Improvement of the Quality of Ginkgo biloba Leaves Fermented by Eurotium cristatum as High Value-Added Feed

Ginkgo biloba leaves are well known for their high content of nutrients and bioactive substances. However, unpleasant smell and a small number of ginkgolic acids greatly reduce the utilization of the leaves. In this work, solid-state fermentation of G. biloba leaves using Eurotium cristatum was studied by investigation of the nutrient changes and its feasibility as a functional feed. E. cristatum could grow on pure G. biloba leaves and the addition of excipients could significantly improve the growth of E. cristatum. The optimal medium was with 10% (w/w) of whole G. biloba seeds and the optimized water content, pH, inoculum size and fermentation time were 45% (w/w), 4.5, 4.76 × 107 CFU/100 g wet medium, and eight days, respectively. Under the optimal conditions, the spore number increased by about 40 times. The content of flavonoids was greatly increased by 118.6%, and the protein and polyprenyl acetates (PPAs) were increased by 64.9% and 10.6%, respectively. The ginkgolic acids, lignin, and cellulose were decreased by 52.4%, 38.5%, and 20.1% than before, respectively. Furthermore, the fermented G. biloba leaves showed higher antioxidant activity and held more aroma substances. Thus, G. biloba leaves fermented by E. cristatum have potential as s high value-added feed. This is the first investigation of E. cristatum fermentation on ginkgo leaves, which will facilitate the use of ginkgo leaves in the feed industry.


Introduction
The use of antibiotics in the feed industry once had positive effects on prevention of animal diseases and improvement of feed reward. However, concern has been raised regarding residue problems, which pose health threats to animals and humans [1]. For that reason, many countries have prohibited the utilization of antibiotics [2,3]. Therefore, effective substitutes are required. Fermented feed has attracted wide attention because of its low-cost, high nutritional value, and multifunctionality. Through microbial action, antinutrient factors, and macromolecular substances, raw materials can be decomposed or transformed into bioactive components such as organic acids, active small peptides, and growth factors [4]. The strains permitted for use in fermented feed include Bacillus subtilis, Saccharomyces, and Aspergillus, among others [3,5]. Bacillus subtilis can improve the growth performance of animals by secreting amylase, protease, and lipase [6]. Saccharomyces has the prominent ability of protein digestion and acid production to prevent antimicrobial-associated diarrhea [7,8]. However,

Materials
The fresh G. biloba leaves obtained from Chinese Herb Transaction Center (Bozhou, China) were picked from August to September. The excipients (G. biloba seeds, corn, rice bran, soybean, barley, wheat) were purchased from Nanjing Guoyao Biotechnology Co. Ltd. (Nanjing, China). The leaves that were prepared by removing the stems and yellow leaves and excipients were dried in an oven at 40 • C for 24 h and at 60 • C for 12 h, respectively, and then were pulverized to 40 mesh using a disintegrator. As for G. biloba seeds, one part was pulverized with hulls (whole G. biloba seeds), another was pulverized by removing hulls. Potato dextrose agar medium (PDA) was purchased from Qingdao Gaokeyuan Haibo Biotechnology Co. Ltd. (Qingdao, China). E. cristatum was deposited in the Fermented Food Laboratory of Nanjing Forestry University. All other chemicals used in this study were of analytical grade and commercially available.

Preparation of Spore Suspension of Eurotium cristatum
To activate Eurotium cristatum, the strain stored at −20 • C was plated on PDA medium, then cultured under constant temperature (28 • C) and humidity (85%) until the plate was covered with golden hyphae (about 72 h). Then, the golden colony of the edge was picked and inoculated into fresh PDA medium. The above operations were repeated 2-3 times.
The activated strains were picked and transferred to a 100 mL triangular flask containing sterile water and several glass beads, then shaken at 220 r/min for 45 min. Spore number was calculated using a hemocytometer plate, and the concentration of the spore suspension was adjusted to 5.6 × 10 6 CFU/mL.

Prefermentation
Preparation of G. biloba leaves medium: 8.5 g of G. biloba leaves powder was mixed with 10 g of deionized water in a 100 mL triangular flask. The mixture was sterilized by autoclaving at 121 • C for 20 min to obtain a solid fermentation medium of G. biloba leaves.
In order to determine whether E. cristatum could grow on pure G. biloba leaves medium without adding any excipients, 850 µL of spore suspension was inoculated into the pure medium, then cultured at 28 • C and relative humidity 85%. The relevant parameters were determined after three days, five days, and seven days to investigate the growth of E. cristatum.

Optimization of Fermentation Medium Components
Ten percent (excipient weight/G. biloba leaves weight) of powder of G. biloba seeds, corn, rice bran, soybean, barley, or wheat were employed as excipients to the G. biloba leaves medium mentioned above. Based on the optimal auxiliary material, the addition amount of excipients was further investigated at 2%, 6%, 10% 14%, 18% (w/w). The other fermentation conditions and operations were as described in Section 2.3.

Determination of Spore Number in the Solid-State Culture
In order to obtain a spore suspension of E. cristatum, 0.1 g of culture was mixed with 10 mL of sterile water in a 50 mL triangular flask containing 10 glass beads and then shaken at 28 • C and 220 rpm for 45 min in a thermostatic shaker. Next, 5 µL of the diluted spore suspension was pipetted into a hemocytometer, and a drop of Medan stain was added. The spore number was measured by microscopy.

Determination of Physicochemical Components and Enzyme Activity
The content of TTLs was determined according to the method developed in our previous work [20]. The content of PAC, flavonoids, and PPAs was determined according to the method developed in our previous work [21]. The content of ginkgolic acids was determined according to the method described by Li et al. [22]. The content of free amino acids (FAAs) was determined according to the National Standard of the People's Republic of China (GB/T8314-2013) [23]. The content of proteins was determined by the Bradford method [24]. The content of reducing sugars was determined by anthrone colorimetry [25]. The lignin content was determined according to the method described by Zhao et al. [26]. The aroma components were determined by GC-MS method [27]. The antioxidant activity was determined according to the method described by Cao et al. [28]. The enzyme activities, including amylase, protease, lipase, pectase, cellulase, and hemicellulase, were determined according to the National Standard of the People's Republic of China (GB1886.174-2016) [29].

Statistical Analysis
The statistical analysis was carried out in the one-way ANOVA program of the SAS system for Windows 8.02 (SAS Institute Inc., Cary, NC, USA). Duncan's multiple-range test was selected as the comparison method in the program and the significance level was set at 0.05.

Results and Discussion
3.1. Prefermentation of Pure G. biloba Leaves with E. cristatum E. cristatum is the dominant fungi species isolated from Fuzhuan Brick Tea, a traditional Chinese food, which is usually applied as a probiotic for human health [11]. Thus, the fungal strain possesses potential advantages for use in the food and feed industry due to its high safety. To determine whether E. cristatum could grow on pure G. biloba leaf medium, the spore suspension of E. cristatum was inoculated into the medium, then the spore number, the content of flavonoids, and enzyme activity as the main indexes were investigated. The contents of TTLs and PAC as the auxiliary indexes were also determined. Figure 1A showed that E. cristatum could grow on pure G. biloba leaf powder, which indicated that G. biloba leaves contain appropriate carbon and nitrogen sources for the growth of E. cristatum. However, the spore number of E. cristatum decreased in the last two days of fermentation. This suggested that the nutrients of G. biloba leaf powder had been consumed by E. cristatum. Therefore, it was necessary to add additional carbon and nitrogen sources to maintain the growth of E. cristatum. During the fermentation, the content of TTLs had no significant change. The content of PAC kept decreasing and the terminal content was 0.12 mg/g. The PAC content in G. biloba leaves originally should be 4-12% [30]. The cause of decline might be that PAC was easily oxidized and decomposed under natural conditions [31]. The content of flavonoids went up gradually, possibly because E. cristatum could produce flavonoids by itself or might transform the polysaccharides of G. biloba leaves to flavonoids [32].
As roughage, G. biloba leaves contain proteins and crude fibers such as cellulose, hemicellulose, pectin, and lignin. It is difficult for animals to assimilate these components by themselves [16]. E. cristatum can secrete abundant enzymes to degrade these macromolecular substances in their metabolic processes, thus investigating the changes of several typical enzymes' activities in the process of fermentation is necessary. The results are shown in Figure 1B. The activities of cellulase, hemicellulase, and pectase greatly increased up to 1952.7 U/g, 1696.1 U/g, and 3935.0 U/g on the third day, respectively. These enzymes work to reduce the lignocellulose content of G. biloba leaves and can be very beneficial to animal digestion of lignocellulose. Compared with other enzymes, the amylase activity was lower but relatively stable, maintaining at about 4.6 U/g. The other five enzymes' activities showed decreases with the extension of fermentation time. At the end of fermentation, the protease, lipase, pectase, cellulase, and hemicellulase activity decreased by 20.8%, 56.9%, 46.8%, 37.6%, and 86.2%, respectively. Due to the limited nutrients in the pure G. biloba leaves, in the late stage of fermentation, the strain's growth viability began to degenerate with the reducing spore number. As a result, the secretion of enzymes also declined. Thus, the ability of E. cristatum to grow on pure G. biloba leaves was restricted. To further promote the growth of E. cristatum for degrading crude fibers and improving the nutrients of feed, it is necessary to add additional carbon and nitrogen sources into G. biloba leaf medium. Processes 2018, 6, x FOR PEER REVIEW 5 of 20

Fermentation of G. biloba Leaves with Addition of Excipients
Considering the cost and safety of the final product, several commonly used excipients for feed were added to the G. biloba leaf powder for promoting the fermentation of E. cristatum. As shown in Table 1, comparison of the spore number indicated the effect of different excipients, in the order of G. biloba seeds, maize, rice bran, wheat, whole G. biloba seeds, soybean meal, and barley. TTLs are usually very low (0.001-0.8%) in G. biloba leaves [33]. The TTLs content remained stable at about 0.65 mg/g during fermentation, which showed that the E. cristatum could not consume it. The variation tendency of PAC was similar to that in the prefermentation. The content of flavonoids increased more obviously than that in the prefermentation. When wheat was added as an excipient, the content of flavonoids reached the highest value. When soybean meal, whole G. biloba seeds, barley, and maize were added, the content of flavonoids remained at the same level. Moreover, when G. biloba seeds and rice bran were used as excipients, the content of flavonoids was slightly lower. There might be two reasons that brought about the distinction of the content of flavonoids between different excipients. Firstly, owing to the addition of excipients, the spore number of E. cristatum increased by nearly 10 times, thus the strains might metabolically synthesize a certain amount of flavonoids. Secondly, different kinds of excipients might also contain different amounts of flavonoids. Regardless of the kind of excipient employed, the content of flavonoids decreased in the prophase of fermentation, possibly because E. cristatum secreted some extracellular enzymes which

Fermentation of G. biloba Leaves with Addition of Excipients
Considering the cost and safety of the final product, several commonly used excipients for feed were added to the G. biloba leaf powder for promoting the fermentation of E. cristatum. As shown in Table 1, comparison of the spore number indicated the effect of different excipients, in the order of G. biloba seeds, maize, rice bran, wheat, whole G. biloba seeds, soybean meal, and barley. TTLs are usually very low (0.001-0.8%) in G. biloba leaves [33]. The TTLs content remained stable at about 0.65 mg/g during fermentation, which showed that the E. cristatum could not consume it. The variation tendency of PAC was similar to that in the prefermentation. The content of flavonoids increased more obviously than that in the prefermentation. When wheat was added as an excipient, the content of flavonoids reached the highest value. When soybean meal, whole G. biloba seeds, barley, and maize were added, the content of flavonoids remained at the same level. Moreover, when G. biloba seeds and rice bran were used as excipients, the content of flavonoids was slightly lower. There might be two reasons that brought about the distinction of the content of flavonoids between different excipients. Firstly, owing to the addition of excipients, the spore number of E. cristatum increased by nearly 10 times, thus the strains might metabolically synthesize a certain amount of flavonoids. Secondly, different kinds of excipients might also contain different amounts of flavonoids. Regardless of the kind of excipient employed, the content of flavonoids decreased in the prophase of fermentation, possibly because E. cristatum secreted some extracellular enzymes which could oxidize and degrade flavonoids. However, E. cristatum might produce pigments or flavonoids at the same time [27]. Thus, in anaphase of fermentation, the content of flavonoids maintained an upward trend with the significant increment of spore number. Nevertheless, flavonoids can be consumed as substances when Aspergillus niger is used as the strain to ferment ginkgo leaves [34], which suggested that E. cristatum fermentation is more advantageous for enhancing the flavonoid content of the media than A. niger. During ginkgo leaves fermentation by A. niger with wheat bran and corncob used as additives, significantly higher enzyme activity was obtained than in the control fermentation [35]. Thus, the addition of excipients had important impacts on enzyme activity. However, these additives also greatly increased the cellulose content of feed, which is an antinutritive factor for monogastric animals. In this study, other additives were used for improving enzymatic activity and full-scale enzyme activities were investigated. As shown in Table 2, enzyme activity positively correlated with the spore number during fermentation. The activity of several enzymes was at a higher level when whole G. biloba seeds severed as excipients. The activities of pectinase, cellulase, and hemicellulase were remarkably increased when G. biloba seeds were added, and the lipase activity was the highest while rice bran was used. The protease activity was always high no matter what kind of excipients were added. For animals, the presence of cellulase and hemicellulase is beneficial for the degradation of cellulose in the feed to reduce the risk of gastrointestinal disease. Lipase can inhibit the absorption of fat and prevent abnormal obesity to keep the animals healthy. Protease breaks down protein into small amino acids and peptides to enable digestion and absorption. Moreover, the presence of enzymes induces the depolymerization of macromolecule constituents, which improves the nutritional and flavor quality of ginkgo leaves [27]. Considering the cost of feed and comprehensive results of various indicators, whole G. biloba seeds were chosen as an excipient, and their addition amount was further optimized. Ten percent (w/w) was the optimal addition amount in terms of spore number and flavonoid content (Figure 2A,B). Table 3 shows that significant increments of six enzymes' activities were observed with increasing excipient from 2% to 10% (w/w). However, continued excipient increase led to a loss of enzyme activity. Thus, combining the spore number with the activity of several enzymes, 10% (w/w) was eventually selected as the optimal addition amount of whole G. biloba seeds.
Processes 2018, 6, x FOR PEER REVIEW 8 of 20 Considering the cost of feed and comprehensive results of various indicators, whole G. biloba seeds were chosen as an excipient, and their addition amount was further optimized. Ten percent (w/w) was the optimal addition amount in terms of spore number and flavonoid content ( Figure  2A,B). Table 3 shows that significant increments of six enzymes' activities were observed with increasing excipient from 2% to 10% (w/w). However, continued excipient increase led to a loss of enzyme activity. Thus, combining the spore number with the activity of several enzymes, 10% (w/w) was eventually selected as the optimal addition amount of whole G. biloba seeds.

Effects of Fermentation Conditions
The pH of the medium has strong effects on many enzymatic processes and transport of various components across the cell membrane [36]. An acidic environment is beneficial for the growth of E. cristatum. Liu et al. reported that E. cristatum can grow between pH 3.0 and 6.0 [37]. In this study, the pH of the medium was optimized in the range of 3.0 to 5.0. The results are shown in Figure 3A,B. The spore number was significantly increased in the pH range from 3.0 to 4.5 (α = 0.05), with the optimal pH being 4.5 ( Figure 3A). When the pH increased further, the spore number decreased. The spore numbers were not significantly different between pH 4.0 and pH 5.0. The content of TTLs was stable and the content of PAC was reduced as mentioned in Section 3.1. The content of flavonoids peaked at pH of 5 and there was no significant difference between pH 4.0 and 4.5 in the later stage of fermentation ( Figure 3B). When the pH was 4.5, the content of flavonoids reached 27.78 ± 0.03 mg/g. However, an obvious decrease of flavonoids content was observed when the pH was 3.0 or 3.5. The content of flavonoids presented a downward trend with the advancement of fermentation. Thus, it can be speculated that flavonoids were not only related to raw materials and E. cristatum, but also the pH value. The acidic environment resulted in the reduction of flavonoids. The highest activity of six enzymes was achieved with a pH of 4.5 (Table 4). During the fermentation, while the extracellular enzymes were certainly influenced by the pH value, the activity changes of intracellular enzymes may be attributed to the variations of growth, reproduction, and metabolism of E. cristatum caused by the pH value. In conclusion, the optimal pH was set as 4.5.

Effects of Fermentation Conditions
The pH of the medium has strong effects on many enzymatic processes and transport of various components across the cell membrane [36]. An acidic environment is beneficial for the growth of E. cristatum. Liu et al. reported that E. cristatum can grow between pH 3.0 and 6.0 [37]. In this study, the pH of the medium was optimized in the range of 3.0 to 5.0. The results are shown in Figure 3A,B. The spore number was significantly increased in the pH range from 3.0 to 4.5 (α = 0.05), with the optimal pH being 4.5 ( Figure 3A). When the pH increased further, the spore number decreased. The spore numbers were not significantly different between pH 4.0 and pH 5.0. The content of TTLs was stable and the content of PAC was reduced as mentioned in Section 3.1. The content of flavonoids peaked at pH of 5 and there was no significant difference between pH 4.0 and 4.5 in the later stage of fermentation ( Figure 3B). When the pH was 4.5, the content of flavonoids reached 27.78 ± 0.03 mg/g. However, an obvious decrease of flavonoids content was observed when the pH was 3.0 or 3.5. The content of flavonoids presented a downward trend with the advancement of fermentation. Thus, it can be speculated that flavonoids were not only related to raw materials and E. cristatum, but also the pH value. The acidic environment resulted in the reduction of flavonoids. The highest activity of six enzymes was achieved with a pH of 4.5 (Table 4). During the fermentation, while the extracellular enzymes were certainly influenced by the pH value, the activity changes of intracellular enzymes may be attributed to the variations of growth, reproduction, and metabolism of E. cristatum caused by the pH value. In conclusion, the optimal pH was set as 4.5.   It is undoubted that the inoculum size determines the final spore number and the content of various nutrients. Restricted to the space of a triangular flask, a large inoculation was not beneficial for fermentation [38]. Thus, the inoculum size should be investigated. As shown in Figure 4A, the spore number of E. cristatum was proportional to the inoculum size ranging from 2.38 × 10 6 to 7.14 × 10 6 CFU. Nonetheless, the spore number decreased with the continued increase of the inoculum size. The flavonoids content was highest when the inoculum size was 7.14 × 10 6 CFU ( Figure 4B). The overall variation trend of flavonoids content was consistent with that of spore number. It suggested that the content of flavonoids in the fermentation mixture was codetermined by G. biloba leaves and Eurotium cristatum. TTLs content and PAC had no obvious changes. The activities of six enzymes were at the maximum when the inoculum size was 7.14 × 10 6 CFU ( Table 5). The spore number directly influenced the enzyme activity because these enzymes are secreted by Eurotium cristatum. Considering all the above changes, the optimal inoculum size was determined to be 7.14 × 10 6 CFU.
It is undoubted that the inoculum size determines the final spore number and the content of various nutrients. Restricted to the space of a triangular flask, a large inoculation was not beneficial for fermentation [38]. Thus, the inoculum size should be investigated. As shown in Figure 4A, the spore number of E. cristatum was proportional to the inoculum size ranging from 2.38 × 10 6 to 7.14 × 10 6 CFU. Nonetheless, the spore number decreased with the continued increase of the inoculum size. The flavonoids content was highest when the inoculum size was 7.14 × 10 6 CFU ( Figure 4B). The overall variation trend of flavonoids content was consistent with that of spore number. It suggested that the content of flavonoids in the fermentation mixture was codetermined by G. biloba leaves and Eurotium cristatum. TTLs content and PAC had no obvious changes. The activities of six enzymes were at the maximum when the inoculum size was 7.14 × 10 6 CFU ( Table 5). The spore number directly influenced the enzyme activity because these enzymes are secreted by Eurotium cristatum. Considering all the above changes, the optimal inoculum size was determined to be 7.14 × 10 6 CFU.   In solid-state fermentation medium, the impacts of water content are important and various. The depletion of water affects the diffusion of gases, solutes, and osmosis caused by excessive metabolites in the vicinity of cells [39]. Too high or too low moisture degree both have negative effects on the growth of E. cristatum. Higher moisture degree will lead to the decrement of porosity and the increment of stickiness, and hinder the transformation of oxygen [40]. Lower moisture degree will lead to the decrease of water-soluble substances that can be utilized by E. cristatum [40]. As shown in Figure 5A,B, the optimal water content was 45%/100 g wet medium in view of spore number and the content of flavonoids. The optimal water content was evidently lower than the previous report by Wang et al. using A. niger as the fermentation strain with optimal water content of 60% [36]. Table 6 shows that the activities of six enzymes were improved in the water content ranging from 35% to 45% (w/w), whereas the activity of the enzymes declined with further increasing water content. The cause of this phenomenon might be that the enzymes' activities are associated with the breathing pattern of strains. If the oxygen is sufficient, E. cristatum tends to adapt aerobic respiration, thus increasing enzyme activity. In summary, 45% (w/w) water content was adequate for the growth of E. cristatum. In solid-state fermentation medium, the impacts of water content are important and various. The depletion of water affects the diffusion of gases, solutes, and osmosis caused by excessive metabolites in the vicinity of cells [39]. Too high or too low moisture degree both have negative effects on the growth of E. cristatum. Higher moisture degree will lead to the decrement of porosity and the increment of stickiness, and hinder the transformation of oxygen [40]. Lower moisture degree will lead to the decrease of water-soluble substances that can be utilized by E. cristatum [40]. As shown in Figure 5A,B, the optimal water content was 45%/100 g wet medium in view of spore number and the content of flavonoids. The optimal water content was evidently lower than the previous report by Wang et al. using A. niger as the fermentation strain with optimal water content of 60% [36]. Table 6 shows that the activities of six enzymes were improved in the water content ranging from 35% to 45% (w/w), whereas the activity of the enzymes declined with further increasing water content. The cause of this phenomenon might be that the enzymes' activities are associated with the breathing pattern of strains. If the oxygen is sufficient, E. cristatum tends to adapt aerobic respiration, thus increasing enzyme activity. In summary, 45% (w/w) water content was adequate for the growth of E. cristatum.  After the above optimization, the fermentation time course was further investigated under the optimized conditions. Figure 6A shows that the spore number was enhanced with the extension of fermentation time until 192 h, at which it began to drop for the following 24 h. The main reason might be that the carbon and nitrogen sources in the medium had been depleted at 192 h. The contents of TTLs and PAC had no remarkable changes as compared with the above-optimized data. The content of flavonoids increased until 120 h and then decreased. It is possible that flavonoids were consumed as energy for the growth of E. cristatum when nutrients were lacking in the medium [32]. As shown in Figure 6B, the protease, lipase, amylase, and pectinase activity maintained an upward trend until 120 h, and then decreased. The cellulase and hemicellulase activity only increased within the first 48 h, possibly because E. cristatum only secretes the two enzymes in the early stage of fermentation. Finally, the optimal pH, inoculum size, water content, and fermentation time were decided to be 4.5, 7.14 × 10 6 CFU, 45%/100 g, and eight days, respectively. Zhang et al. reported that G. biloba leaves fermented by Aspergillus niger could improve feed efficiency, intestinal morphology, digestion, and absorption of broilers as supplementation in the starter and grower diets [5]. In our work, G. biloba leaves fermented by E. cristatum possessed considerable improvement of flavonoids content. Further studies are necessary to investigate whether this improvement would have positive impacts on animals.
Processes 2018, 6, x FOR PEER REVIEW 16 of 20 After the above optimization, the fermentation time course was further investigated under the optimized conditions. Figure 6A shows that the spore number was enhanced with the extension of fermentation time until 192 h, at which it began to drop for the following 24 h. The main reason might be that the carbon and nitrogen sources in the medium had been depleted at 192 h. The contents of TTLs and PAC had no remarkable changes as compared with the above-optimized data. The content of flavonoids increased until 120 h and then decreased. It is possible that flavonoids were consumed as energy for the growth of E. cristatum when nutrients were lacking in the medium [32]. As shown in Figure 6B, the protease, lipase, amylase, and pectinase activity maintained an upward trend until 120 h, and then decreased. The cellulase and hemicellulase activity only increased within the first 48 h, possibly because E. cristatum only secretes the two enzymes in the early stage of fermentation. Finally, the optimal pH, inoculum size, water content, and fermentation time were decided to be 4.5, 7.14 × 10 6 CFU, 45%/100 g, and eight days, respectively. Zhang et al. reported that G. biloba leaves fermented by Aspergillus niger could improve feed efficiency, intestinal morphology, digestion, and absorption of broilers as supplementation in the starter and grower diets [5]. In our work, G. biloba leaves fermented by E. cristatum possessed considerable improvement of flavonoids content. Further studies are necessary to investigate whether this improvement would have positive impacts on animals.

Component Analysis of the Fermented and Unfermented G. biloba Leaves
Except for TTLs, PAC, and flavonoids, G. biloba leaves also contain proteins, free amino acids, PPAs, reducing sugars, ginkgolic acids, lignin, and aroma components. Ginkgolic acids are unwanted constituents in G. biloba leaves and are sought to be reduced by fermentation. The other ingredients were desired to be maintained or enhanced after fermentation. The results are listed in Table 7. It was observed that the content of proteins was increased by 64.9%, which was 14.82% higher than after fermentation with A. niger [27]. The content of free amino acids was decreased by 91.3%. The changes demonstrated that the great mass of free amino acids was consumed by the E. cristatum and stored in the form of proteins [41]. For the feed, the value of proteins was reflected in not only the amount, but also the composition of various amino acids. The higher the percentage of essential amino acids account for, the higher nutritional value the protein has. The consumption of the semiessential amino acids can alleviate the wastage of essential amino acids [42]. The content of reducing sugars decreased by 47.1%, which might be due to the fact that macromolecular carbohydrates added to the medium were not sufficient, resulting in the consumption of the soluble sugars in the fermentation process. When the fermentation was completed, the content of PPAs improved from the original 40.08 mg/g to 44.34 mg/g. As naturally occurring substances, PPAs have obvious curative effects on immune diseases such as hypertension, hyperlipidemia, and diabetes [43]. Surprisingly, the content of ginkgolic acids dropped to less than half of the initial amount. The components were also greatly reduced via solid-state fermentation of ginkgo leaf residues by Candida tropicalis and Aspergillus oryzae [16]. Ginkgolic acids are considered as a carcinogen and mutagen, which can cause skin, mucous membrane, and other allergic inflammatory responses [22]. Thus, the fermented G. biloba leaves would possess higher safety than the unfermented leaves. The content of lignin and cellulose also declined after the fermentation, owing to their degradation via the enzymes secreted by E. cristatum, which suggested that the fermented leaves possessed lesser nutrient resistance to animals than the unfermented leaves and confirmed fungal fermentation was available for ginkgo leaves. Fungal fermentation usually possess higher delignocellulose ability when a plant material rich in lignocellulose is used as the medium than bacterial fermentation due to the fungal extracellular enzymatic system and hyphal penetration power [44]. Table 8 shows that the antioxidant activity was improved in comparison with that before fermentation as a whole, even when four different extraction solvents were employed. For ABTS free radical scavenging capability, 80% (v/v) methanol was the best extraction solvent. For DPPH free radical scavenging capability, 80% (v/v) acetone was the best extraction solvent. The enhancement of antioxidant activity was valuable for the feed to prevent the nutrients from oxidizing. The aroma components also made a difference during the fermentation. Before fermentation, there were 30 kinds of aroma components detected in the G. biloba leaf powder, and most of them were alcohols and a small number of esters. After fermentation, there were also 30 kinds of aroma components, but the alcohols decreased, whereas the aldehydes and ketones increased. A. niger-fermented ginkgo leaves had more aroma components than the control and presented a pleasant fragrance, but the specific aroma components were not investigated [27]. The transformations of these aroma components may be concerned with auto-oxidation of E. cristatum, but the specific change mechanisms remain to be studied.