Effects of Solid-State Fermentation with the Ganoderma spp . and Coriolus versicolor on the Total Phenol Contents and Antioxidant Properties on Black Soybean

Aim of this study was to evaluate the infuence of solid-state fermentation of 4 strains (( Ganoderma oregonense (GO), Ganoderma lucidum (GL), Ganoderma sinensis (GS), and Coriolus versicolor (CV))) on the nutrients, functional ingredients, and antioxidant activity in black soybean. As a result, after fermentation by 3 strains of Ganoderma spp . and CV, the contents of crude protein, crude fat, reducing sugar, soluble protein, triterpenoid, and total phenol contents in black soybean signifcantly increased ( P < 0 . 01 ) , particularly in CV-black soybean. Te contents of crude protein, crude fat, reducing sugar, soluble protein, and total phenol were 122.92%, 132.69%, 290.08%, 122.58%, and 142.47% higher than those of the control groups, respectively. But the total sugar and crude fber contents decreased signifcantly ( P < 0 . 01 ) . Furthermore, analysis of antioxidant capacity showed that all the fermented products had scavenging efects on DPPH, hydroxyl, and superoxide anion radicals. In addition, the DPPH scavenging ability of CV fermented products was highest, the hydroxyl radical scavenging ability of GS fermented products was highest, and the superoxide anion radical scavenging ability of GO fermented products was highest, which were 9.43, 7.65, and 3.39 folds higher than those of control groups, respectively. All results evidenced that the fermented strains and the fermented time had remarkable infuences on the nutritional ingredient and antioxidant activity of fermented products in black soybean. Also, there was a signifcant correlation between total phenolic content and antioxidant activities. In conclusion, fermented black soybean with fungus has the potential to be developed into a functional food and nutraceutical material.


Introduction
Ganoderma lucidum is a well-known edible and medicinal mushroom in Japan as Reishi, in China as Lingzhi, and in Europe and the United States as Youngzhi.Te potential of those medicinal mushrooms in treating several diseases has been exploited by traditional folk medicines for centuries.Modern biochemistry and pharmacology studies have shown that it contains many active components, such as nucleosides, furans, sterols, alkaloids, triterpenoids, and polysaccharide [1].Polysaccharide produced by G. lucidum is a type of carcinostatic agent, which has antitumor and hypoglycemic activities [2].In addition, the usage of Ganoderma products is without any toxicity, and there are no apparent side efects, they have no particular efects on a specifc organ in the human body, and they can improve normalization of organ function [3].Terefore, Ganodermabased products have attracted a great deal of attention for years.At present, the Ganoderma-based products could be generally divided into three types: fruit bodies-based, mycelia-based, and spore powder-based products [4].Among these, mycelia-based products are the most promising for industrial production.Compared with the artifcial cultivation of the fruit bodies, the production of Ganoderma products by fermentation technology holds obvious advantages [5,6].
Solid-state fermentation (SSF) is defned as a fermentation process using a nonsoluble material that acts both as a physical support and as a source of nutrients in the absence of free-fowing liquid [7].Many reports have shown that SSF of higher fungi would enhance the nutrition and antioxidant properties of the cereal extracts [8,9].Black soybean acted as a kind of ideal comprehensive nutritional food of legume starches and has become an indispensable part of people's daily life.Te most valuable compounds found were isofavones, anthocyanins, and phenols [10].Tis grain would be a good resource to provide much protein for the human body.However, neither cooking nor popping resulted in an efective utilization in the nutrition of soybean [11].In the present study, we chose soybean acting as substrate to produce Ganoderma mycelia-based production.Te objective of our work was to assay the infuences of SSF with three strains of Ganoderma spp.and Coriolus versicolor on the total phenolic contents and antioxidant properties of soybean.In particular, the relationship between the indicators tested and the fermentation time was analyzed.Tis study would lay a foundation for phenolic contents and antioxidant properties production by fermenting soybean with diferent fungi. 2 H 2 O, and 3% CaCO 3 .Te initial pH of the salt solution was adjusted to 6.5 with 1M NaOH (aqueous solution).Te medium is packed in polypropylene bags (12 cm × 24 cm × 0.05 cm).Te bags of medium were sealed with plastic rings and sterilized for 60 minutes at 121 °C (the moisture content was about 44%).

Preparation of Solid Fermentation
Products.After sterilization, each bag was surface inoculated with mycelia from PDA medium (three discs, 10 mm in diameter) and incubated at 27 °C in the dark.An uninoculated bag served as control.Te fermented grains were harvested at 0, 7, 14, 21, 28, and 35 days after the mycelia extended into the whole medium.Ten, the samples were dried to a constant weight at 45 °C and then ground to pass through a 2 mm sieve prior to determining the nutritional components and antioxidant properties.was mixed with 1 mL of ethanolic solution containing DPPH radical.Te mixture was shaken vigorously and left to stand for 30 min without light.Ten, the antioxidant activity of the mixture prepared using the methods was described by Xu et al. [12], and its absorbance was monitored at 517 nm.Te scavenging activity (R 1 ) was calculated according to the following equation: 2 Journal of Chemistry

Determination of Main
where R 1 is the radical scavenging of peptides, A 0 is the optical density of the control sample, A 1 is the optical density of the extract, and A 2 is the blank control.

Superoxide Anion Radical Scavenging Ability.
Te superoxide anion radical scavenging ability of ethanolic extracts was determined using the nitro blue tetrazolium (NBT) method as described by Xu et al. [12].Te test sample 1 g was combined with 1.0 mL of sodium carbonate (50 mM), 0.4 mL of NBT (24 mM), and 0.2 mL of EDTA (0.1 mM).0.4 mL of hydroxylamine hydrochloride was added to initiate the reaction, and then the reaction mixture was incubated at room temperature for 15 min.Ascorbic acid was used as the reference compound, and its absorbance was monitored at 560 nm.Te scavenging activity was obtained according to equation (1).

Statistical Analysis.
Data are expressed as the mean-± standard deviation.Statistical signifcance between the groups was determined by a paired t test and one-way ANOVA for repeated measures.Te results of P < 0.05 were considered statistically signifcant.Te data were assessed using SPSS (version 15.0, SPSS Inc., Chicago, Illinois).

Phytochemical Composition of Fermented Products.
Table 2 shows the phytochemical composition of fermented products varied with the fermentation fungi.Te crude protein content in the fermented black soybean with GO, GL, CV, and GS strains demonstrated 19.57%, 10.26%, 22.93%, and 12.7% higher than that in unfermented grains, respectively.Te soluble protein of fermented products increased 15.03%, 20.04%, 32.69%, and 5.31% than that in unfermented grains, respectively.Te contents of the triterpenoid fermented product enhanced 38.98%, 37.9%, 37.63%, and 31.45%than control.Te signifcant increased contents (101.53%,122.52%, 290.02%, and 119.08%) were found in the reducing sugar production of fermented product.In addition, the total polyphenol contents in fermented products indicated signifcantly higher (16.69%, 29.07%, 42.47%, and 32.73%) than those in the control, respectively.Furthermore, the crude fat content of fermented products improved, which were up to 20.82%∼ 22.9%, whereas the content of total sugar in fermented products was signifcantly lower than that in unfermented grains.Te dietary fber in unfermented black soybean was 31.7%; after fermentation with 4 strains, it reduced 27.76%, 40.73%, 23.38%, and 35.24%, respectively.
In conclusion, the black soybean was fermented for 35 days at 27 °C with the strains of GO, GL, GS, and CV; the contents of crude protein, soluble protein, reducing sugar, crude fat, triterpene, and total polyphenol were signifcantly higher than those observed in unfermented grains (the control).Meanwhile, the contents of total sugar and dietary fber were signifcantly lower than those of the control group (P < 0.05).

DPPH Radical Scavenging Ability of Fermentation
Products.As shown in Figure 1, after fermented by 4 strains (GO, GL, GS, and CV), it was a signifcant positive correlation between DPPH radical scavenging ability in fermented products and fermentation time, and the correlation coefcient was 0.915, 0.912, 0.983, and 0.946, respectively.After being fermented for 35 days, DPPH radical scavenging ability in black soybean reached the maximum, which was 51.02%, 40.04%, 25.05%, and 37.73%, respectively, and it was 9.43, 7.19, 4.12, and 6.72 folds higher than that of unfermented black beans (control).By reason of the foregoing, the improvement degree of DPPH radical scavenging ability in fermented black beans of four strains was GO > GL > GS > CV.

Superoxide Anion Radical Scavenging Ability of Fermentation Products.
After being fermented by four strains (GO, GL, CV, and GS), a signifcant positive correlation (P < 0.01, r � 0.931, 0.928, 0.963, and 0.914) was observed between superoxide anion radical scavenging ability and fermented time in Figure 2. Te superoxide anion radical scavenging ability of black soybean reached the maximum value (25.98% and 18.84%) after fermented by GO and CV strains for 35 days, which was 3.39 and 2.18 folds higher than that of unfermented black soybean (control), respectively.Meanwhile, after being fermented by GL and GS strains for 30 days, the superoxide anion radical scavenging ability in black soybean reached the maximum value (15.63% and 22.38%), which was 1.64 and 2.78 times higher than that of unfermented black beans (control), respectively.In contrast, the degree of improvement of superoxide anion radical scavenging ability by fermented black soybean of four strains was GO > GS > CV > GL.

Hydroxyl Radicals Scavenging Ability of Fermentation
Products.Variation of the hydroxyl radical scavenging ability of fermentation of black soybean by 4 edible fungi is presented in Figure 3.It evidenced a signifcant positive correlation (0.917, 0.918, 0.794, and 0.870) between the hydroxyl radical scavenging ability of fermented products and fermented time.After being fermented by four strains for 35 days, hydroxyl radical scavenging ability of black Journal of Chemistry soybean reached the maximum value (12.07%, 17.22%, 14.67%, and 36.31%), which was 1.87, 3.1, 2.49, and 7.65 folds higher than that showed in unfermented black beans (control), respectively.On the contrary, the improvement degree of black beans fermented by 4 strains on the hydroxyl radical scavenging ability was GS > GL > CV > GO.

Te Correlation between the Total Phenolic Contents and
Antioxidant Activity.Pearson's correlation coefcient can be used to measure the strength of a linear correlation between two continuous variables, and usually the absolute value of the correlation coefcient is less than 0.40.Moderate correlation was defned as the absolute value of the correlation coefcient between 0.40 and 0.69, and strong correlation is defned as an absolute correlation coefcient greater than 0.70.Te correlation of total phenolic content, with DPPH, hydroxyl, and superoxide anion radical scavenging ability, was shown in Table 3. Te correlation coefcient was higher (r 2 � 0.878 and 0.935, respectively.P < 0.01), with DPPH and superoxide anion fermented with GS than the others, while the correlation coefcient was highest (r 2 � 0.987, P < 0.01) between total phenolic content and hydroxyl radical scavenging ability fermented with GL.Tis means there was a signifcant correlation between total phenolic content and antioxidant activities.It could indicate the major contribution of phenolic compounds to the antioxidant activities of the extracts.

Discussions
After SSF on black soybean for 35 days by 3 strains of Ganoderma spp.(GO, GL, and GS) and CV, the contents of crude protein, crude fat, reducing sugar, soluble protein, triterpenoid, and total phenol signifcantly enhanced (P < 0.01); meanwhile, the total sugar and crude fber content decreased signifcantly (P < 0.01).Te ability of DPPH radical scavenging, superoxide anion radical scavenging, and hydroxyl radical scavenging of fermented products also increased signifcantly (P < 0.01), and there was signifcant positive correlation between the antioxidant capacity and fermentation time.Tose results have been  found in the production of other substances, such as soybeans, buckwheat, and millet [12].Te phenomenon is in accordance with the exhibit on the higher fungus, Agaricus spp.[8,9].Te total sugar and cellulose content of black soybean decreased signifcantly after fermentation by four strains, which were 35.7%-53.2%and 23.45%-40.7%lower than those of the control, respectively.Reducing sugar content increased signifcantly, 2-3.9 folds higher than that of unfermented black soybean.Tose results have shown that the 4 strains in our study had the synthesis ability of reducing sugar.Te strains could decompose macromolecular material, such as starch and cellulose, into small molecules (reducing sugar) of black beans through the amylase, cellulose enzyme in the growth of mycelium of the Ganoderma spp., and CV.Compared with 3 strains of Ganoderma, the total sugar content of fermentation products by CV decreased sharply (53.2%), while reducing sugar content increased obviously (390.1%); it showed that CV had the highest ability to break down macromolecule sugar into the reducing sugar.However, it was not easy to decompose the fber of the black soybeans; therefore, crude fber content was 23.4% lower than that of control after the fermentation.When cereals were fermented by edible fungi, the reducing sugar content in the fermentation products is 2-100 times than that of unfermented cereals, and the reducing sugar content in our study is 2-3.9 folds than that of the control; the phenomenon may be attributed to the diferent starch structures of black soybean and other cereals [8].
Black soybean is rich in protein.After fermentation, the protein content and soluble protein content of black bean increased signifcantly, which were 12.7%∼22.93%and 5.31∼32.69%higher than the control, respectively.CV could make the protein and soluble protein of black bean increase higher than other strains, which were 22.93% and 32.69%, respectively.Tough the increasing rate was not the highest (compared with other cereal fermentation) [8], the protein content of fermented black beans could be up to 45.41%, soluble protein was 15.1% of the total weight, and soluble protein in the proportion of the total protein also increased from 30.81% to 33.25%.Some researchers reported that protein content of the fruit body of CV is 13.2% (wild versicolor) [15], the protein content of mycelium is 43.1% [16], and the protein content of the fruit body of Ganoderma spp. is 8.8%-17%, which were 27%-30% in mycelium [17]; it showed that the crude protein content of the fruit body of CV was higher than that in other strains of Ganoderma, so CV had a strong ability of protein synthesis in the solid-state fermentation process.
Black beans are rich in phytochemicals, such as anthocyanins, phenolic acids, favanols, and saponins, which have good anti-infammatory, antioxidation, and antitumor  Journal of Chemistry activities and neuroprotective efects.Epidemiological and intervention studies have shown that there was positive correlation between regular consumption of soy products (including black beans) and the occurrence of multiple chronic diseases, such as Alzheimer's disease and diabetes mellitus [18][19][20].Te content of triterpenoids in black beans increased 31.45%-38.98%higher than control after fermentation, and there was no signifcant diference in the content of triterpenoids among the 3 strains (GO, GL, and CV) (P < 0.01), which were all above 5.1 g/100 g, but they were higher than that of GS.Te content of total phenol after fermentation also increased signifcantly, and the fermentation efect of CV was the best, which increased 42.47% higher than the control, and the content reached to 330.85 mg/100 g.Our results indicated that the antioxidant properties of fermented black soybeans were stronger than that of the control.Furthermore, the total phenols of fermented cereals improved compared with the control.Mushrooms can produce a variety of secondary metabolites, such as polyphenols and polysaccharides, which have strong antioxidant activity [21,22].Generally, phenol compounds have strong antioxidant activity.Te content of total phenols is one of the standards to evaluate the antioxidant activity of the edible and medicinal plants [23,24].So, the antioxidant properties of the samples should be in accordance with their total phenol contents.In our study, the total phenol contents were enhanced; meanwhile, the antioxidant activity increased after SSF.Tose results showed that there was a positive correlation between polyphenol content and antioxidant activity during SSF in our study.In this study, three methods were used to determine the antioxidant activity.Te results showed that the scavenging ability of fermented black beans on DPPH•, O 2− •, and OH − • was also signifcantly increased (P < 0.01), which was consistent with the results of Xu et al. [7], Chen et al. [8], and Kang et al. [9].In our study, the DPPH scavenging ability of CV products was the highest, which was consistent with the result that the total phenol content of CV products was the highest.Te ability of hydroxyl radical scavenging in GS products was the highest, and the superoxide anion radical scavenging ability of GO products was the highest during fermentation between 28 and 35 days.It showed that solidstate fermentation could improve the antioxidant performance of the substrate, and the activity changed with the fermentation time and strains.
Te phenolic compounds act as antioxidants in plants [25].Lee et al. [26] found that the contents of total phenols were moderately to highly associated with antioxidant properties.Teoretically, the antioxidant properties of the samples should be in accordance with their total phenols [9].In our study, the correlations between antioxidant activities (measured by DPPH, hydroxyl, and superoxide anion assays) and total phenolics contents were analyzed by means of Pearson's correlation test.Results showed that the antioxidant activity has high correlation with the contents of total phenolics.Xu et al. [7] also suggested the highest correlation between total phenolic content and antioxidant activity, such as DPPH, hydroxyl, and superoxide anion.Our results agreed with those.Phenolic compounds are considered to be the most important antioxidants of plant materials.Tey constitute one of the major groups of compounds acting as a primary antioxidant or free radical terminators.Te antioxidant activity of phenolic compounds is based on their ability to donate hydrogen atoms to free radicals [27].In addition, they possess ideal structural properties for free radical scavenging properties [28].On the contrary, diferent reports are found from the literature, whereby some authors suggested the phenolic compounds did not correlate with the antioxidant activity [8].Tey believed that the type and quantity of phenolic compounds and the presence of nonphenolic antioxidants may also contribute to the antioxidant activity of the extracts.Nonphenolic antioxidants in fermented products with fungi, such as vitamin C, vitamin E, and beta-carotene might also be accountable in enhancing the antioxidant activity.
Soybean products play an important role in people's diets because they are high in protein, dietary fber, and other micronutrients and low in fat.Due to their relatively low price and high protein quality, beans are an important plant protein substitute for animal protein in many developing countries [21].Compared with other beans, black bean has the highest content of phenolic compounds and antioxidant activity [22], which has attracted extensive attention.In the fermentation process, microbes can use the protein and nonprotein nitrogen, glycogen of substrate compound, a large number of polysaccharides, and protein of edible fungus.At the same time, the exocrine, such as peptides, enzymes, proteins, and sugars, could increase the nutritional component contents in the fermented products.After fermentation,the lipid, protein, and carbohydrate retained, the vitamins increased, but the contents of cellulose and phytic acid decreased.Fermentation on grains could degrade nutrients into small molecules which could be easily eliminated by people, and the taste of the coarse grain was improved [23,24].
At present, the solid fermentation technology of Ganoderma has been applied to the development of functional food in China, such as Ganoderma lucidum polysaccharide oral liquid [29], nutrition and health care fermentation tea [30,31], Ganoderma lucidum sauce [32], and Ganoderma lucidum dietary fber convenient food [33].In those experiments, Ganoderma spp.and CV were used as the original strain, and black beans were used as the substrate for fermentation culture, and the nutrient composition and antioxidative activity of black beans were improved through the fermentation of Ganoderma lucidum mycelia.Te fermentation products can be used as raw materials of functional food after drying and crushing.

Conclusion
Tis study was the frst to report on the SSF of black soyabean by Ganoderma spp.and CV.Te following conclusions can be drawn from this study: (1) After solid-state fermentation by 3 strains of Ganoderma and CV, the contents of crude protein, crude fat, reducing sugar, soluble protein, triterpenoid, and total phenol in black beans increased signifcantly (P < 0.01), 6 Journal of Chemistry while total sugar and crude fber content decreased significantly (P < 0.01).( 2) Te fermentation efect of CV was the best; the contents of crude protein, crude fat, reducing sugar, soluble protein, and total phenol in black beans products all increased with highest rate except triterpenoid, which were 22.92%, 32.69%, 290.08%, 22.58%, and 42.47% higher than that of the control.(3) Te fermentation time of black beans had remarkable infuences on the antioxidant activity of fermented product.(4) Solid-state fermentation can improve the antioxidant capacity of black bean, the DPPH scavenging ability of CV fermentation products of black bean was the highest, the hydroxyl radical scavenging ability of GS fermentation products was the highest, and the superoxide anion radical scavenging ability of GO fermentation products was the highest, which was 9.43 times, 7.65 times, and 3.39 times higher than that of the control, respectively.( 4) Tere was a signifcant correlation between total phenolic content and antioxidant activities.

Figure 1 :Figure 2 :
Figure 1: DPPH radical scavenging ability of fermentation products.Data were expressed as means ± SD (n � 3).Results marked with the superscript lowercase letter within each group difer, P < 0.05.

Table 1 )
were obtained from Shanxi Agricultural University, China, and cultured on potato agar dextrose (PDA) at 25 °C.

Table 1 :
Strain number of Ganoderma spp.and Coriolus versicolor.

Table 2 :
Phytochemical composition of fermented product.Note.Results marked with the superscript lowercase letter within each group difer, P < 0.05; results marked with the superscript capital letter within each group difer signifcantly, P < 0.01.

Table 3 :
Correlation between total phenolic contents and antioxidant activity in the investigated extracts.