Improvement of Physiological Active Substance of Wheat Dried Distillers ' Grains with Solubles Fermented by Preussia aemulans under Optimum Fermentation Conditions

In this study, a new fungus named Preussia aemulans was isolate from Cordyceps sinensis fruiting body. The agricultural waste of wheat dried distillers' grains with solubles (DDGS) was utilized as a culture medium for Preussia aemulans. By using orthogonal experiment, the fermentation conditions of polysaccharide, polyphenol and adenosine were optimized. Under the optimum fermentation conditions of polysaccharide, polyphenol and adenosine, the content of polysaccharide, polyphenol, adenosine and protein were 32.68 ± 3.24 mg/g dry matter, 7.91 ± 0.2 mg/g dry matter, 1.36 ± 0.24 μmol/g dry matter and 104.41 ± 6.65 mg/g dry matter, respectively. Based on the nutritional profile, the fermented wheat DDGS could be utilized as functional material of feed or food.


Introduction
Cordyceps, one of the well-known traditional Chinese medicines is a large family which contains more than 350 types of fungi, such as Cordyceps militaris, Cordyceps ophioglossoides, Cordyceps pseudomilitaris, etc. (Russell, & Paterson, 2008).Cordyceps sinensis (Berk.)Sacc. is a parasitic fungus and has long been used to treat multitude of ailments, promote longevity, increase athletic power and improve quality of life.The physiological biological activities of cordycepin were reported, such as inhibition of DNA and RNA synthesis, antitumor activity and enhancement of cell differentiation, etc. (Sun, Ling, Lu, & Zhang, 2003).
Wheat dried distillers' grains with solubles (DDGS); the by-product of ethanol factory has increased in recent years.In Japan, 0.8 million tons of wheat DDGS is disposed every year, and most of wheat DDGS was dumped into ocean or burned.However, according to the London Convention, the ocean disposal was prohibited since 1996.Because of the high content of protein, fiber and reduced starch (Cozannet et al., 2010;Widyaratne & Zijlstra, 2007), the wheat DDGS has been primarily utilized in ruminant diets (Greter, Penner, Davis, & Oba, 2008;Penner, Yu, & Christensen, 2009).Furthermore, based on the nutritional profile, it was considered that the wheat DDGS could be used as a culture medium to culture microbial.
In this study, the wheat DDGS was used as a culture medium for Preussia aemulans (P.aemulans) which was isolated from Cordyceps sinensis fruiting body.The optimum fermentation conditions of polysaccharide, polyphenol and adenosine were investigated, and the nutritional value was detected.

Isolation and Cultivation of P. aemulans
The fruiting body of Cordyceps sinensis was purchased from Qinhai, China, and the isolated P. aemulans mycelium (SIID11759-01) was identified by TechnoSuruga laboratory co., ltd, Japan.The stroma of Cordyceps sinensis fruiting body was sterilized with ethanol three times, air-dried, cut into small segment and transferred to slant tube fermentor to incubate for 7 days, at room temperature.
The white mycelium appeared on the surface during slant fermentation.Then, mycelium was transferred to agar medium, which contained (per liter): 20 g of surcose, 10 g of peptone, 20 g of agar powder, 1.5 g of MgSO 4 , 3 g of KH 2 PO 4 .After 7 days of the culture, when white mycelium appeared on the surface of the medium, the mycelium was transferred into the liquid medium, which was containing (per liter): 20 g of surcose, 10 g of peptone, 4 g of potato powder, 1.5 g of MgSO 4 , 3 g of KH 2 PO 4 .The Cordyceps sinensis mycelium was incubated in a 200 mL of flask with 100 mL of PDA liquid medium, and the mixture was stationary cultured for 7 days.

Orthogonal Experiment
The wheat DDGS was obtained from Kyushu, Japan.The carbon nitrogen ratio and pH of wheat DDGS were 11.79 and 4.03 ± 0.02 respectively.The culture medium was contained 5 g of wheat DDGS and 100 mL of distilled water.The adding dosage of sucrose, pH, the fermentation time and the shaking condition were regarded as correlated factors of the fermentation conditions.And the inoculum size of P. aemulans was 5% (v/v).The optimum fermentation conditions of polysaccharide, polyphenol and adenosine were obtained by an orthogonal layout L 9 (3 4 ).The levels of the factors were shown in Table 1.After the fermentation, the yield of mycelium was different among the nine experiments, the brown mixture of fermented wheat DDGS and the mycelium was dried (40 ) and grounded to powder for the furthe ℃ r analysis.

Determination of the Polysaccharide Content
The polysaccharide content was measured by modified phenol-sulfuric acid method according to Masuko, et al., (2005).The fermented wheat DDGS powder was extracted by boiling water for 2 hours.After filtration, the supernatant was precipitate by 87.5 % of ethanol at 4 for 12 hours.Then, the supernatant was centrifuged at ℃ 9500 rpm for 10 min.The precipitate was washed twice by 99.5% of ethanol and dried at room temperature to remove residual ethanol.Then, the precipitate was dissolved in distilled water and used for polysaccharide analysis.The color reaction was initiated by mixing 1 mL of crude polysaccharide solution with 0.5 mL of phenol solution and 2.5 mL of concentrated sulfuric acid, and the reaction mixture was kept in a 100 water ℃ bath for 15 min.After cooling its temperature to room temperature, the optical density (OD) of the mixture was determined at 490 nm and the crude exopolysaccharide content was calculated with D-glucose as the standard.

Determination of Polyphenol Content
The polyphenol content of the fermented wheat DDGS powder was estimated according to Folin-Ciocalteu, colorimetric method with some modifications (Mau, Lin, & Song, 2002).The fermented wheat DDGS powder was extracted by methanol for 24 hours in a shaking incubator at ambient temperature.After filtration, the supernatant (0.5 mL) was mixed with 0.5 mL of the Folin-Ciocalteu reagent.Three minutes later, 0.5 mL of 20% Na 2 CO 3 was added, and the mixture was made up to 5 mL with distilled water.After being kept it in dark for 90 min, the OD of the mixture was measured at 725 nm.The polyphenol content was calculated with gallic acid as the standard, and expressed as milligram gallic acid equivalent (mg GAE/g extract).

Determination of Adenosine and Cordcepin Contents
According to Ikeda, Nishimura, Sun, Wada and Nakashima (2008), the adenosine and cordycepin content were simultaneously determined in this experiment with some modifications.The fermented wheat DDGS powder was extracted with deionized water (1/10 W/V) by using ultrasonic-assisted extract method for 1 h (50 W) at ambient temperature.Then, the supernatant was collected and filtered by filter (0.45 μm, whatman) for HPLC determination.The samples were analyzed by the HPLC with Capcell-Pak C 18 column (4.6 mm I.D. × 150 mm, particle size of 5 μm) in a flow rate of 1.0 mL/min, the column temperature was set at 30 and the UV detection ℃ was operated at 260 nm.The mobile phase was a mixture of acetonitrile and water (5:95, v/v).

Determination of Protein Content
The protein content was determined by the Protein Quantification Kit-Rapid (Shi, Yang, Li, Wang, & Zhang, 2011).Briefly, 200 mg of the fermented wheat DDGS powder was mixed with 4 mL of phosphate buffer (pH: 7.6) and kept homogenate by pulp refiner for 2 min.After 10 min, 6 μL of samples (10 mg/mL) and 300 μL of Coomassie Brilliant Blue (CBB) were added into a 96-well plate separately, then the OD of the mixture was measured at 595 nm and the protein content was calculated by a Bovine serum albumin (BSA) solution as the standard.

Determination of Free Amino Acid
The method of determining free amino acid was according to Shi, Yang, Guan, Wang and Zhang (2012) with some modifications.The fermented wheat DDGS powder was extracted by 80 % ethanol in 80 water bath for ℃ 20 min.The supernatant was collected.After repeated the previous steps twice, the sediment was washed by 80 % ethanol.Then all of the collected supernatant was centrifuged and filtered.The supernatant was evaporated to dryness and dissolved with distilled water, then keeping at 4℃ for 12 h.The solution was mixed with TCA solution at the ratio of 4:1 and placing at 4℃ for 10 min.The pH was adjusted with NaOH and HCl at the range of 2-3.Finally the supernatant was filtered by 0.45 μm filter and assessed by an auto amino acid analyzer (JLC -500/V2, Jeol Ltd., Tokyo, Japan) in Chemical Analysis Center (university of Tsukuba).

Statistical Analysis
The obtained data were analyzed by student's t-test, and results were expressed as mean ± SD.Statistic difference was considered to be significant at p<0.01 (**).

The yield of polysaccharide
The polysaccharide yield of the fermented wheat DDGS was shown in Table 2.The highest yield of polysaccharide in the orthogonal experiment was 31.45 ± 2.46 mg/g dry matter.The optimum Levels of factors were 3 % adding dosage of the surcose (ADS 3), 10 days of the fermentation time (FT 1), 8 of pH (PV 3), 50 round/min of shaking condition (SC 2), respectively.The R value of various factors indicated that the adding dosage of the surcose (3 %) was the highest among these factors.And the significant levels were shown in Table 3, the results were indicated that all of the factors significantly related with the yield of polysaccharide.Further, the ADS 3, FT 1, PV 3 and SC 2 conditions were demonstrated, the mean polysaccharide content of the fermented wheat DDGS was reached to 32.68 ± 3.24 mg/g dry matter.Compared with the polysaccharide content of the unfermented wheat DDGS (2.23 ± 0.15 mg/g dry matter), the polysaccharide content of optimum fermentation conditions (PSOFC) was increased 14 folds during the fermentation by P. aemulans.Note 1: ADS, Adding dosage of sucrose; FT, Fermentation time; SC, Shaking condition, respectively.Mean values were mean of three determinations with standard deviation (±).I j , II j , III j , were the polysaccharide contents of level 1, level 2 and level 3, respectively; R means the maximum of I j , II j and III j minus the minimum of I j , II j and III j , respectively.

The yield of Polyphenol
As shown in Table 4, the highest mean yield of polyphenol was 8.81 ± 0.46 mg/g dry matter, and the optimum Levels of factors were 3 % adding dosage of the surcose (ADS 3), 15 days of the fermentation time (FT 2), 6 of pH (PV 1), 50 round/min of shaking condition (SC 2), respectively.The R value of various factors indicated that the fermentation time (15 days) was the highest among these factors.And the significant levels were shown in Table 5, the results were indicated that the polyphenol content of the fermented wheat DDGS was significantly related to the pH, shaking condition and the fermentation time.The adding dosage of surcose exhibited little influence on the polyphenol content.Further, the ADS 3, FT 2, PV 1 and SC 2 conditions were demonstrated, the mean polyphenol content of the fermented wheat DDGS was reached to 9.46 ± 0.34 mg/g dry matter.
Compared with the polyphenol content of unfermented wheat DDGS (1.71 ± 0.06 mg/g dry matter), the polyphenol content of optimum fermentation conditions (PPOFC) was increased 6 folds by P. aemulans.Mean values were mean of three determinations with standard deviation (±).I j , II j , III j , were the polyphenol contents of level 1, level 2 and level 3, respectively; R means the maximum of I j , II j and III j minus the minimum of I j , II j and III j , respectively.

The Yield of Adenosine
The adenosine yield of the fermented wheat DDGS was shown in Table 6.The highest mean yield of adenosine was 1.89 ± 0.11 μmol/g dry matter.The optimum Levels of factors were ADS 1, FT 2, PV 2 and SC 2, they were named 1 % adding dosage of the surcose, 15 days of the fermentation time, 7 of pH, 50 round/min of shaking condition, respectively.The R value of various factors indicated that the fermentation time (15 days) was the highest among these factors.And the significant levels were shown in Table 7, the results were indicated that the adenosine content of the fermented wheat DDGS was significantly related to the fermentation time, shaking condition and pH.The adding dosage of surcose exhibited little influence on the adenosine content.Further, the ADS 1, FT 2, PV 2 and SC 2 conditions were demonstrated, the mean adenosine content of the fermented wheat DDGS was reached to 1.93 ± 0.21 μmol/g dry matter.Compared with the adenosine content of unfermented wheat DDGS (0.18 ± 0.02 μmol/g dry matter), the adenosine content was increased 10 folds by P. aemulans, under the optimum fermentation conditions of adenosine (ADOFC).Note 5: ADS, Adding dosage of sucrose; FT, Fermentation time; SC, Shaking condition, respectively.Mean values were mean of three determinations with standard deviation (±).I j , II j , III j , were the adenosine contents of level 1, level 2 and level 3, respectively; R means the maximum of I j , II j and III j minus the minimum of I j , II j and III j , respectively.

The Optimum Fermentation Condition of Polysaccharide, Polyphenol and Adenosine (PPAOFC)
According to the results of orthogonal experiment, the optimum fermentation conditions of polysaccharide, polyphenol and adenosine were different.Therefore, it was necessary to discuss the optimum fermentation condition of polysaccharide, polyphenol and adenosine (PPAOFC).The polysaccharide contents of PSOFC (32.68 ± 3.24 mg/g dry matter), PPOFC (9.76 ± 0.68 mg/g dry matter) and ADOFC (5.68 ± 0.8 mg/g dry matter) were shown in Figure 1 A, by using Duncan's multiple range test, the polysaccharide content of PSOFC, was significantly higher than that of PPOFC and ADOFC.Further, polyphenol and adenosine contents of PSOFC, PPOFC and ADOFC were compared, the significant levels and contents were shown in Figure 1 B and C. The polyphenol contents of PSOFC, PPOFC and ADOFC were 7.91 ± 0.2, 9.46 ± 0.34 and 7.99 ± 0.58 mg/g dry matter, respectively.The adenosine contents of PSOFC, PPOFC and ADOFC were 1.36 ± 0.24, 0.81 ± 0.11 and 1.94 ± .021μmol/gdry matter, respectively.As the results, PSOFC was the optimum fermentation condition of polysaccharide, polyphenol and adenosine for producing polysaccharide, polyphenol and adenosine.

The Changes of Protein and Total Free Amino Acid Contents of Fermented Wheat DDGS under PPAOFC
As shown in Figure 2, the protein content of the fermented wheat DDGS on PPAOFC and unfermented wheat DDGS, were 104.41 ± 6.65 and 67.86 ± 5.42 mg/g dry matter, respectively.And the protein content was increased 1.5 folds under PPAOFC by P. aemulans.The total free amino acid content was shown in Figure 3.After the fermentation, the total amino acid was decreased 64% to 98.92 ± 10.63 μmol/g dry matter.However, the total free amino acid content of unfermented wheat DDGS was 274.78 ± 19.56 μmol/g dry matter, which was much higher than that of fermented wheat DDGS and other functional materials, such as the unfermented and fermented soybean residue (10.93 ± 0.27 and 225.13 ± 18.41 μmol/g dry matter) by Lentinus edodes (Shi et al., 2012).Based on the protein content of fermented wheat DDGS, it was considered that the decrease of free amino acid of unfermented wheat DDGS was utilized by P. aemulans to converted to protein.

The Cordycepin Content of Fermented Wheat DDGS under PPAOFC
According to previous studies, the cordycepin content that contained in cordyceps sinensis was lower than that of other cordyceps fungi.And the content of natural cordyceps sinensis fruiting body was much higher than that of culture mycelium (Li et al., 2004;Sun, Ling, Lu, Zhang, & Zhang, 2003).In this study, cordycepin has not been detected under PPAOFC.The optimum fermentation condition of cordycepin content by P. aemulans should be explored in the future.

Conclusions
The optimum fermentation condition of polysaccharide, polyphenol and adenosine (PPAOFC) was determined in this study, the fermentation condition were 3 % adding dosage of the surcose, 10 days of the fermentation time, 8 of pH, 50 round/min of shaking condition, respectively.In the PPAOFC, the polysaccharide, polyphenol, adenosine content were increased 14, 6 and 10 folds comparing with the unfermented condition, and the content were 32.68 ± 3.24 mg/g dry matter, 7.91 ± 0.2 mg/g dry matter and 1.36 ± 0.24 μmol/g dry matter, respectively.After the fermentation, the protein content was increased 1.5 folds, and reached to 104.41 ± 6.65 mg/g dry matter.
Therefore, based on the nutrient substance, the fermented wheat DDGS could be utilized as animal feed and food additives in further.

Figure 2 .
Figure 2. The protein contents of unfermented (UFC) and the optimum fermentation condition of polysaccharide, polyphenol and adenosine (PPAOFC) Unfermented wheat DDGS was control.Data were expressed as means ± S.D. (n=3) ( ** p < 0.01 in comparison with the control).

Figure 3 .
Figure 3.The total free amino acid contents of unfermented (UFC) and the optimum fermentation condition of polysaccharide, polyphenol and adenosine (PPAOFC) Unfermented wheat DDGS was the control.Data were expressed as means ± S.D. (n=3) ( ** p < 0.01 in comparison with the control).

Table 7 .
The variance analysis of L 9 (3 4 ) orthogonal test of adenosine content