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. 2020 Aug 17;58(5):1927–1936. doi: 10.1007/s13197-020-04704-y

Solid state fermentation of fenugreek (Trigonella foenum-graecum): implications on bioactive compounds, mineral content and in vitro bioavailability

Sanju Bala Dhull 1,✉, Sneh Punia 1, Rajesh Kumar 2, Manoj Kumar 3, Kiran Bala Nain 4, Kanchan Jangra 1, Chanchal Chudamani 1
PMCID: PMC8021647  PMID: 33897029

Abstract

In the present study, solid-state fermentation (SSF) of four fenugreek cultivars viz. HM-57, AFG-2, RMT-1 and RMT-303 were carried out using Aspergillus awamori and its effect on antioxidant properties, phenolic content and bioactive compounds were studied. Macro (Ca, K, and Na) as well as micro (Fe, Zn, and Cu) elements and in vitro bioavailability of the unfermented fenugreek (UFF) and Aspergillus-fermented fenugreek (AFF) samples were assessed with standard methods. On 5th day, total phenolic and condensed tannin contents showed significant (p ≤ 0.05) increase for all cultivars. Further, HPLC analysis confirmed formation of some new bioactive (vanillin, benzoic acid and catechin) compounds. Similarly, extracts from all AFF also showed an increase in the antioxidant potential such as inhibition of DPPH, hydroxyl free radical scavenging, reducing power, and total antioxidant capacity up to 5th day of SSF. Mineral in AFF were found with enhanced values when compared with respective UFF. In vitro bioavailability of Fe, Zn and Ca was also improved during SSF. Results from the present study may be helpful to food industry in developing new health foods and may provide a rational for development of functional ingredient in preparation of novel nutraceuticals.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04704-y) contains supplementary material, which is available to authorized users.

Keywords: Fenugreek, Solid state fermentation, Aspergillus awamori, Antioxidant, Mineral bioavailability

Introduction

Legumes are the third largest family of angiosperms belonging to Leguminosae. They provide a range of essential nutrients including protein, low glycemic index carbohydrates, dietary fibre, minerals and vitamins which make it main human dietary component worldwide. Apart from nutritional significance, they are also loaded with natural antioxidant which has been proved by a number of reports including experimental, epidemiological, and clinical researches (Xu and Chang 2007; Dhull et al. 2020a). Trigonella foenum-graecum, commonly known as fenugreek, is a unique legume crop having protein (25–35%), fat (5.0–7.5%), lysin (5.7 g/16 g N), soluble dietary fiber (20–25%) and insoluble dietary fibre (25–30%) along with several minerals and vitamins (calcium, iron and β-carotene) (Naidu et al. 2011; Dhull and Sandhu 2018; Dhull et al. 2019). Being rich in phytochemicals like phenols, flavonoids, carotenoids, alkaloids, tannins, amino acids and essential fatty acids, it has been tagged as antidiabetic, anticarcinogenic, hypocholesterolemic, antioxidant and immunological booster (Wani and Kumar 2018; Dhull et al. 2020b).

In a healthy normal human body, our natural defense systems (glutathione, catalase, and superoxide dismutase etc.) keep a check on the pro-oxidants. But, environmental stress and unhealthy lifestyle, creates an imbalance of these natural antioxidants and free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are spontaneously generated resulting in severe oxidative stress and ROS-mediated damage of tissues in human body (Bursal and Gülçin 2011; Köse et al. 2015; Türkan et al. 2020).

To elevate the nutritional quality as well as antioxidant profile of many cereals and legumes, SSF has been focused these days (Sandhu et al. 2016; Salar et al. 2017; Magro et al. 2019; Dhull et al. 2020a). Fermentation is preferred over other processes for production of polyphenolic compounds which might be due to faster growth rate of microorganisms, low cost, easy downstream processing and environment-friendly nature of the process. Fermenting microbes produce a number of enzymes (protease, amylase, lipase, phytase etc.) which can hydrolyze carbohydrate, proteins and lipids into easy digestible components with an appealing taste and texture. Meanwhile, several anti-nutritional factors such as phytate, tannin and protease inhibitor are markedly degraded by these enzymes (Soetan and Oyewole 2009) which also increased the interest in SSF to improve the absorption and bioavailability of certain minerals present in seeds.

It has been reported previously that the protein and fat content, free phenolics and antioxidant property of a variety of legumes such as pigeon pea, African yam bean, bambara groundnut and kidney bean (Oboh et al. 2009) has been improved during SSF. Lentil SSF with fungi Aspergillus awamori significantly modulated the phenolic profile of fermented samples (Dhull et al. 2020a).

Nevertheless, limited attempts have been carried out until now to modulate the nutritional and functional quality of Indian fenugreek cultivars, effect of SSF on bioactive compounds, mineral content and in vitro bioavailability have not been discussed so far. As, the selection of suitable substrate and microorganism is crucial for maximal recovery of bioactive compounds from natural resources, A. awamori (generally recognized as safe) was selected for SSF on the basis of suggestions for its use and benefits as starter culture (Bhanja et al. 2009; Sandhu et al. 2016; Dhull et al. 2020a). Therefore, the study was undertaken to study the effect of SSF using A. awamori on antioxidant potential, phenolic profile and mineral bioavailability of different fenugreek cultivars.

Materials and methods

Substrate

Certified fenugreek cultivars (cv.) namely HM-57 (CCSHAU, Hisar), AFG-2 (NRCSS, Ajmer), RMT-1 and RMT-303 (SKNAU, Jobner) were procured, cleaned thoroughly and packed in airtight containers for further study.

Starter culture for SSF

Fermenting microorganism i.e. Aspergillus awamori (MTCC 548), was purchased from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India and initially maintained on czapekdox agar (CDA) as well as broth (CDB) (30 ± 2 °C). Four days old mycelia suspended in aqueous 0.1% (w/v) solution of Tween 80 was used to inoculate the fenugreek seed samples after autoclaving (Dhull et al. 2020a).

Solid state fermentation (SSF)

SSF of fenugreek seeds were carried out as per the method described by Salar et al. (2017). Dried seeds (50 g) were soaked overnight in CDB in 250 mL Erlenmeyer flasks. After decanting excess CDB, the seeds were autoclaved at 121 °C for 15 min and then cooled to room temperature. Spore suspension (5 mL) prepared above was sprayed on the autoclaved seeds, thoroughly mixed and then incubation at 25 ± 2 °C was done for 7 days. The control i.e. unfermented fenugreek seeds were prepared without the inoculation with spores.

Preparation of extracts

At regular intervals of 24 h, Aspergillus-fermented fenugreek (AFF) samples from fermentation flask were withdrawn and dried in hot air oven (40 ± 2 °C, 48 h). After grinding, defatting and air drying, the flour was extracted with ethanol as described by Dhull et al. (2020a), filtered and evaporated under vacuum. For analysis, solvent and extract were mixed in the ratio of 2:1 and stored in dark at − 4 °C till further use (Bhanja et al. 2009).

Total Phenolic Content (TPC) and Condensed Tannins Content (CTC)

The Folin–Ciocalteu reagent method described by Gao et al. (2002) was adapted for TPC determination of AFF and UFF extracts. Absorbance were recorded at 765 nm and using standard calibration curve, the results were calculated and expressed as mg gallic acid equivalents per gram (mg GAE/g) of sample (Gülçin et al. 2019, 2020; Bursal et al. 2019). Triplicate observations were recorded for each analysis.

The method described by Julkunen-Titto (1985) was explored to estimate the CTC of the samples. At 500 nm the absorbance were recorded where catechin (0.05–1 mg/mL) was used for standard calibration curve. The results were expressed as milligrams of catechin equivalent per g (mgCE/g) dry weight basis (dwb). Triplicate observations were recorded for each analysis.

Antioxidant potential

DPPH radical scavenging activity

The 2,2-diphenyl-1-picrylhydrazyl(DPPH) scavenging activity was determined using the assay described by Yen and Chen (1995) in which 100 µM DPPH was added to 100 µL of extract, mixed and after 30 min, the changes in absorbance was observed at 517 nm. The following expression was used to calculate percent (%) DPPH scavenging activity:

DPPH Scavenging Activity%=Ac-AE/Ac×100

where AC and AE are the absorbance of control and extracts, respectively.

Hydroxyl free radical scavenging activity (HFRSA)

The HFRSA of sample extracts was analyzed using the method described by Smirnoff and Cumbes (1989). An extract (100 µL) and Smirnoff reagent (3 mL) were mixed thoroughly and then incubated (37 °C, 30 min). The HFRSA was calculated using the following formula:

HFRSA%=Ac-AE/Ac×100

where AC and AE are the absorbance of control and extracts, respectively.

Reducing power assay (RPA)

The method described by Oyaizu (1986) was adapted using quercetin as standard to estimate the RPA of the sample extracts. Briefly, 100 µL of extract and 100 µL of 1% solution of potassium ferricyanide were mixed and then incubated (50 °C, 30 min). To the mixer, 1% trichloroacetic acid (100 µL) and 0.1% ferric chloride (100 µL) were added, mixed and again incubated (20 min, ambient temperature). Before recording the absorbance at 700 nm, the volume of mixer was made to 10 mL using distilled water. The values were recorded as mg quercetin equivalents/g (mg QE/g) on dry weight basis of the sample.

Total antioxidant capacity (TAC)

TAC was evaluated using the method of Prieto et al. (1999). To analyze the antioxidant activity, reagent was prepared by mixing equal ratio (1:1:1) of 0.6 M concentrated sulphuric acid, 4 mM ammonium molybdate and 28 mM sodium hydrogen orthophosphate. 3 mL of this reagent was added to 100 µL of sample extract, mixed and incubated in waterbath (95 °C, 90 min). Using ascorbic acid as standard, absorbance was noted at 695 nm and the values were recorded as mg ascorbic acid equivalents/g (mg AAE/g) on dry weight basis of the sample.

High performance liquid chromatography (HPLC)

The AFF (5th day sample) and UFF extracts were analyzed using HPLC (Shimadzu 10 AVP HPLC system) as per the procedure described by Dhull et al. (2016). The samples were analyzed using 2% v/v acetic acid (solvent A) and methanol: acetonitrile (40:50 v/v) mixture (solvent B) at a rate of 0.6 mL/min. The analytes were noted at 280 nm using 10 µL injection volumes. Standard graph were prepared and the equation generated by the data was used to further quantify the studied bioactive constituents.

Mineral content

Atomic absorbance spectrophotometer (AAS) (AA-7000, Shimadzu, Tokyo, Japan) was used in atomic absorption mode to analyze the iron (Fe), copper (Cu), zinc (Zn), and calcium (Ca) content while emission mode to analyze the potassium (K) and sodium (Na) content of UFF and AFF flour samples following the methods of AOAC (2005).

In vitro bioavailability

The method described by Sadh et al. (2017) was used to analyze the in vitro bioavailability of iron, zinc and calcium. Briefly, 5 g of each UFF and AFF samples were mixed with saliva solution (9 mL, pH 6.5) containing organic, inorganic components and α-amylase (700 mg/L of saliva solution) in a flask and incubated in water bath (37 °C, 95 rpm, 5 min). Gastric juice (13.5 mL) with organic, inorganic solutions, mucin (6 g/L of gastric juice), bovine serum albumin (2 g/L of gastric juice), and pepsin (2 g/L of gastric juice) from porcine stomach were then added to the flask and adjusted to pH 1.1 using HCl and further incubated (37 °C, 1 h). Further, after adding freshly prepared duodenal juice and bile solutions, pancreatic lipase colipase, cholesterol esterase, phospholipase A2, taurocholate salts and incubation, the mineral content under simulated gastro-intestinal conditions in permeate was determined by AAS. Bioavailability of minerals (iron, zinc and calcium) was calculated as:

Bioavailability%=D/C×100

where D = Mineral content in the dialysate (permeate) and C = Mineral content of sample.

Statistical analysis

For all the experiments except for HPLC analysis (n = 2), the data reported as mean ± standard deviation (n = 3). Further, analysis of variance (ANOVA) with a significance level of 5% was applied using the commercial statistical package (SPSS Inc, Chicago, IL). Further, Duncan’s test was applied to determine significant difference between mean values.

Results and discussion

SSF effect on total phenolic and condensed tannin contents

The changes in TPC and CTC during SSF of Aspergillus-fermented fenugreek (AFF) compared with unfermented fenugreek (UFF) counterpart are tabulated in Tables 1 and 2. TPC of UFF cultivars ranged between 26.9 and 42.3 mg GAE/g dwb. Fenugreek cultivars were studied for their TPC after fermentation. For all cultivars, TPC increased significantly (p ≤ 0.05) during fermentation in comparison to their corresponding non-fermented counterparts. The increase in TPC was observed till 5th day of fermentation and thereafter it decreased. On 5th day of incubation the highest and the lowest TPC was observed for cv. HM-57 (58.8 mg GAE/g) and cv. AFG-2 (40.4 mg GAE/g), respectively and % increase in TPC from initial concentration was 39.0–73.9% in comparison to their counterpart UFF for different cultivars. Dhull et al. (2020c) reported TPC for different fenugreek cultivars ranging from 38.9 to 45.4 mg GAE/g dwb for aqueous ethanol and 13.4–21.8 mg GAE/g dwb for methanol extracts. In another recent study, Dhull et al. (2020a) reported a linear increase in TPC during SSF of lentils using A. awamori with the highest values observed on 6th day. However, Magro et al. (2019) found continuous increase with maximum values of TPC after 96 h of fermentation of lentil with A. oryzae and A. niger. Similar results were obtained for SSF of pearl millet by A. sojae (Salar et al. 2017) and wheat by A. awamorinakazawa (Sandhu et al. 2016). This enhancement may be attributed to the fact that during fermentation, some active hydolases including β-glucosidase, β-xylosidase and α-arabinofuranosidase are produced by fermenting fungi such as A. awamori. These enzymes may crack the phenol-glycoside linkages, increase the bioactive compound mobilization, liberate phenolic compounds and therefore, enhance the polyphenol content of fermenting substrate (Dhull et al. 2020a). On the 6th day of SSF, the TPC of AFF samples were decreased and the values were in the range of 35.2 to 55.3 mg GAE/g dwb (Table 1). As the fermentation progressed and fungal biomass increased, there will be depletion in media nutrients which can induce stress response in fermenting fungi. This response results in the activation of oxidative enzymes which can polymerize the released phenolics and decrease the total content (Vattem et al. 2004). Additionally, certain phenolic compounds are degraded to some aliphatic compounds which can further add on to this decrease.

Table 1.

Effect of SSF on total phenolic content of extracts from different unfermented and Aspergillus-fermented fenugreek cultivars

Fermentation time HM-57 (mg GAE/g dwb) Variation (%) AFG-2 (mg GAE/g dwb) Variation (%) RMT-1 (mg GAE/g dwb) Variation (%) RMT-303 (mg GAE/g dwb) Variation (%)
Control 42.3 ± 0.21bs – 26.9 ± 0.21ap – 29.1 ± 0.25aq – 30.0 ± 0.21ar –
1st Day 44.6 ± 0.19cr + 5.4 30.0 ± 0.15 bp + 11.5 31.1 ± 0.35bq + 6.9 30.9 ± 0.18bpq + 3.0
2nd Day 45.1 ± 0.20dr + 6.6 33.1 ± 0.13cp + 23.0 35.3 ± 0.19cq + 21.3 32.9 ± 0.15 bp + 9.7
3rd Day 48.4 ± 0.31es + 14.4 35.1 ± 0.31dq + 30.4 36.1 ± 0.17dr + 24.0 33.3 ± 0.19cp + 11.0
4th Day 57.0 ± 0.18gs + 34.7 38.4 ± 0.17ep + 42.7 50.0 ± 0.25gr + 71.8 46.4 ± 0.25fq + 54.7
5th Day 58.8 ± 0.23 h + 39.0 40.4 ± 0.19fp + 50.2 50.6 ± 0.22hq + 73.9 49.8 ± 0.22gpq + 66.0
6th Day 55.3 ± 0.27 fs + 30.7 35.2 ± 0.25dp + 30.8 47.5 ± 0.27fr + 63.2 37.4 ± 0.24 eq + 24.7
7th Day 41.7 ± 0.19ar − 1.4 33.8 ± 0.23cp + 25.6 43.8 ± 0.15es + 50.5 35.3 ± 0.31dq + 17.7
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Mean ± SD, n = 3, followed by different superscripts (a–h) in a column differ significantly (p ≤ 0.05) and show variation among Aspergillus-fermented fenugreek extracts of same cultivars for different days. Superscripts p, q, r and s in a row show variation among different cultivars. Variation (%) denotes the percentage increase/decrease from control samples for corresponding properties

Table 2.

Effect of SSF on condensed tannin content of extracts from different unfermented and Aspergillus-fermented fenugreek cultivars

Fermentation time HM-57 (mg CE/g dwb) Variation (%) AFG-2 (mg CE/g dwb) Variation (%) RMT-1 (mg CE/g dwb) Variation (%) RMT-303 (mg CE/g dwb) Variation (%)
Control 3.5 ± 0.11br – 2.9 ± 0.12bq – 4.0 ± 0.10bs – 2.4 ± 0.13ap –
1st Day 4.0 ± 0.10cr + 14.3 3.6 ± 0.11cdq + 24.1 4.9 ± 0.15cs + 22.5 3.1 ± 0.12 bp + 29.1
2nd Day 4.4 ± 0.12dq + 25.7 3.9 ± 0.10dp + 34.5 5.1 ± 0.11cdr + 27.5 3.9 ± 0.10cp + 62.5
3rd Day 4.5 ± 0.13dpq + 28.6 4.3 ± 0.13ep + 48.3 5.3 ± 0.12dr + 32.5 4.7 ± 0.11dq + 95.8
4th Day 5.1 ± 0.10 eq + 45.7 4.7 ± 0.12fp + 55.9 6.0 ± 0.11er + 50.0 5.1 ± 0.11deq + 112.5
5th Day 7.9 ± 0.12gr + 125.7 5.5 ± 0.17gp + 89.6 6.9 ± 0.13fq + 72.5 5.7 ± 0.12epq + 137.5
6th Day 5.6 ± 0.15fr + 60.0 3.4 ± 0.15cp + 17.2 4.9 ± 0.12cq + 22.5 4.8 ± 0.13dq + 100.0
7th Day 2.3 ± 0.17ap − 34.3 2.2 ± 0.13ap − 24.1 3.7 ± 0.10ar − 7.5 3.0 ± 0.15bq + 25.0
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Mean ± SD, n = 3, followed by different superscripts (a–h) in a column differ significantly (p ≤ 0.05) and show variation among Aspergillus-fermented fenugreek extracts of same cultivars for different days. Superscripts p, q, r and s in a row show variation among different cultivars. Variation (%) denotes the percentage increase/decrease from control samples for corresponding properties

The CTC content of UFF ranged between 2.4 and 4.0 mg CE/g, which upon fermentation increased and the range of CTC on 5th day was 5.5–7.9 mg CE/g, with the highest and the lowest being observed for cv. HM-57 and cv. AFG-2, respectively (Table 2). After 5th day, CTC started to decrease which might be attributed to activation of degrading enzymes with the progressed fermentation. Some of previous studies also found substantial increase in CTC of Aspergillus-fermented lentil (Dhull et al. 2020a) and pearl millet (Salar et al. 2017).

SSF effect on antioxidant potential

A number of secondary metabolites with strong biological activity are produced during SSF which can lead to enhanced antioxidant activity of substrates. This antioxidant potential can be measured using a number of assays which have different theoretical principle for analyzing different antioxidants. Therefore, no single assay can reflect the true antioxidant potential of any sample (Gülçin 2006a, 2006b, 2007, 2012, 2020). In the current study, four different assays including DPPH radical scavenging assay, Hydroxyl free radical scavenging activities (HFRSA), Reducing power assay (RPA) and Total antioxidant capacity (TAC) assay were employed to analyze the antioxidant potential of all UFF and AFF extracts and the results are presented in Fig. 1a–d. Significant (p ≤ 0.05) improvement in the antioxidant capacities of all AFF extracts were observed in comparison with their non-fermented counterparts.

Fig. 1.

Fig. 1

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Effect of SSF on antioxidant properties of fenugreek cultivars: a DPPH inhibition activity; b Hydroxyl free radical scavenging activity (HFRSA); c Reducing power assay (RPA); d Total antioxidant capacity (TAC)

DPPH radical scavenging assay determines the electron or hydrogen giving capacity of an extract. Among cultivars, UFF do not differed significantly (p ≤ 0.05) and the values ranged from 91.6 to 93.4% with cv.HM-57 and cv.AFG-2 showing the highest and the lowest values, respectively (Fig. 1a). Dhull et al. (2020c) also suggested similar results for DPPH radical scavenging activities of fenugreek cultivars ranging from 81.8 to 87.6% and 82.2 to 93.2% for aqueous ethanol and methanol extracts, respectively. Some previous studies reported an increase in TPC during SSF mainly contributing to increased antioxidant potential of the fermenting substrates (Sandhu and Punia 2017; Dhull et al. 2020a). Among all cultivars, the maximum DPPH inhibition was found on the 5th day of fermentation with the values ranging from 96.6 to 97.8%. Dhull et al. (2020a) and Salar et al. (2017) reported highest DPPH inhibition on 6th day of fermentation for lentils and pearl millets, respectively. Meanwhile, one another study reported maximum inhibition activity in lentils after 24 h of fermentation by A. oryzae and after 48 h by A. niger (Magro et al. 2019). Many hydrolytic enzymes produced by the fungi catalyze the release of aglycones from the substrate and contribute in increased phenolics, anthocyanin as well as antioxidant potential of fermented flours (Bhanja et al. 2009; Lee et al. 2007). Although, a number of factors such as fermenting substrate, starter microorganism and fermenting conditions significantly control the fermentation process and the metabolites produced during SSF which simultaneously affect the antioxidant profile of substrate (Dhull et al. 2020a).

HFRSA of all UFF and AFF were analyzed and the results are reported in Fig. 1b. Among UFF samples, the values ranged from 16.2 to 18.6%, cv.RMT-1 and cv.RMT-303 showed the highest and the lowest activities, respectively (Fig. 1b). For all AFF samples, the maximum HFRSA was observed on 5th day of the process with cv. RMT-1 showing the highest value (44.91%) with 141.0% increase in comparison to their unfermented counterpart. The varietal difference in antioxidant properties of extracts during fermentation might be due to variation in nutrient as well as anti-nutrient composition of cultivars which finally affect the fermentation process. Kim et al. (2011) observed a significant increase in the activity of β-glucosidase enzyme during fermentation which can transform poorly active isoflavone glucosides to highly active isoflavone aglycone antiradicals.

Figure 1c represents the change in RPA during SSF of fenugreek cultivars and showed continuous improvement in the values up to 5th day of fermentation. On 5th day, the values for RPA was observed from 9.9 to 11.1 mg QE/g with an increase of 27.5–160.5% which started decreasing thereafter (Fig. 1c). Similarly, Dhull et al. (2020a) and Salar et al. (2017) reported an increase in RPA in Aspergillus-fermented lentils and pearl millet. Lee et al. (2007) concluded that reducing power of any reductant is directly related to its hydrogen donating capability. The reductants react with free radicals and terminates the radical chain reactions, thus result in increased RPA of fermenting substrate extracts (Lin et al. 2006).

For UFF extracts of cv.HM-57, cv.AFG-2, cv.RMT-1 and cv.RMT-303, the TAC values were 20.3, 15.9, 21.1 and 19.9 mg AAE/g, respectively. Like other assays, it increased gradually and significantly (p ≤ 0.05) up to 5th day of fermentation with values ranging from 24.1 to 27.1 mg AAE/g (Fig. 1d). Aspergillus species are excellent producers of many hydrolytic enzymes such as amylases, proteases, lipases and cellulases. During fermentation, these enzymes possibly can hydrolyze polymers and release the conjugated phenols in cell walls of legumes, increasing their solubility as well as concentration which in turn enhance the antioxidant potential of the fermented substrate. But as the fermentation progressed after a certain time, increased fugal biomass resulted in nutrient depletion and stress induced activation of oxidative enzymes which can polymerize the released phenolics (Vattem et al. 2004). Additionally, decrease in the activity of some enzymes (α-amylase, β-glucosidase) which can mobilize the bound bioactive compounds with progressive fermentation also suggested dependence of antioxidant assays on the enzymes activity (Salar et al. 2017).

HPLC analysis of bioactive compounds

Qualitative and quantitative HPLC analysis of both UFF as well as AFF with maximum phenolic content i.e. 5th day sample extracts of all four cultivars were carried out and the results are presented in Table 3 and supplementary Fig. S1(a–h). Ten standards (benzoic acid, p-coumaric acid, ascorbic acid, vanillin, quercetin, catechin, resorcinol, catechol, cinnamic acid, gallic acid) were used during the HPLC analysis. Among UFF extracts, nine out of ten tested bioactive compounds were detected in cv. HM-57, eight compounds were found in cv. AFG-2 while seven compounds were detected in cv. RMT-1 and cv. RMT-303 each. Out of the tested ten standards, cinnamic acid in cv. HM-57, vanillin and benzoic acid in cv. AFG-2, catechin, vanilin and cinnamic acid in cv. RMT-1 and catechin, p-coumaric acid and benzoic acid in cv. RMT-303, were found absent. However AFF extracts (5th day) of cv. AFG-2 showed the presence of all the ten bioactive compounds, whereas cv. HM-57, cv. RMT-303 and cv. RMT-1 showed nine, eight and seven bioactive compounds, respectively.

Table 3.

Quantification profile of bioactive compounds using HPLC from different unfermented (UFF) and 5th day Aspergillus-fermented (AFF) fenugreek cultivars

Phenolic compounds (mg/g dwb) HM-57 AFG-2 RMT-1 RMT-303
UFF AFF UFF AFF UFF AFF UFF AFF
Ascorbic acid 3.54b 4.65d 3.35a 4.17c 4.13c 5.27f 5.02e 12.66 g
Gallic acid 1.41a 7.41f 5.99d 6.23e 5.47c 6.10de 3.71b 11.79 g
Catechin 1.39b 1.81d 1.62c 2.35e ND 0.23a ND ND
Resorcinol 11.95f 17.9 g 4.89b 5.11c 8.76e ND 4.14a 6.53d
Catechol 11.80c 14.38e 12.08d 14.67f 1.89a 2.34b 1.96a 2.11ab
Vanilin 1.52b 2.34d ND 1.21a ND 1.61c 5.65e 7.38f
p-Coumaric acid 1.10b 1.14b 13.6c 14.4d 0.46a ND ND ND
Quercetin 4.31e 4.78f 1.18a 2.51c 1.43b 9.71 h 4.17d 4.94 g
Benzoic acid 2.80c 3.11d ND 0.94a 6.92e 13.89f ND 1.09ab
Cinnamic acid ND ND 4.22c 5.08d ND ND 1.22a 1.74b
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The values followed by different superscripts in a row differ significantly (p ≤ 0.05)

ND—Not detected

Ascorbic acid, gallic acid, catechol, and quercetin were found mainly in both UFF and AFF extracts. For all AFF extracts, quantitative results (Table 3) showed an increase in the values of most of the bioactive compounds compared with their respective UFF sample extracts. A number of earlier studies on SSF also reported improvement of nutritional properties, polyphenolic profile and antioxidant properties of various legumes, pulses, cereals etc. (Lin et al. 2006; Lee et al. 2007; Oboh et al. 2009; Sandhu et al. 2016; Salar et al. 2017; Dhull et al. 2020a). Also, the AFF extracts showed appearance of some new bioactive compounds (such as vanillin and benzoic acid for cv. AFG-2, catechin and vanillin for cv. RMT-1 and benzoic acid for cv. RMT-303) which were not originally present in their respective UFF counterparts. This also confirmed the microbial synthesis as well as liberation of some bound bioactive compounds during the fermentation process. The enzymatic activities, carbohydrate–cleavages, glycosidic or other fragmentary-cleavages are increased during fermentation which all lead to release of different compounds with strong antioxidant potential (Dhull et al. 2020a). Meanwhile, compounds such as resorcinol and p-coumaric acid originally present in UFF sample extract (for cv. RMT-1) were not detected in its AFF extract which can be attributed to the degradation of some phenolic compounds during the SSF.

Effect of SSF on mineral contents and in vitro bioavailability

Minerals perform many key functions starting from the bone formation to nerve impulse transmittance which are essential for a healthy and normal life. The UFF and 5th day AFF were analyzed for their macro (Ca, Na, K) and micro (Fe, Cu, Zn) minerals and the results are presented in Table 4. Till 5th day, an increase in mineral values was observed, thereafter it started to decrease. Zn content for UFF and AFF ranged from 20.6 to 23.0 ppm and 24.9 to 27.5 ppm, respectively, showed 14.3–20.9% increase in its concentration after SSF. Among all AFF samples, Cu content exhibited the highest increase of 27.5–35.6% in its values and the maximum value was recorded for cv.RMT-1. Among all minerals, K do not showed significant improvement in its concentration after SSF. Bio-transformed lentils (Dhull et al. 2020a) and black eyed pea (Chawla et al. 2017) seed were also reported with increased mineral concentrations compared with unfermented samples.

Table 4.

Mineral (Fe, Zn, Ca, Cu, Na, K) content different unfermented (UFF) and 5th day Aspergillus-fermented (AFF) fenugreek cultivars

HM-57 AFG-2 RMT-1 RMT-303
UFF (ppm) AFF (ppm) UFF (ppm) AFF (ppm) UFF (ppm) AFF (ppm) UFF (ppm) AFF (ppm)
Fe 335.3 ± 1.1a 350.5 ± 1.2b 332.2 ± 1.5a 346.2 ± 1.2ab 335.26 ± 1.1a 351.26 ± 01.2b 335.57 ± 0.9a 350.75 ± 1.5b
Zn 23.0 ± 1.0ab 26.3 ± 1.0c 22.8 ± 0.8ab 27.5 ± 1.1d 22.7 ± 1.2ab 27.1 ± 1.2d 20.6 ± 0.9a 24.9 ± 1.9b
Ca 1880.0 ± 6.6d 1940.9 ± 8.2e 1825.7 ± 9.6b 1885.9 ± 7.7d 1798.0 ± 6.6a 1868.9 ± 8.3c 1888.9 ± 6.6d 1935.9 ± 9.2e
Cu 9.8 ± 1.0a 12.5 ± 1.1b 9.5 ± 1.0a 12.8 ± 1.1b 10.4 ± 1.3ab 14.1 ± 1.1c 10.3 ± 1.3ab 13.9 ± 1.0c
Na 857.2 ± 3.5 g 869.2 ± 4.5 h 691.7 ± 3.5e 702.3 ± 5.4f 592.3 ± 4.8a 601.5 ± 3.2b 625.2 ± 5.2c 635.2 ± 3.3d
K 7820.8 ± 6.3 g 7836.5 ± 5.3 h 7785.7 ± 6.1e 7802.3 ± 5.2f 7286.5 ± 5.2c 7301.2 ± 5.2d 6947.1 ± 6.0a 6960.8 ± 5.7b
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Mean ± SD, n = 3, followed by different superscripts (a–h) in a row differ significantly (p ≤ 0.05)

The bioavailability i.e., part of dietary mineral absorbed and utilized by human body for different physiological functions, always differed from the total amount of that mineral present in any food. In vitro bioavailability of trace minerals such as Fe, Zn and Ca were analyzed using gastrointestinal, simulation method and compared with respective free form of inorganic salts i.e. iron and zinc sulfate heptahydrate and calcium carbonate, respectively. The results (only for cv.HM-57) are presented in Fig. 2. All AFF samples showed significantly (p ≤ 0.05) improved bioavailability of Fe, Zn and Ca in comparison to their respective UFF as well as inorganic salts (data not shown). A number of anti-nutritional factors such as phytates present in legumes form complexes with these trace minerals and remarkably decrease their availability during absorption (Gupta et al. 2015). Different enzymes produced during SSF degrade these anti-nutritional factors, hence improve the bioavailability and digestibility of minerals in fermented samples. On the other hand, squat solubility and precipitation of inorganic salts at gastric and intestinal pH resulted in their low bioavailability in the duodenum (Chawla et al. 2019).

Fig. 2.

Fig. 2

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Mineral bioavailability of inorganic salts, unfermented fenugreek (UFF) and 5th day Aspergillus-fermented fenugreek (AFF) samples from cv. HM-57

Conclusion

SSF with A. awamori is a fast, low cost, safe and effective way to modulate the total phenolics, condensed tanins and antioxidant properties of fenugreek seeds which therefore, may prove useful for large scale industrial usage. SSFof fenugreek seeds was carried out by taking generally recognized as safe fungal strain i.e., Aspergillus awamori (MTCC 548). For all fenugreek cultivars, the antioxidant profile was significantly improved after SSF. HPLC analysis confirmed the appearance of some new phenolic compounds (vanillin, benzoic acid and catechin). For all fermented samples, the in vitro bioavailability of trace minerals i.e., Fe, Zn and Ca showed significant improvement compared to their unfermented counterparts. Thus, it may be concluded that after conducting some toxicity studies further for any inhibitory substances, Aspergillus-fermented fenugreek seeds may be consumed as health food or utilized as functional food ingredient.

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Compliance with ethical standards

Conflict of interest

There is no conflict of interest among authors regarding this manuscript.

Human and animal rights

No human and animal subjects were used in the present study.

Footnotes

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References

  1. AOAC (2005) Official methods of analysis. In: The association of official analytical chemists, 18th edn. North Fredrick Avenue Gaithersburg, Maryland, p 481
  2. Bhanja T, Kumari A, Bannered R. Enrichment of phenolics and free radical scavenging property of wheat koji prepared with two filamentous fungi. Bioresour Technol. 2009;100:2861–2866. doi: 10.1016/j.biortech.2008.12.055. [DOI] [PubMed] [Google Scholar]
  3. Bursal E, Gülçin İ. Polyphenol contents and in vitro antioxidant activities of lyophilised aqueous extract of kiwifruit (Actinidia deliciosa) Food Res Int. 2011;44(5):1482–1489. doi: 10.1016/j.foodres.2011.03.031. [DOI] [Google Scholar]
  4. Bursal E, Aras A, Kılıç Ö, Taslimi P, Gören AC, Gülçin İ. Phytochemical content, antioxidant activity, and enzyme inhibition effect of Salvia eriophora Boiss and Kotschy against acetylcholinesterase, α-amylase, butyrylcholinesterase, and α-glycosidase enzymes. J Food Biochem. 2019;43(3):12776. doi: 10.1111/jfbc.12776. [DOI] [PubMed] [Google Scholar]
  5. Chawla P, Bhandari L, Sadh PK, Kaushik R. Impact of solid state fermentation (Aspergillus oryzae) on functional properties and mineral bioavailability of black eyed pea (Vigna unguiculata) seed flour. Cereal Chem. 2017;94:437–442. doi: 10.1094/CCHEM-05-16-0128-R. [DOI] [Google Scholar]
  6. Chawla P, Kumar N, Kaushik R, Dhull SB. Synthesis, characterization and cellular mineral absorption of nanoemulsions of Rhododendron arboreum flower extracts stabilized with gum arabic. J Food Sci Technol. 2019;56(12):5194–5203. doi: 10.1007/s13197-019-03988-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dhull SB, Sandhu KS. Wheat–fenugreek composite flour noodles: effect on functional, pasting, cooking and sensory properties. Curr Res in Nutr Food Sci. 2018;6:174–182. doi: 10.12944/CRNFSJ.6.1.20. [DOI] [Google Scholar]
  8. Dhull SB, Kaur P, Purewal SS. Phytochemical analysis, phenolic compounds, condensed tannin content and antioxidant potential in Marwa (Origanum majorana) seed extracts. Resour Technol. 2016;2:168–174. doi: 10.1016/j.reffit.2016.09.003. [DOI] [Google Scholar]
  9. Dhull SB, Punia S, Sandhu KS, Chawla P, Kaur R, Singh A. Effect of debittered fenugreek (Trigonella foenum graecum L.) flour addition on physical, nutritional, antioxidant and sensory properties of wheat flour rusk. Legume Sci. 2019;2:21. doi: 10.1002/leg3.21. [DOI] [Google Scholar]
  10. Dhull SB, Punia S, Kidwai MK, et al. Solid-state fermentation of lentil (Lens culinaris L.) with Aspergillus awamori: effect on phenolic compounds, mineral content, and their bioavailability. Legume Sci. 2020 doi: 10.1002/leg3.37. [DOI] [Google Scholar]
  11. Dhull SB, Sandhu KS, Punia S, Kaur M, Chawla P, Malik A. Functional, thermal and rheological behavior of fenugreek (Trigonella foenum–graecum L.) gums from different cultivars: a comparative study. Int J Biol Macromol. 2020;159:406–414. doi: 10.1016/j.ijbiomac.2020.05.094. [DOI] [PubMed] [Google Scholar]
  12. Dhull SB, Kaur M, Sandhu KS. Antioxidant characterization and in vitro DNA damage protection potential of some Indian fenugreek (Trigonella foenum-graecum) cultivars: effect of solvents. J Food Sci Technol. 2020 doi: 10.1007/s13197-020-04380-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gao L, Wang S, Oomah BD, Mazza G (2002) Wheat quality: antioxidant activity of wheat mill streams. In: AACC International, pp 219–233
  14. Gulcin İ. Antioxidants and antioxidant methods: an updated overview. Arch Toxicol. 2020 doi: 10.1007/s00204-020-02689-3. [DOI] [PubMed] [Google Scholar]
  15. Gülcin İ. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006;78(8):803–811. doi: 10.1016/j.lfs.2005.05.103. [DOI] [PubMed] [Google Scholar]
  16. Gülcin I. Antioxidant activity of food constituents: an overview. Arch Toxicol. 2012;86(3):345–391. doi: 10.1007/s00204-011-0774-2. [DOI] [PubMed] [Google Scholar]
  17. Gülçin İ. Antioxidant activity of caffeic acid (3, 4-dihydroxycinnamic acid) Toxicology. 2006;217(2–3):213–220. doi: 10.1016/j.tox.2005.09.011. [DOI] [PubMed] [Google Scholar]
  18. Gülçin I. Comparison of in vitro antioxidant and antiradical activities of l-tyrosine and L-Dopa. Amino Acids. 2007;32(3):431–438. doi: 10.1007/s00726-006-0379-x. [DOI] [PubMed] [Google Scholar]
  19. Gulcin I, Kaya R, Goren AC, Akincioglu H, Topal M, Bingol Z, et al. Anticholinergic, antidiabetic and antioxidant activities of cinnamon (cinnamomum verum) bark extracts: polyphenol contents analysis by LC-MS/MS. Int J Food Prop. 2019;22(1):1511–1526. doi: 10.1080/10942912.2019.1656232. [DOI] [Google Scholar]
  20. Gülçin İ, Gören AC, Taslimi P, Alwasel SH, Kılıc O, Bursal E. Anticholinergic, antidiabetic and antioxidant activities of Anatolian pennyroyal (Mentha pulegium)-analysis of its polyphenol contents by LC-MS/MS. Biocatal Agric Biotechnol. 2020;23:101441. doi: 10.1016/j.bcab.2019.101441. [DOI] [Google Scholar]
  21. Gupta RK, Gangoliya SS, Singh NK. Reduction of phytic acid andenhancement of bioavailable micronutrients in food grains. J Food Sci Technol. 2015;52:676–684. doi: 10.1007/s13197-013-0978-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Julkunen-Titto R. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J Agric Food Chem. 1985;33:213–217. doi: 10.1021/jf00062a013. [DOI] [Google Scholar]
  23. Kim J, Choi JN, Kang D, Son GH, Kim YS, Choi HK, Kwon DY, Lee CH. Correlation between antioxidative activities and metabolite changes during Cheonggukjang fermentation. Biosci Biotechnol Biochem. 2011;75:732–739. doi: 10.1271/bbb.100858. [DOI] [PubMed] [Google Scholar]
  24. Köse LP, Gülcin I, Gören AC, Namiesnik J, Martinez-Ayala AL, Gorinstein S. LC–MS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes. Ind Crop Prod. 2015;74:712–721. doi: 10.1016/j.indcrop.2015.05.034. [DOI] [Google Scholar]
  25. Lee IH, Hung YH, Chou CC. Total phenolic and anthocyanin contents, as well as antioxidant activity, of black bean koji fermented by Aspergillus awamori under different culture conditions. Food Chem. 2007;104:936–942. doi: 10.1016/j.foodchem.2006.12.049. [DOI] [Google Scholar]
  26. Lin CH, Wei YT, Chou CC. Enhanced antioxidative activity of soybean koji prepared with various filamentous fungi. Food Microbiol. 2006;23:628–633. doi: 10.1016/j.fm.2005.12.004. [DOI] [PubMed] [Google Scholar]
  27. Magro AEA, Silva LC, Rasera GB, de Castro RJS. Solid-state fermentation as an efficient strategy for the biotransformation of lentils: enhancing their antioxidant and antidiabetic potentials. Bioresour Bioprocess. 2019;6:38. doi: 10.1186/s40643-019-0273-5. [DOI] [Google Scholar]
  28. Naidu MM, Shyamala BN, Naik JP. Chemical composition and antioxidant activity of the husk and endosperm of fenugreek seeds. LWT Food Sci Technol. 2011;44:451–456. doi: 10.1016/j.lwt.2010.08.013. [DOI] [Google Scholar]
  29. Oboh G, Ademiluyi AO, Akindahunsi AA. Change in the polyphenol distribution and antioxidant activity during fermentation of some underutilized legumes. Food Sci Technol Int. 2009;15:41–46. doi: 10.1177/1082013208101022. [DOI] [Google Scholar]
  30. Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Jpn J Nutr Diet. 1986;44:307–315. doi: 10.5264/eiyogakuzashi.44.307. [DOI] [Google Scholar]
  31. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999;269:337–341. doi: 10.1006/abio.1999.4019. [DOI] [PubMed] [Google Scholar]
  32. Sadh PK, Chawla P, Bhandari L, Kaushik R, Duhan JS. In vitro assessment of bio-augmented minerals from peanut oil cakes fermented by Aspergillus oryzae through Caco-2 cells. J Food Sci Technol. 2017;54(11):3640–3649. doi: 10.1007/s13197-017-2825-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Salar RK, Purewal SS, Sandhu KS. Fermented pearl millet (Pennisetum glaucum) with in vitro DNA damage protection activity, bioactive compounds and antioxidant potential. Food Res Int. 2017;100:204–210. doi: 10.1016/j.foodres.2017.08.045. [DOI] [PubMed] [Google Scholar]
  34. Sandhu KS, Punia S. Enhancement of bioactive compounds in barley cultivars by solid substrate fermentation. J Food Measure. 2017;11(3):1355–1361. doi: 10.1007/s11694-017-9513-6. [DOI] [Google Scholar]
  35. Sandhu KS, Punia S, Kaur M. Effect of duration of solid state fermentation by Aspergillus awamorinakazawa on antioxidant properties of wheat cultivars. LWT Food Sci Technol. 2016;71:323–328. doi: 10.1016/j.lwt.2016.04.008. [DOI] [Google Scholar]
  36. Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes. Phytochem. 1989;28:1057–1060. doi: 10.1016/0031-9422(89)80182-7. [DOI] [Google Scholar]
  37. Soetan KO, Oyewole OE. The need for adequate processing to reduce the antinutritional factors in plants used as human foods and animal feeds: a review. Afr J Food Sci. 2009;3:223–232. [Google Scholar]
  38. Türkan F, Atalar MN, Aras A, Gülçin İ, Bursal E. ICP-MS and HPLC analyses, enzyme inhibition and antioxidant potential of Achillea schischkinii Sosn. Bioorg Chem. 2020;94:103333. doi: 10.1016/j.bioorg.2019.103333. [DOI] [PubMed] [Google Scholar]
  39. Vattem DA, Lin YT, Labbe RG, Shetty K. Phenolic antioxidant mobilization in cranberry pomace by solid-state bioprocessing using food grade fungus Lentinusedodes and effect on antimicrobial activity against select food borne pathogens. Innov Food Sci Emerg Technol. 2004;5:81–91. doi: 10.1016/j.ifset.2003.09.002. [DOI] [Google Scholar]
  40. Wani SA, Kumar P. Fenugreek: a review on its nutraceutical properties and utilization in various food products. J Saudi Soc Agric Sci. 2018;17:97–106. doi: 10.1016/j.jssas.2016.01.007. [DOI] [Google Scholar]
  41. Xu BJ, Chang SKC. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. J Food Sci. 2007;72:S159–S166. doi: 10.1111/j.1750-3841.2006.00260.x. [DOI] [PubMed] [Google Scholar]
  42. Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their mutagenicity. J Agric Food Chem. 1995;43:27–32. doi: 10.1021/jf00049a007. [DOI] [Google Scholar]

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