantages. Solid‐state fermentation (SSF) reproduces the natural microbiological process that can be utilized in a controlled way to produce the desired product. In the present study, modulation of phenolic compounds, antioxidant potential, and mineral content during SSF of three lentil cultivars, namely, HM‐1, LL‐931, and Sapna, were explored. The total phenolic content (TPC) for 6th day Aspergillus‐fermented lentil (AFL) flour increased by 79.2% for cv. HM‐1, 78.8% for cv. LL‐931, and 122.8% for cv. Sapna. High‐performance liquid chromatography (HPLC) results also showed that SSF not only improved the phenolic content of lentil cultivars but also resulted in the formation of some new phenolic compounds (resorcinol and cinnamic acid). The condensed tannin content, DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) inhibition activity, hydroxyl free radical scavenging activity, reducing power activity, and total antioxidant capacity of aqueous ethanolic extracts from all AFLs also increased significantly (p ≤ 0.05) up to 6th day of fermentation. Mineral content differed significantly (p ≤ 0.05), with AFL extracts exhibiting higher mineral content than their unfermented counterparts. Among different minerals, Cu content of all AFL extracts was the highest with an increase of 46.4% to 60.0% upon fermentation. All minerals showed a significant (p ≤ 0.05) increase in their concentrations upon fermentation except for K in which the increase was less than 0.1%. However, in vitro bioavailability of iron and zinc was significantly (p ≤ 0.05) higher in AFL as compared with their unfermented counterparts, with the highest level being observed on the 6th day of fermentation. Thus, biotransformed lentils could be utilized in the preparation of functional foods and novel nutraceuticals for their health‐promoting properties.

Environmental factors and unhealthy human lifestyle generate free radicals including reactive oxygen species (ROS) and reactive nitrogen species (RNS) spontaneously through metabolism (Aseervatham, Sivasudha, Jeyadevi, & Arul, 2013). In a normal healthy body, these prooxidants are kept in check through various antioxidant defense systems, such as glutathione, catalase, and superoxide dismutase (Ighodaro & Akinloye, 2018). Exposure to unhealthy environmental conditions and habits result in an imbalance of antioxidants, which causes severe oxidative stress leading to reactive oxygen species-mediated tissue damage (Videla, 2009).
To deal with this excess stress, body requires additional strategies such as diet supplements loaded with exogenous antioxidants, which can help in scavenging free radicals (Aseervatham et al., 2013;Dhull & Sandhu, 2018). The use of synthetic antioxidants has been restricted because of their suspected carcinogenic effects (Valentão et al., 2002). Therefore, there is increasing interest in the intake of natural antioxidants such as vitamin C, polyphenols, and flavonoids from plant sources in place of synthetic antioxidants.
Phenolic compounds are primarily produced by plants, but some other sources can also produce these compounds as secondary metabolites such as green algae (Onofrejová et al., 2010), yeasts (Banach & Ooi, 2014), endophytes (de Carvalho, Silva, Chagas-Paula, Luiz, & Ikegaki, 2016, and mushroom basidiomycetes (Palacios et al., 2011;Reis, Martins, Barros, & Ferreira, 2012). Fermentation has been receiving considerable attention for the extraction and production of phenolic compounds due to the fast growth rate, cost effectiveness, easy cultivation, recovery, and eco-friendly nature of microbial generation of phenolic compounds (Fowler & Koffas, 2009). During solid-state fermentation (SSF), various enzymes such as amylases, proteases, and lipases are produced by fermenting microorganisms. These enzymes hydrolyze carbohydrates, protein, and lipids to more digestible compounds with a pleasant aroma, flavor, and texture. Moreover, many antinutritional compounds such as phytic acid, tannins, protease inhibitors, etc., are significantly reduced during fermentation (Soetan & Oyewole, 2009).
Though many studies on modulation of nutritional and functional attributes of lentils due to germination and fermentation have been carried out, to the best of our knowledge, changes in phenolic acids and mineral bioavailability of Indian lentil cultivars due to SSF have not been discussed so far. In this perspective, the present study was carried out to compare the phenolic profile of lentil cultivars and to analyze the changes in antioxidant potential through biotransformation of lentil cultivars with Aspergillus awamori. Moreover, the effect of fermentation on the mineral content and bioavailability of iron and zinc in fermented lentil flours were also evaluated.

| Substrate
Certified seeds of three lentils cultivars, namely, HM-1, LL-931, and Sapna, were procured from Chaudhary Charan Singh Haryana Agricultural University, Hisar, India. The seeds were thoroughly cleaned and stored in airtight containers until further use.

| Starter culture for SSF
Starter culture, that is, A. awamori (MTCC 548), purchased from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India, was maintained on czapekdox agar (CDA) and czapekdox broth (CDB) at 30 ± 2 C. Spore suspension was prepared by washing a 4-day-old mycelium with an aqueous solution of 0.1% (W/V) Tween 80, which was further used for inoculating the autoclaved lentil grains.

| Solid-state fermentation
The method described by Salar, Purewal, and Sandhu (2017) was adapted for SSF of lentil grains. Shade dried lentil grains (50 g) were taken in Erlenmeyer flasks (250 ml) as substrate for each of SSF and soaked in CDB at room temperature overnight. Next day excess of CDB was decanted; lentil grains in the Erlenmeyer flasks were autoclaved (121 C for 15 min) and then cooled. The spore suspension prepared with double distilled water was sprayed (5 ml) on to the surface of autoclaved grains, mixed thoroughly and incubated at 25 ± 2 C for a period of 7 days. The lentil grains were stirred and mixed after 24 and 36 h of inoculation to release the fermentation heat. The unfermented lentils were prepared without the addition of spore suspension.

| Extraction of bioactive compounds from unfermented and fermented lentils
Aspergillus-fermented lentil (AFL) samples were withdrawn out of the Erlenmeyer flasks at every 24-h intervals and dried in an oven at 40 C for 48 h. The dried AFL and unfermented control (UFL) were ground in an electric grinder (Sujata, India). All flour samples (AFL and UFL) were defatted with hexane (1:5 w/v, 5 min, thrice) at ambient temperature and air dried. The defatted samples (1 g) were extracted with aqueous ethanol (50%) in the ratio (1:3 w/v) at 60 C for 60 min in a water bath. After extraction, samples were filtered (Whatman No. 1) and then vacuum evaporated.

| Total phenolic and condensed tannin contents
For the determination of the total phenolic content (TPC) of AFL and UFL extracts, method as given by Gao, Wang, Oomah, and Mazza (2002) was adapted. Briefly, 100 μl of extracts and 0.5 ml of FC reagents were mixed in a 10-ml volumetric flask followed by mixing with 1.5-ml aqueous solution (20%, w/v) of anhydrous sodium carbonate, vortexing and incubation for 15 min at room temperature.
After incubation, the flask was filled with distilled water to volume.
The absorbance was read at 765 nm, and the results were calculated from the standard calibration curve and expressed as mg gallic acid equivalents per gram (mg GAE/g) of sample.
The condensed tannin content (CTC) of the extracts was estimated according to the method given by Julkunen-Titto (1985). The absorbance against blank was recorded at 500 nm. Catechin was used to make standard curve (0.05 to 1 mg/ml). The results were expressed as milligrams of catechin equivalent per g (mgCE/g) dry weight basis (dwb). All analyses were performed in triplicates.

| 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity of sample extracts was analyzed by the adapting the method described by Yen and Chen (1995). Briefly, 100 μl of extract was taken in spectrophotometric cell and then 3 ml of 100-μM DPPH was added. The changes in absorbance at 517 nm were recorded after 30 min. Percent (%) DPPH scavenging activity was calculated using the following formula: where A C and A E are the absorbance of control and extracts, respectively.

| Hydroxyl free radical scavenging activity
The hydroxyl free radical scavenging activity (HFRSA) of sample extracts against hydroxyl radicals was evaluated using the method of Smirnoff and Cumbes (1989). Briefly, 100 μl of extracts was mixed with 3 ml of Smirnoff reagent and incubated at 37 C for 30 min. Extraction phase was used as a negative control to check the antioxidant potential of different extracts. The percentage of HFRSA by extracts was calculated using the following formula: where A C and A E are the absorbance of control and extracts, respectively.

| Reducing power activity
The reducing power activity (RPA) of the extracts was estimated by adapting the method of Oyaizu (1986). Briefly, 100 μl of extract was mixed with 100 μl of potassium ferricyanide (1%) and incubated at 50 C for 30 min. After that, 100-μl trichloroacetic acid (1%) and 100-μl ferric chloride (0.1%) were added, and the mixture was further incubated for 20 min at ambient temperature. The mixture was diluted with double distilled water to prepare final volume of 10 ml, and the absorbance was recorded at 700 nm. Quercetin was used as standard to compare the reducing power potential of extracts, and the results were expressed as mg quercetin equivalents per gram (mg QE/g) of sample.

| Total antioxidant capacity
The method described by Prieto, Pineda, and Aguilar (1999) was adapted using ascorbic acid as standard to evaluate the total antioxidant capacity (TAC) of sample extracts. The reagent to assess antioxidant activity of extracts was prepared by mixing concentrated sulfuric acid (0.6 M), ammonium molybdate (4 mM), and sodium hydrogen orthophosphate (28 mM) in the ratio of 1:1:1. Further, 100 μl of sample extract was mixed with 3 ml of prepared reagent and incubated in water bath at 95 C for 90 min. Absorbance was recorded at 695 nm using ascorbic acid as standard to compare the antioxidant activity of extracts and the results were expressed as mg ascorbic acid equivalents per gram (mg AAE/g) of sample.

| High-performance liquid chromatography
High-performance liquid chromatography (HPLC) was carried out to detect the presence of specific bioactive compounds in AFL (6th day sample) and UFL by the method as described by Dhull, Kaur, and Purewal (2016

| Mineral estimation and in vitro bioavailability
The UFL and AFL flour samples were analyzed for their calcium, iron, zinc, and copper contents in atomic absorption mode and sodium and potassium contents in emission mode using atomic absorbance spec-

| Statistical analysis
The data were reported as the mean ± standard deviation of three replicates (n = 3) for all experiments except for HPLC analysis (n = 2).
Further, using the commercial statistical package (SPSS Inc, Chicago, IL), an analysis of variance with a significance level of 5% was done, and Duncan's test was applied to determine significant difference between mean values.

| Effect of SSF on total phenolics and condensed tannin content
The effect of fermentation time on TPC and CTC of UFL and AFL extracts is shown in  (Salar et al., 2017) and wheat with A. awamorinakazawa (Sandhu, Punia, & Kaur, 2016) also resulted in an increase in their phenolic contents. This increase has been reported to the production of some active hydrolases such as β-glucosidase, β-xylosidase, and α-arabinofuranosidase by A. awamori. This cracked the linkages between phenolics and their glycosides, increasing the mobilization of the bioactive constituents, liberating phenolics and enhancing the polyphenol content of substrate (Ajila et al., 2012;Bhanja et al., 2009;Gottschalk, Oliveira, & Bon, 2010). On the 7th day of fermentation, the TPC of all AFL extracts decreased to the range of 28.2% to 30.5% (Table 1). This decrease could be attributed to the stress induced response by the fungus due to the depletion in media nutrients resulting in the activation of oxidative enzymes that can polymerize the released phenolics (Vattem, Lin, Labbe, & Shetty, 2004). Degradation of some phenolic compounds such as gallic acid to aliphatic compounds can also lead to this decrease (Bhat, Singh, & Sharma, 1998). Schmidt and Furlong (2012) reported that the fermentation substrate, the starter fungus as well as different fermentation conditions have significant effect on the phenolic acid profile.

| Effect of SSF on antioxidant potential
Note. Mean ± SD, n = 3, followed by different superscripts (a-h) in a column differ significantly (p ≤ 0.05) and show variation among Aspergillus-fermented lentil extracts of same cultivars for different days. Superscripts p, q, and r in a row show variation among different cultivars. Variation (%) denotes the percentage increase from control samples for corresponding properties. produce hydrolytic enzymes catalyzing the release of aglycones, thereby increasing the phenolics, anthocyanin content, and the antioxidant capability of fermented flours (Bhanja et al., 2009;Lee, Hung, & Chou, 2007. HFRSA until 10th day of SSF in pearl millet. In an earlier study it was observed that enzymatic activity of β-glucosidase increased significantly during fermentation, transforming less active isoflavone glucosides to more active isoflavone aglycones antiradicals (Kim et al., 2011).
RPA of AFL extracts continued to improve up to 6 days of SSF with an increase of 26.0% to 30.4%, and thereafter, it decreased ( Figure 1c). Salar et al. (2017) reported an increase in RPA up to 8 days during SSF of pearl millet, which they attributed to increased xylanase activity upon fermentation. Also, the hydrogen donating ability of reductants was found to be directly related with the reducing power (Lee et al., 2008). During fermentation, the reductant formation starts, which terminates the radical chain reaction by reacting with the free radicals, resulting in high RPA of the fermented extracts (Lin, Wei, & Chou, 2006).
Therefore, there is a possibility of hydrolysis of polymers during fermentation, thereby releasing the conjugated phenolic compound present in the cell walls of legumes, making these compounds soluble, thereby increasing their concentration and antioxidant potential of the extracts.

| HPLC of bioactive compounds
Among different legumes, lentils have been reported to have the highest oxygen radical absorption and antioxidant potential (Fratianni et al., 2014) that can be attributed to a wide range of phenolic as well as nonphenolic antioxidants (Xu & Chang, 2008). Different standards (ascorbic acid, quercetin, vanillin, resorcinol, p-coumaric acid, catechin, cinnamic acid, and gallic acid) were selected for bioactive constituent screening in the extracts. The results of HPLC qualitative as well as quantitative analysis of UFL and AFL are presented in Figure 2a-f and Table 2, respectively. Ascorbic acid and two phenolic compounds (quercetin and catechin) were observed mainly in both UFL and AFL extracts. The amount of bioactive compounds was found to be higher in AFL in comparison with their control counterparts. Some previous studies also demonstrated the role of SSF in the modulation of nutrient profile, polyphenolic content, and antioxidant potential of different legumes, cereals, and other natural sources (Oboh et al., 2008;2009;Juan & Chou, 2010;Wu & Chou, 2009;Sandhu et al., 2016;Salar et al., 2017). Also, some new phenolic compounds not originally detected in UFL extracts were found to be present in AFL extracts such as resorcinol (for cv. HM-1 and LL-931) and cinnamic acid (for cv. LL-931), which provided clear evidence in support of microbial synthesis of these phenolic compounds. During microbial fermentation, carbohydrate cleavage due to different enzymatic activities are increased which result in change in the glycosides or fragmentary cleavage and release of potent antioxidants (Chiou et al., 2013;McCue & Shetty, 2003). On the other hand, compounds such as vanillin and gallic acid originally present in UFL extracts were not observed in their AFL counterparts. Bhat et al. (1998) also postulated that gallic acid may be degraded to aliphatic compounds during the process of fermentation resulting in a decrease in phenolic content.

| Effect of SSF on mineral content and bioavailability
A number of key functions such as building strong bone to transmitting nerve impulse are performed by minerals for a healthy and lengthy life. Different macro (Ca, K, Na) and micro (Fe, Zn, Cu) minerals were analyzed in the extracts using AAS but the results for control (UFL) and 6th day fermented AFL are only reported (Table 3).
Among different lentil cultivars, mineral content differed significantly (p ≤ 0.05), with AFL extracts exhibiting higher mineral content than their unfermented counterparts. The mineral content of AFL increased until the 6th day of fermentation, thereafter it decreased. Zn content ranged from 31.8 to 32.5 ppm for UFL and 34.2 to 35.5 ppm for AFL, thereby showing an increase of 7.5% to 9.2% in concentration. Cu content of all AFL extracts was the highest with an increase of 46.4% to 60.0%, the highest increase was observed for cv. LL-931. All minerals showed significant increase in their concentrations upon fermentation except for K in which the increase was less than 0.1%. Sadh, Chawla, Bhandari, Kaushik, and Duhan (2017) and Chawla, Bhandari, Sadh, and Kaushik (2017) also reported increase in mineral content in biotransformed peanut oil cakes and black-eyed pea seed flour, respectively.
The total quantity of mineral present in a food does not reflect total amount of available mineral and adsorbed by human body, as only a certain quantity is bioavailable (Jafari & McClements, 2017).
The bioavailability of a mineral can be defined as the fraction of the consumed mineral, which absorbed and utilized in different physiological functions of body (Fairweather-Tait & Hurrell, 1996). in vitro bioavailability of iron and zinc of AFL was significantly (p ≤ 0.05) higher in comparison with their unfermented counterparts (Table 3)  fermentation). The bioavailability of minerals is highly dependent on its release from the food matrix that is affected by digestion and absorption rate of target mineral by intestinal cells as well as its concentration in food. Lower digestibility of iron and zinc in unfermented flours is mainly attributed to the presence of antinutritional factors such as phytates and polyphenols in legume flours that form complexes with Zn and Fe and remarkably reduce their absorption (Gupta, Gangoliya, & Singh, 2015;Kiewlicz & Rybicka, 2020). After fermentation, these antinutritional factors complexing with proteins and minerals are reduced, thereby increasing their bioavailability in fermented flours (Adegbehingbe, 2015).

| CONCLUSIONS
SSF using A. awamori (MTCC 548) significantly improved the antioxidant profile of fermented flours from all lentil cultivars. Fermentation with A. awamori is a cost effective, reliable, and efficient method to improve the TPC, CTC, and antioxidant potential of lentils in a short period of time and, therefore, may prove to be an important process for industrial usage. New phenolic compounds (resorcinol, cinnamic acid) synthesis was also confirmed by HPLC.
Different macro (Ca, K, Na) and micro (Fe, Zn, Cu) mineral content improved during fermentation. However, in vitro bioavailability of iron and zinc of AFL was significantly (p ≤ 0.05) higher in comparison with their unfermented counterparts. Thus, biotransformed Aspergillus-fermented lentils could be used in preparation of nutritious functional foods.

ACKNOWLEDGMENT
The authors acknowledge Department of Food Science & Technology, Chaudhary Devi Lal University, Sirsa for providing necessary infrastructure for this research work. No funds were received from any agency for this work.

CONFLICT OF INTEREST
The authors have no conflicts of interest with respect to this manuscript.

COMPLIANCE WITH ETHICAL REQUIREMENTS
This article does not contain any studies with human and animal subjects.

DATA AVAILABILITY STATEMENT
Due to technical limitations, the full dataset is unable to be published at this time. However, it is available upon request from the authors.  Note. Mean ± SD, n = 3, followed by different superscripts (a-f) in a row differ significantly (p ≤ 0.05). Variation (%) denotes the percentage increase from control samples for corresponding properties.