Optimization of Solid-State Fermentation Conditions of Astragali Radix Residues and Changes of Its Nine Constituents Content Before and After Fermentation

Background: For the last few years huge quantity of herb residues are discharged in China, and so far there is not a good solution. The current methods of disposal and utilization of herb residues (stacking, incineration, and landll) were not only great harmful to the ecological environment, but also cause the waste of medicinal herb resources. The fermentation can further decompose and transform the active ingredients and nutrients in herb residue, so the fermentation technology has good application prospect in the treatment of herb residues. In this study, we investigated the applicability of fermentation processes with the Aspergillus niger (ACCC 30583) strain for the reuse of Astragali Radix residues (ARR) by comparing the content changes of its nine constituents before and after fermentation. The contents of total protein and crude fat in the ARR were determined via near-infrared scanning; the iron, copper, zinc, and manganese contents were assayed with atomic absorption spectroscopy, the calycosin-7-glucoside and astragaloside (cid:0) content were determined by HPLC, and the polysaccharide content was determined via phenol sulfuric acid spectrophotometry. Results: The optimum fermentation conditions for ARR with ACCC 30583 was culture medium content 60%, fermentation time 5 days, and fermentation temperature 28 ℃ . Compared with the residues before fermentation, the contents of total protein, calycosin-7-glucoside, astragaloside (cid:0) , Astragali Radix polysaccharides (ARP), and manganese increased signicantly, the iron content decreased signicantly, and the crude fat, zinc, and copper contents exhibited no signicant changes. Conclusions: The solid fermentation of ARR with the Aspergillus niger (ACCC 30583) strain effectively promoted the separation of astragaloside (cid:0) and ARP from ARR, which provided methodological basis for the effective reuse of herb residues.

Effect of cellulase culture medium amount on Aspergillus niger-mediated fermentation Six portions of ARR were placed in different triangular asks, and different amounts of cellulase culture solution produced by Aspergillus niger (30583) were added. The contents of cellulase culture medium were 30%, 40%, 50%, 60%, 70%, and 80%. The mixtures were fermented in a constant temperature incubator at 28 ℃ for 5 d. All experiments were conducted in triplicate. Astragaloside IV precipitation was measured after fermentation.

Effect of temperature on fermentation
The six portions of ARR were mixed with cellulase culture solution produced by Aspergillus niger (30583) in a ratio of 1 : 1, and then those mixtures were respectively fermented at 24, 26, 28, 30 and 32 ℃ for 5 d.
All experiments were conducted in triplicate. Astragaloside IV precipitation was measured after fermentation.

Effect of time on fermentation
Six portions of ARR were placed in different triangular asks, and different amounts of cellulase culture solution produced by Aspergillus niger (30583) were added. The mixtures containing 50% culture medium were prepared and fermented for 3 4 5 6 7 8 d at 28 ℃. All experiments were conducted in triplicate. Astragaloside IV precipitation was measured after fermentation.

Orthogonal experiment
According to single-factor analyses of the effect of culture medium content, fermentation time, or temperature on the precipitation of astragaloside IV during the solid fermentation of ARR, the experimental culture medium contents were selected to be 50%, 60%, and 70%, the fermentation times were 4, 5, and 6 d, and the fermentation temperatures were 26, 28, and 30 ℃. A three-factor and threelevel L9 (3 3 ) orthogonal experiment was then conducted to determine the best conditions for solid fermentation of ARR.
Determination of nine components in ARR before and after fermentation Astragaloside IV content determination via HPLC HPLC was performed with the following chromatographic conditions (C18 column): mobile phase, acetonitrile : water = 30 : 70; ow rate = 1 mL min -1 ; detection wavelength, 210 nm; column temperature, 30 °C; injection volume, 10 μL. Here, we investigated whether the linear relationship met the quantitative requirements and also if the experimental evaluation parameters of precision, stability, and recovery were reliable.
Preparation of reference and test solutions Calycosin-7-glucoside was accurately weighed and mixed with methanol to obtain a standard solution (500 g mL -1 ). Samples were then obtained from the Aspergillus niger culture medium and ARR mixtures, after which they were centrifuged at 4000 r min -1 for 10 min. The supernatant was recovered and concentrated to 10 mL with a rotary evaporator. The extraction procedure was carried out four times with saturated n-butanol (40 mL per extraction). The n-butanol solution was then mixed and thoroughly washed with ammonia water twice (40 mL per wash). Afterward, the ammonia solution was discarded, the n-butanol solution was allowed to evaporate, and the resulting residue was mixed with 5 mL of deionized water. D101 type macroporous adsorption resin columns (inner diameter 1.5 cm, column height 12 cm) were used to elute the samples with 50 mL of water. The eluent was then discarded and eluted with 70% ethanol. This second eluent was collected, evaporated, and dried, and the residue was dissolved with methanol, with a constant methanol capacity of 10 mL.
Calycosin-7-glucoside content determination via HPLC HPLC was performed with a C18 chromatographic column; gradient elution was conducted with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B. The elution conditions were: 0-20 min from 20% A to 40% A; 20-30 min 40% A; 30-40 min from 40% A to 20% A; ow rate, 1.0 mL min -1 ; detection wavelength, 260 nm; column temperature, 30°C; injection volume, 10 μL. As described above, we investigated whether the linear relationship met the quantitative requirements and also if the experimental evaluation parameters of precision, stability, and recovery were reliable.
Reference and test solution preparation: Calycosin-7-glucoside was accurately weighed and mixed with methanol to make a standard solution (500 g mL -1 ). A 1 g sample of ARR was accurately weighed before and after fermentation and placed in an appropriately labeled round-bottom ask, after which 50 mL of methanol were added; the mixture was weighed once again. After re ux heating for 4 h and cooling to room temperature, the weight was measured. Methanol was used to compensate for lost weight, after which the mixture was thoroughly shaken and ltered. The ltrate was discarded and evaporated in a water bath. The residue was dissolved in methanol and transferred to a 5 mL volumetric bottle; the volume was adjusted with methanol. The resulting preparation was used as a test solution for all downstream experiments.
Determination of AstragaliRadix polysaccharide content via the phenol-sulfuric acid method An anhydrous glucose reference was accurately weighed and mixed with ultrapure water to make a 1 mg mL -1 glucose reference solution; a standard curve was then constructed. ARR powder samples (5 g) were taken before and after fermentation. These samples were then mixed with pure water (50 mL), heated to boil for 1 h, and ltered. The residue was extracted twice again as above, and then all the ltrates were combined and dried at 100 ℃ water bath. The samples were then precipitated with 60% and 80% ethanol for 5 h in sequence and centrifuged at 4000 r min -1 for 5 min. The deposit was diluted to 50 mL with ultrapure water; when mixed fully, a 2-mL sample of solution was taken and diluted to 100 mL with ultrapure water, which was ARR test solution, and its concentration was 2 mg mL -1 (2 mg of ARR powder per mL of test solution).
The reference and test solutions (2 mL) were measured and transferred to appropriately labeled test tubes with stoppers. A phenol solution (3%, 2 mL) and a sulfuric acid solution (98%, 9 mL) were then added successively; the mixture was then shaken vigorously. Immediately after cooling to room temperature, the absorbance was measured at a 490 nm wavelength using a TU-1901 dual-beam UVvisible spectrophotometer. The Astragali Radix polysaccharide content was then calculated.
Total protein and crude fat determination via near-infrared scanning ARR powder with an 80-mesh particle size was transferred into a glass dish and measured with a nearinfrared scanner. The ARR powder samples before and after fermentation (i.e., sampled in triplicate) were analyzed in duplicate, after which the results were averaged.

Metal element content determination via atomic absorption spectrometry
Metal elemental composition was measured in accordance with the GB/T13885-2003 national standard "Determination of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc contents in feed." A series of standard working solutions were prepared with 1 g L -1 iron, copper, zinc, and manganese stock solutions.
The ARR powder (1 g) was weighed before and after fermentation, after which it was mixed with a hydrochloric acid solution (1 : 10; 100 mL), fully dissolved, and left undisturbed prior to ltration. The ltrate was then analyzed with an atomic absorption spectrometer to determine copper, iron, zinc, and manganese contents in the sample.

Statistical analysis
The SPSS 19.0 software was used for data analysis. All measurement data were expressed as mean ± standard deviation (mean ± SD). Comparisons before and after fermentation were performed via the paired t-test. A p < 0.05 indicated a signi cant difference and p < 0.01 indicated an extremely signi cant difference.

Optimization of ARR solid fermentation conditions
Effect of cellulase culture medium amount on Aspergillus niger-mediated fermentation Fig. 1 illustrates the relationship between the amount of astragaloside IV in the ARR and the content of the cellulase culture solution produced by Aspergillus niger. Fig. 1 demonstrates that, with the continuous increase of culture solution content, the amount of astragaloside IV increases rst and then decreases, and the culture solution accounts for 60% of the total volume of the fermentation product (i.e., ARR + Aspergillus niger culture solution), reaching a maximum at 0.4% (0.44 ± 0.0015 mg g -1 ). This demonstrates that the optimal moisture content for Aspergillus niger solid fermentation ARR was 60%.
Effect of temperature on fermentation Fig. 2 illustrates the relationship between the amount of astragaloside IV and fermentation temperature during ARR solid fermentation. It can be seen from Fig. 2 that when the fermentation temperature is between 24 and 28 °C, the amount of astragaloside IV signi cantly increases with increased temperature, reaching a maximum at 0.4 °C (0.46 ± 0.020 mg g -1 ). Moreover, when the fermentation temperature is 28-32 °C, the amount of astragaloside IV decreases as the temperature increases. Therefore, the optimal temperature for solid fermentation of ARR was found to be 28 °C.

Effect of time on fermentation
It can be seen from Fig. 3 that the amount of astragaloside IV increases gradually as fermentation time increases. Particularly, astragaloside IV concentrations changed substantially during the rst 3-5 d of fermentation, with less pronounced changes occurring thereafter. The optimal duration for solid fermentation of ARR was thus found to be 5 d.

Orthogonalexperiment results
Tables 1 and 2 summarized the results of the orthogonal experiment, whereby three factors and three levels (culture medium content, fermentation time, and fermentation temperature) were evaluated.
According to Table 1, the R values of the culture broth content, fermentation time, and fermentation temperature were 0.108, 0.024, and 0.057, respectively. Notably, the order of the three factors affecting the solid fermentation of ARR was: moisture content > temperature > fermentation time. Moreover, based on the Average value in the Table 1, the best fermentation conditions for solid fermentation of Aspergillus niger with ARR were the following: a water content of 60%, fermentation time 4 d, and fermentation temperature 28 °C.

Contents of nine components inARRbefore and after fermentation
Astragaloside and calycosin-7-glucoside content According to the astragaloside IV and calycosin-7-glucoside peaks measured by liquid chromatography, the standard curves of the two substances were calculated as follows: Y = 28.85x -1.825 (R 2 = 0.999), with a linear range of 0.12-0.54 μg, and Y = 475.64x + 12.039 (R 2 = 0.997), with a linear range of 0.45-4.05 μg, respectively. The contents of astragaloside IV and calycosin-7-glucoside in ARR before and after fermentation (Table 3) were calculated according to Fig. 4 and Fig. 5.
Astragali Radix polysaccharide content According to our tests, the standard curve equation for ARP was Y = 0.008x + 0.010, R 2 = 0.999, with a linear range of 0-100 μg mL -1 . The polysaccharide contents in ARR before fermentation was 36.34 ± 3.12 mg g -1 , which increased signi cantly to 49.90 ± 3.48 mg g -1 after fermentation (p < 0.01). Fig. 6 illustrates the differences between polysaccharide contents in ARR before and after fermentation.
Total protein and crude fatcontents Table 4 summarizes the total protein and crude fat contents of ARR before and after fermentation.
Notably, we observed that under the same humidity conditions, the total protein and crude ash contents in the ARR after fermentation was signi cantly higher than before fermentation (p < 0.05), whereas the crude fat content remained largely constant before and after fermentation (p > 0.05).

Contents of four metallic elements
Linear regression was performed for each element, with the concentration as the abscissa and the absorbance as the ordinate. It can be seen from Table 5 that the concentration of each element exhibited a good linear relationship with the absorbance.
The results demonstrated that the iron content in ARR after fermentation was signi cantly lower than before fermentation (p < 0.01), and the manganese content was signi cantly higher than before fermentation (p < 0.01) ( Table 6).

Selection of fermentation strain ACCC 30583
Aspergillus niger is a common species of Aspergillus, which is widely distributed in food, plant products, and soil around the world. Notably, some Aspergillus strains are utilized in industrial fermentation processes [11][12][13]. The industrial use of Aspergillus niger is generally regarded as being highly safe. According to the FDA, Aspergillus niger can be directly used in food production or feed [14]. Aspergillus niger hyphae are relatively developed, with fast asexual growth and propagation speed, as well as strong adaptability, and thus are often used for fermentation feed in the breeding industry [15]. The overarching purpose of this study was to use the fermented ARR as an animal feed additive. Before that, our research team had screened 11 strains of cellulase-producing fungi and bacteria. An enzyme activity test determined that among the 11 strains, Aspergillus niger ACCC 30583 and Bacillus 01784 had the highest enzyme production activity. We then investigated the in uence of the Aspergillus niger ACCC 30583 and Bacillus 01784 culture media on the precipitation of Astragaloside , the active component of ARR. Our results showed that Astragaloside yields were higher in Aspergillus niger ACCC 30583 fermentation, and was therefore deemed the optimal strain for ARR fermentation. Our team then investigated the effects of fermentation temperature, time, carbon source composition, carbon source concentration, inoculum amount, and nitrogen source concentration on the cellulase production activity of Aspergillus niger ACCC 30583, and determined the optimal cellulase production conditions through a four-factor (nitrogen source concentration, carbon source concentration, inoculum amount, and fermentation time) orthogonal experiment [10]. In this study, a three-level orthogonal experiment was carried out to determine the optimal conditions for ARR solid fermentation. We concluded that the optimal culture medium content, fermentation time, and fermentation temperature were 60%, 4 days, and 28 ℃, respectively. Under these conditions, the fermentation of ARR by the Aspergillus niger ACCC 30583 strain effectively promoted the separation of astragaloside from the residues. Additionally, another potential advantage of using ARR fermented by Aspergillus niger as a feed additive is that Aspergillus niger produces many kinds of enzymes during the fermentation process, among which proteases can decompose the protein in feed and facilitate animal digestion (i.e., by compensating endogenous enzyme de ciencies), stimulate the secretion of endogenous enzymes, accelerate the digestion and absorption of nutrients, and improve feed utilization e ciency [16].

Importance of ARR as a test substance
Astragali Radix (Huangqi), which is widely used in medicine or food, is thought to reinforce qi and strengthen the spleen, and is therefore suitable to treat diseases associated with qi failure and blood de ciencies [17]. Pharmacological studies have demonstrated that Astragali Radix has a wide range of clinical applications, including anti-tumor, antioxidative, anti-diabetes, antibacterial, and antiviral and immunostimulant properties [18][19][20][21]. However, due to extraction technology limitations, the extraction e ciency of Astragali Radix is low, resulting in ARR with a large amount of bioactive substances. Huang et al. used HPLC to detect the astragaloside contents in Astragali Radix and ARR, and demonstrated that ARR contained 72.08% of the total astragaloside content in Astragali Radix [22]. Moreover, Zhou et al. used HPLC to detect the astragaloside content in ARR discarded by Dali Pharmaceutical Co. Ltd. and found that the astragaloside content was as high as 0.74 μg g -1 [23]. The results of this study showed that the astragaloside content in residues extracted twice via the regular water extraction method was 0.13 mg g -1 . In addition to functional components such as astragaloside , ARR also contain an abundance of nutrients such as crude fat, crude protein, amino acids, and minerals, which could be used as growth enhancers in animal feed [24]. Previous studies have shown that ARR is a natural and safe feed additive, which can accelerate animal growth and improve immunity and meat quality [25][26]. Thus, the potential of ARR as feed additives for animal production should be studied further.
Astragaloside IV as the basis for judging the effectiveness of ARR fermentation Saponins are among the most representative active components of Astragali Radix. Currently, more than 40 saponins, such as astragaloside and isoastragaloside, have been isolated from ARR, among which astragaloside has the best biological activity [27] and is thus often referred to as a "super Astragali Radix polysaccharide." Astragaloside has broad pharmacological applications, such as neuroprotection and liver protection, as well as anti-cancer and anti-diabetes effects [28]. Zang et al. demonstrated that astragaloside IV is a commonly used Chinese patent medicine for patients with chronic heart failure [29]. Moreover, Leng et al. reported that astragaloside IV has protective effects against endothelial dysfunction in diabetic rats [30]. Astragaloside IV has also been reportedly used as a quality-control marker of Astragali Radix in the Chinese Pharmacopoeia (2005 version) [31]. Therefore, astragaloside IV is arguably the most representative component of Astragali Radix.

Analysis of test results
Our results demonstrated that the contents of bioactive substances such as astragaloside IV, calycosin-7glucoside, and ARP in ARR increased signi cantly after Aspergillus niger-mediated fermentation. This may be because Astragali Radix contains more lignin and cellulose, which provides nutrition for the growth of Aspergillus niger, thereby allowing these microorganisms to produce lignin-degrading enzymes and cellulase, which can decompose the cell walls of Astragali Radix. Thus, astragaloside , iso avones, and polysaccharides in Astragali Radix can be separated from cell walls or protein complexes. The total protein content in ARR was notably increased by fermentation, which was directly related to the many enzymes produced by Aspergillus niger (i.e., enzymes are proteins themselves). The increase in crude ash may be mainly attributed to metabolic waste after fermentation. The mechanisms by which elemental metal contents in ARR were modi ed by fermentation remain unclear. It has been speculated that Aspergillus niger can use iron and other minerals contained in ARR to participate in the synthesis of its own organic compounds during its growth process, and the hydrochloric acid used in the analytical digestion process cannot disrupt the bonds between the iron and the aforementioned compounds, resulting in a decrease in the amount of iron detected [32][33]. Similarly, the increase of manganese content may also be due to chemical changes or associations before and after fermentation. However, this theory needs to be further con rmed.
In conclusion, Aspergillus niger-mediated fermentation greatly improved the separation of active substances and nutrients in ARR, thereby facilitating the repurposing of these residues as animal feed additives. Moreover, our proposed method has important implications for the treatment of other Chinese herbal medicine residues after extraction.   Table 2 Analysis of variance of culture medium content, fermentation time, and fermentation temperature via a 3-factor and 3-level orthogonal test  Table 3 Determination of astragaloside IV and calycosin-7-glucoside in Astragali Radix residues before and after fermentation Group Astragaloside IV (μg g -1 ) Calycosin-7-glucoside (μg g -1 ) Before fermentation 0.13 ± 0.011 6.1 ± 0.77 After fermentation 0.63 ± 0.090* 8.15 ± 1.12* Note: * indicates a signi cant difference between pre-and post-fermentation (p < 0.05) Table 4 Near-infrared scanning results of total protein and crude fat contents in Astragali Radix residues before and after fermentation (%)     Chromatogram of astragaloside IV before and after fermentation (A: astragaloside IV standard; B: before fermentation; C: after fermentation) Figure 5 Chromatogram of calycosin-7-glucoside before and after fermentation (A: calycosin-7-glucoside standard; B: before fermentation; C: after fermentation)