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4. Soybean fermentation using mushroom mycelia
The legume soybean is highly proteinaceous (36% protein in dried beans), rich in major nutrients essential for human nutrition and can potentially be a good replace- ment for animal-derived proteins [38–41]. It can be used both in fermented and
Figure 4.
Activation of NGF synthesis with wild H. erinaceum and H. ramosum mycelia [9]. NGF levels in various parts of the brain were measured after 14 days of repeated oral administration of H. erinaceum and H. ramosum mycelia (300 mg/kg). 1, Cortex; 2, striatum; and 3, hippocampus. Data are expressed as the mean ± SEM.
*p < 0.05, compared with vehicle (Student’s test).
non-fermented forms [42]. While soybeans are rich in flavonoid groups such as genis- tein, daidzein, and glycitein isoflavones that have tremendous health benefits [43], they are not easily absorbed and incorporated in their natural glycosylated forms unless hydrolyzed by the microflora of the intestine through their beta-glucosidase production [44]. Isoflavones have health benefits against several diseases and hormone-related issues [45–48]. The easily absorbable form of flavones is the aglycon form, which is abundant in fermented sources of soybean, such as tempeh, miso, and natto [49].
Mushroom mycelia can be used as a source of beta glucosidase to convert iso- flavone glycosides to their aglycon form. For example, G. lusidum, belonging to the basidiomycetes group, has been shown to increase serum concentration of the aglycon form of isoflavones in soybeans [50].
Studies from our laboratory investigated the health effects of fermentation using mushrooms, such as G. lucidum, H. erinaceum, and H. ramosum [10]. We measured DPPH scavenging activity, total phenolic content, antioxidant activity, alpha glu- cosidase inhibition, and isoflavone concentration, few major health parameters of paramount importance, in soybeans fermented with different mushroom types and compared them with non-fermented soybeans.
Soybean fermentation was carried out as described in Suruga et al. [10]. We found that G. lucidum was more effective in quickly fermenting soybeans compared to the other two mushroom types (Figure 6).
4.1 Antioxidant activity of fermented soybean 4.1.1 Methods
The DPPH radical scavenging activity and total phenolic content of fermented soybeans were analyzed using the methods described in Subsections 2.3.1 and 2.4.1.
Oxygen radical absorbance capacity (ORAC) was determined using the OxiSelect™
ORAC Activity Assay Kit (Cell Biolabs Inc., San Diego, CA, USA) [51]. The assay was performed as described in Suruga et al. [10]. Briefly, fluorescence activity of the reac- tion mixture with antioxidant and fluorescein solution was measured after adding the free radical initiator. Increasing Trolox concentrations were used for the standard
Figure 5.
Effect of varying concentrations of wild H. ramosum mycelia on NGF synthesis in different parts of mouse brain [9]. 1, Cortex; 2, striatum; and 3, hippocampus. Data are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01, compared with vehicle (Student’s test).
curve, and extracts were quantified and expressed as μmol Trolox equivalents/g of dry fermented soybean powder.
4.1.2 Total phenolic content and antioxidant activity of fermented soybean by mushroom mycelia
Total phenolic content was higher in all the fermented extracts compared to non- fermented control soybeans. Both DPPH radical scavenging activity and antioxidant activity were higher in fermented soybeans than in non-fermented ones (Table 1).
4.2 Alpha-glucosidase inhibitory activity of soybeans fermented with mushroom mycelia
4.2.1 Methods
Yeast alpha-glucosidase inhibitory activity was measured using methods reported before [52] with modifications as described in Suruga et al. [10]. Briefly, yeast
alpha-glucosidase was incubated with fermented soybean extract solutions and then p-nitrophenyl α-D-glucopyranoside (pNP-glucoside) was added and absorbance was determined at 400 nm. For the mammalian reaction, alpha glucosidase from rat
Figure 6.
G. lucidum was faster in fermenting soybeans compared to other types (a) Control (non-fermented soybeans); (b) G. lucidum; (c) H. erinaceum; (d) H. ramosum [10].
intestinal acetone powder [53] was incubated with fermented soybean extract and the amount of glucose released was measured. We also used maltose as the substrate and calculated the % inhibition rate of alpha glucosidase [54].
4.2.2 Fermented soybean showed higher inhibition of alpha-glucosidase activity compared to non-fermented ones
Comparison of control (non-fermented soybeans) to soybeans fermented with mush- room mycelia showed that significant alpha-glucosidase inhibition could be achieved in the fermented soybeans using both pNP-glucoside and maltose (Figure 7A and B). Yeast alpha-glucosidase inhibition was the highest with H. ramosum compared to G. lucidum and H. erinaceum, while the mammalian alpha-glucosidase inhibition was significantly higher with G. lucidum fermentation (Figure 7A-C).
4.3 Comparison of isoflavone concentrations in soybeans fermented with mycelia versus non-fermented soybeans
4.3.1 Methods: high-performance liquid chromatography (HPLC) analysis
We followed the methods described in Kudou et al. [55] for measuring isofla- vone concentrations in fermented and non-fermented soybeans. An LC-6A system (Shimadzu, Kyoto, Japan) equipped with a PEGASIL-ODS (4.6 mm i.d. × 250 mm) HPLC column (Senshu Scientific, Tokyo, Japan) was used for analysis. HPLC param- eters used for the measurement of different isoflavones, such as genistein, daidzein, glycitein, daidzin, and glycitin, both in the glycosylated and in aglycone forms, are detailed in Suruga et al. [10].
4.3.2 Methods: liquid chromatography/mass spectrometry (LC/MS) analysis
An ACQUITY UPLC apparatus (Waters MS Technologies, Manchester, UK) equipped with a reversed-phase Acquity UPLC CHS C18 column with a particle size of 2.1 mm × 100 mm × 1.7 μm (Waters MS Technologies) was used for LC/MS analysis. Parameters of analysis are documented in detail in Suruga et al. [10].
Control G. lucidum H. erinaceum H. ramosum
Total phenolic content (mg/g dry powder)
1.547 ± 0.068 2.304 ± 0.035 2.074 ± 0.066 2.160 ± 0.014
DPPH radical scavenging activity (μmol Trolox/g dry powder)
1.847 ± 0.073 4.246 ± 0.010 2.246 ± 0.061 2.367 ± 0.173
ORAC (μmol Trolox/g dry powder)
49.763 ± 2.856 60.090 ± 1.506 66.147 ± 1.898 72.897 ± 2.113
Table 1.
Total phenolic content and antioxidant activity of soybeans fermented by mushroom mycelia [10].
4.3.3 Fermentation with mushrooms decreased the concentrations of glycosylated forms of isoflavones and increased the concentrations of aglycon forms
LC/MS profile was shown in Figure 8. The concentration of glycosylated forms of isoflavones, such as daidzin, glycitin, and genistin was about 95.6% in non-fermented soybeans, while it was reduced to 52.5, 15.8, and 17.6% in soybeans fermented by the G. lucidum, H. erinaceum, and H. ramosum mycelia, respectively. The aglycone forms of these isoflavones, on the other hand, increased from 4.4% in the non-fermented controls to 47.5, 84.2, and 82.4% in soybeans fermented by G. lucidum, H. erinaceum, and H. ramosum mycelia, respectively. LC/MS profile shown in Figure 8 corroborate these results. Based on the retention time and MS data, molecular formula and iden- tity of compounds corresponding to 11 of the 12 peaks have been predicted: peak #1, daidzin; peak #2, glycitin; peak #3, 8-hydroxydaidzein; peak #4, genistin; peak #5,
Figure 7.
Inhibition of alpha-glucosidase activity soybeans fermented by mushroom mycelia. (A) Yeast alpha-glucosidase inhibition using pNP-glucoside as substrate, (B) mammalian alpha-glucosidase inhibition using maltose as substrate, and (C) mammalian alpha-glucosidase inhibition using sucrose as substrate. Results are expressed as mean ± SD (n = 3). N.D.: not detectable. 1: p < 0.01 vs. control, 2: p < 0.01 vs. G. lucidum, 3: p < 0.01 vs.
H. erinaceum, 4: p < 0.01 vs. H. ramosum, and 5: p < 0.05 vs. H. ramosum [10].
6″-O-malonyldaidzin; peak #7, 6″-O-acetyldaidzin; peak #8, 6″-O-malonylgenistin;
peak #9, daidzein; peak #10, glycitein; peak #11, 6″-O-acetylgenistin; and peak #12, genistein, respectively.