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

Abstract

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.

1. 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, both of them lack cellulase production, meaning that degradation of lignin and cellulose is difficult. As a mycelial fungus, Aspergillus can secret large amounts of protease and cellulase to improve the nutritive value of various substrates, which is especially suitable for solid-state fermentation of cellulose material [9,10]. E. cristatum, the dominant fungi species in Fuzhuan Brick Tea, is the teleomorph of Aspergillus [11]. Scholars have found that E. cristatum and its fermentation metabolites have many functions, including regulation of intestinal flora, bacteriostasis, antioxidation, and antineoplasticity, etc. [12,13]. Thus, E. cristatum has great potential when applied in the feed industry.

G. biloba L. is the sole living species of the once large plant division Ginkgophyta, which is commonly called an “archaic living fossil” [14,15]. Leaves of G. biloba L. are famous for their high content of flavonoids [16]. In addition, G. biloba leaves also contain many other components, including terpene trilactones (TTLs), procyanidine (PAC), and PPAs, as well as ginkgolic acids, which are considered to be allergenic compounds [14,17]. Numerous studies have shown that G. biloba leaves extract can be used to treat cognitive disorders, ischemic heart disease, arrhythmias, cancer, and diabetes [15]. Since the 1960s, G. biloba L. has been widely cultivated in China and the annual production of G. biloba leaves is about 40,000 tons [16]. The planting areas are mainly distributed in Sichuan, Jiangsu, Shandong, and Zhejiang provinces [5]. At present, the output of G. biloba leaves substantially exceeds the demand because the development and utilization of G. biloba leaves is currently unfavored. It is urgent to develop a way to take full advantage of this resource. Cao et al. have reported that G. biloba leaves can serve as a biological feed additive to promote the growth and immunity of yellow feather broilers [18]. Yang et al. have reported that G. biloba leaves of different proportions have different impacts on the growth performance of broilers [19]. Therefore, it is feasible that G. biloba leaves can be applied in the feed industry.

In this work, the fungal solid-state fermentation of G. biloba leaves using E. cristatum for the production of fermented feed was investigated for the first time. The idea that E. cristatum could effectively ferment tea leaves was hypothesized to be transferrable to ginkgo leaves. The results demonstrated that E. cristatum could grow on pure G. biloba leaves and the addition of excipients could significantly improve the growth of Eurotium cristatum. The results showed that G. biloba leaves fermented by E. cristatum were promising as a fermented feed.

2. Materials and Methods

2.1. 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.

2.2. 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 × 10⁶ CFU/mL.

2.3. 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.

2.4. 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.

2.5. Optimization of Fermentation Conditions

After obtaining the optimal excipient and its optimal addition amount, the effects of different pH values (3.0, 3.5, 4.0, 4.5, and 5.0), inoculum size (2.38 × 10⁶, 4.76 × 10⁶, 7.14 × 10⁶, 9.52 × 10⁶, and 11.9 × 10⁶ CFU), water content (35, 45, 55, 65, and 75%/100 g wet medium, the water content of G. biloba leaves was about 10%), and fermentation time (1, 2, 3, 4, 5, 6, 7, 8, and 9 days) on the growth of E. cristatum were further optimized. In these cases, phosphate buffer instead of ultrapure water was added to the medium. The other operations and fermentation conditions were as described in Section 2.3.

2.6. 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.

2.7. 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].

2.8. 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.

3. 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.

3.2. 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. Effects of the excipients on the spore number, TTLS, PAC, and flavonoids in the fermented G. biloba leaves.

Excipient	Time (d)	Spore Number (×108 CFU)	TTLs (mg/g)	PAC (mg/g)	Flavonoids (mg/g)
Maize	3	1.05 ± 0.04n	0.62 ± 0.02c	0.05 ± 0.01g	24.98 ± 0.03e
	5	2.44 ± 0.03e	0.63 ± 0.02bc	0.09 ± 0.02f	23.44 ± 0.01g
	7	2.75 ± 0.03c	0.62 ± 0.03bc	0.04 ± 0.02g	25.32 ± 0.03d
Rice bran	3	1.25 ± 0.02m	0.66 ± 0.01b	0.24 ± 0.03c	18.11 ± 0.03j
	5	2.22 ± 0.04f	0.65 ± 0.02bc	0.24 ± 0.02c	16.15 ± 0.01k
	7	2.47 ± 0.01de	0.65 ± 0.01bc	0.14 ± 0.01de	19.48 ± 0.01hi
Soybean meal	3	0.91 ± 0.01o	0.62 ± 0.01c	0.09 ± 0.04f	25.78 ± 0.01d
	5	1.73 ± 0.03k	0.61 ± 0.01c	0.12 ± 0.02de	24.69 ± 0.01e
	7	1.93 ± 0.02i	0.62 ± 0.01c	0.12 ± 0.01de	26.65 ± 0.04c
Barely	3	0.61 ± 0.01q	0.65 ± 0.02bc	0.16 ± 0.01d	24.28 ± 0.05f
	5	1.58 ± 0.04l	0.64 ± 0.01bc	0.16 ± 0.02d	23.69 ± 0.01g
	7	1.85 ± 0.01j	0.65 ± 0.01bc	0.04 ± 0.04h	24.82 ± 0.01d
Wheat	3	0.93 ± 0.02o	0.65 ± 0.01bc	0.39 ± 0.03a	29.32 ± 0.09a
	5	2.03 ± 0.02g	0.65 ± 0.01bc	0.28 ± 0.01b	26.61 ± 0.05c
	7	2.52 ± 0.04d	0.66 ± 0.01b	0.29 ± 0.08ab	27.98 ± 0.02b
Whole G. biloba seeds	3	0.80 ± 0.01p	0.60 ± 0.01c	0.21 ± 0.03c	25.86 ± 0.03d
	5	1.90 ± 0.03i	0.62 ± 0.01c	0.11 ± 0.02e	24.15 ± 0.01f
	7	2.02 ± 0.03g	0.61 ± 0.01c	0.04 ± 0.01g	27.65 ± 0.01b
G. biloba seeds	3	1.87 ± 0.02j	0.72 ± 0.01a	0.34 ± 0.04a	19.19 ± 0.01i
	5	2.90 ± 0.01b	0.73 ± 0.01a	0.34 ± 0.01a	18.11 ± 0.04j
	7	3.10 ± 0.04a	0.72 ± 0.01a	0.36 ± 0.02a	19.78 ± 0.01h

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

, 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. Effects of the excipients on the activities of several enzymes in fermented G. biloba leaves.

Excipient	Time (d)	Enzyme Activity (U/g)
		Amylase	Lipase	Protease	Pectinase	Cellulase	Hemicellulase
Maize	3	4.67 ± 0.01a	142.22 ± 2.06e	336.69 ± 9.93fg	5586.39 ± 4.81d	2900.75 ± 74.39b	4136.11 ± 31.82c
	5	4.65 ± 0.01ab	98.52 ± 1.22h	314.07 ± 9.49h	5572.50 ± 30.05d	2133.14 ± 25.05f	2920.83 ± 41.67h
	7	4.65 ± 0.02ab	73.33 ± 0.89j	190.06 ± 6.62l	4900.28 ± 76.98g	845.38 ± 66.17l	927.78 ± 52.43m
Rice bran	3	4.68 ± 0.01a	124.44 ± 1.82f	317.64 ± 10.96h	5094.72 ± 41.94f	1804.89 ± 91.42gh	3663.89 ± 63.65e
	5	4.65 ± 0.01ab	103.70 ± 1.88	307.12 ± 6.18hi	4641.94 ± 19.25h	1219.08 ± 58.07j	2233.33 ± 55.12j
	7	4.64 ± 0.01bc	93.33 ± 0.35i	199.78 ± 9.49l	3203.06 ± 4.81k	582.77 ± 74.39m	573.61 ± 31.82n
Soybean meal	3	4.67 ± 0.02a	182.22 ± 0.97b	327.86 ± 12.10gh	5697.50 ± 41.67d	1850.34 ± 118.45g	4962.50 ± 55.12b
	5	4.64 ± 0.03abc	93.33 ± 2.05i	321.81 ± 6.18gh	5711.39 ± 24.06d	1567.53 ± 43.05j	3761.11 ± 12.03d
	7	4.64 ± 0.04abc	53.33 ± 1.22j	258.51 ± 9.99k	4594.72 ± 12.73h	719.13 ± 97.53m	1177.78 ± 52.43l
Barely	3	4.63 ± 0.02bc	195.55 ± 2.12a	377.56 ± 8.09bc	4111.39 ± 20.97i	2436.14 ± 133.32e	3268.06 ± 52.43g
	5	4.60 ± 0.01c	127.41 ± 1.94f	371.81 ± 5.63c	3994.72 ± 31.55i	1804.89 ± 62.57g	2316.67 ± 72.17j
	7	4.59 ± 0.06c	94.81 ± 2.06hi	359.90 ± 9.75de	3122.50 ± 8.33l	1264.53 ± 64.46j	184.72 ± 12.03o
Wheat	3	4.64 ± 0.03abc	148.15 ± 2.91d	360.50 ± 8.33de	5108.61 ± 9.62f	2678.54 ± 42.15c	3400.00 ± 20.83f
	5	4.63 ± 0.01bc	120.00 ± 1.22g	291.25 ± 9.11i	4833.61 ± 75.61g	1703.89 ± 35.03h	1420.83 ± 20.83k
	7	4.62 ± 0.01bc	56.30 ± 0.46k	262.38 ± 8.73j	3786.39 ± 79.20j	633.28 ± 72.72m	858.33 ± 20.83m
Whole G. biloba seeds	3	4.68 ± 0.02a	184.44 ± 5.08b	384.11 ± 5.78b	6066.94 ± 41.11c	2592.69 ± 4.96d	5191.67 ± 83.33b
	5	4.64 ± 0.05ac	139.26 ± 2.05e	361.29 ± 7.03d	5644.72 ± 127.29d	1284.73 ± 21.90j	2747.22 ± 86.74i
	7	4.64 ± 0.01bc	97.78 ± 1.22hi	343.63 ± 8.84ef	4830.83 ± 8.33g	890.83 ± 8.06l	1163.89 ± 43.37l
G. biloba seeds	3	4.60 ± 0.02c	186.67 ± 5.56ab	398.99 ± 6.62a	6789.17 ± 22.05a	3183.55 ± 4.96a	5629.17 ± 75.12a
	5	4.59 ± 0.01c	164.44 ± 2.05c	373.39 ± 12.52bc	6578.06 ± 45.90b	1658.44 ± 4.96i	2920.83 ± 90.81h
	7	4.57 ± 0.03c	166.68 ± 3.22c	346.61 ± 7.54ef	5214.17 ± 41.67e	951.43 ± 12.12k	1462.50 ± 126.72k

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

, 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. Effects of varying amounts of whole G. biloba seeds on the activities of several enzymes in fermented G. biloba leaves.

Whole G. biloba Seeds (%, w/w)	Time (d)	Enzyme Activity (U/g)
		Amylase	Lipase	Protease	Pectinase	Cellulase	Hemicellulase
2	3	4.40 ± 0.01f	124.44 ± 4.15d	287.88 ± 6.76d	4091.94 ± 5.24k	832.25 ± 31.54l	1504.17 ± 36.08k
	5	4.40 ± 0.01f	108.89 ± 2.90e	292.04 ± 5.85d	4169.72 ± 19.25j	640.35 ± 8.75m	1344.44 ± 12.03l
	7	4.39 ± 0.01f	83.71 ± 0.98i	293.43 ± 5.85d	4189.17 ± 11.02j	539.35 ± 15.15n	1212.50 ± 19.82m
6	3	4.51 ± 0.02c	148.15 ± 5.34c	315.85 ± 5.28c	4880.83 ± 2.08g	1700.86 ± 23.14g	2295.83 ± 21.11g
	5	4.52 ± 0.01c	120.00 ± 2.36d	314.46 ± 6.52c	4872.50 ± 2.08g	1599.85 ± 15.15h	2156.94 ± 18.13i
	7	4.52 ± 0.02c	100.00 ± 1.04f	314.46 ± 5.97c	4922.50 ± 3.61f	1296.85 ± 15.15k	1990.28 ± 31.82j
10	3	4.62 ± 0.02a	192.59 ± 1.78a	378.35 ± 5.63a	6014.17 ± 2.08c	2933.07 ± 15.15a	4997.22 ± 25.89a
	5	4.60 ± 0.03ab	140.00 ± 2.93c	374.78 ± 6.76a	6080.83 ± 5.51b	2549.26 ± 38.13b	4281.94 ± 45.91b
	7	4.61 ± 0.04ab	102.22 ± 2.36f	377.96 ± 5.63a	6166.94 ± 1.20a	2347.26 ± 31.54c	3677.78 ± 104.17d
14	3	4.58 ± 0.03ab	174.07 ± 2.36b	343.83 ± 6.32b	5272.50 ± 4.17e	2306.86 ± 8.75c	4316.67 ± 73.49b
	5	4.56 ± 0.01b	128.89 ± 1.04c	336.09 ± 6.18b	5250.28 ± 7.89e	2024.06 ± 30.30e	3941.67 ± 67.90c
	7	4.57 ± 0.01b	95.56 ± 5.34g	339.27 ± 6.85b	5341.94 ± 1.20d	1912.96 ± 8.75f	3525.00 ± 43.37e
18	3	4.46 ± 0.04d	142.96 ± 5.34c	299.19 ± 8.22d	4608.61 ± 3.18h	1700.86 ± 8.75g	3677.78 ± 20.83d
	5	4.46 ± 0.02d	120.74 ± 2.36d	297.40 ± 6.76d	4525.28 ± 4.81i	1620.05 ± 8.75h	2983.33 ± 31.93f
	7	4.45 ± 0.01e	88.15 ± 1.21h	298.99 ± 6.13d	4555.83 ± 3.61i	1559.45 ± 8.75j	2288.89 ± 21.34g

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

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.

3.3. 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. Effects of pH on the activities of several enzymes in fermented G. biloba leaves.

pH	Time (d)	Enzymes Activity (U/g)
		Amylase	Lipase	Protease	Pectinase	Cellulase	Hemicellulase
3	3	4.36 ± 0.03c	144.44 ± 2.22d	316.65 ± 5.28d	3800.28 ± 29.27h	956.48 ± 25.43f	3865.28 ± 3.18h
	5	4.35 ± 0.01d	115.56 ± 2.22f	317.24 ± 6.18d	3755.83 ± 8.33i	774.68 ± 50.87g	3573.61 ± 5.24k
	7	4.37 ± 0.02c	57.78 ± 2.22i	314.46 ± 5.54d	3741.94 ± 25.46i	491.87 ± 25.43h	3156.94 ± 5.24m
3.5	3	4.41 ± 0.02c	160.75 ± 3.39c	354.94 ± 5.54c	4236.39 ± 12.73g	1148.38 ± 16.65d	4212.50 ± 2.08f
	5	4.41 ± 0.04c	123.70 ± 3.39e	352.36 ± 5.63c	4214.17 ± 22.05g	961.53 ± 25.43f	3816.67 ± 3.61i
	7	4.41 ± 0.01c	76.30 ± 3.39h	352.76 ± 7.03c	4222.50 ± 22.05g	799.93 ± 16.65g	3115.28 ± 7.89n
4	3	4.52 ± 0.01b	186.67 ± 2.22b	371.41 ± 6.85ab	5375.28 ± 17.35e	1365.53 ± 34.66c	4754.17 ± 2.08b
	5	4.53 ± 0.03b	146.67 ± 2.22d	370.81 ± 7.03ab	5325.28 ± 41.11e	1289.78 ± 25.43d	4358.33 ± 4.17e
	7	4.53 ± 0.02b	95.56 ± 2.22g	371.61 ± 5.54ab	5291.94 ± 37.58f	981.73 ± 16.65f	3740.28 ± 9.39j
4.5	3	4.61 ± 0.03a	212.59 ± 7.80a	388.08 ± 5.85a	6361.39 ± 12.73a	1643.28 ± 34.66a	5191.67 ± 4.17a
	5	4.60 ± 0.01a	182.22 ± 2.22b	385.50 ± 5.85a	6378.06 ± 34.69a	1451.38 ± 16.65b	4594.44 ± 3.18d
	7	4.60 ± 0.01a	146.67 ± 2.22d	387.68 ± 7.32a	6330.83 ± 8.33b	1128.18 ± 25.43e	3865.28 ± 3.18h
5	3	4.51 ± 0.02b	182.22 ± 2.22b	363.67 ± 7.62b	5608.61 ± 17.35c	1340.28 ± 34.66c	4670.83 ± 10.42c
	5	4.51 ± 0.02b	147.41 ± 4.63d	369.23 ± 5.54ab	5561.39 ± 17.35d	1128.18 ± 25.43e	4073.61 ± 3.18g
	7	4.51 ± 0.02b	98.52 ± 3.39g	367.64 ± 6.44b	5555.83 ± 14.43d	961.53 ± 25.43f	3538.89 ± 4.34l

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

). 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 × 10⁶ to 7.14 × 10⁶ 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 × 10⁶ 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 × 10⁶ CFU (

Table 5. Effects of inoculum size on the activities of several enzymes in fermented G. biloba leaves.

Inoculum Size (×106 CFU)	Time (d)	Enzymes Activity (U/g)
		Amylase	Lipase	Protease	Pectinase	Cellulase	Hemicellulase
2.38	3	4.55 ± 0.02cd	151.11 ± 2.22d	321.01 ± 6.52g	4819.72 ± 20.97g	1148.38 ± 16.65g	4302.78 ± 4.34h
	5	4.56 ± 0.01cd	115.56 ± 2.22f	318.43 ± 5.85g	4730.83 ± 16.67h	966.58 ± 16.65j	4031.94 ± 3.18i
	7	4.53 ± 0.02d	101.48 ± 3.39g	304.35 ± 7.32h	4655.83 ± 16.67i	744.38 ± 34.66k	3413.89 ± 3.18m
4.76	3	4.61 ± 0.02ab	195.56 ± 2.22b	378.55 ± 6.73c	6080.83 ± 30.05b	1400.88 ± 25.43c	5059.72 ± 3.18c
	5	4.60 ± 0.01b	149.63 ± 3.39de	373.59 ± 6.85cd	6094.72 ± 12.73b	1299.88 ± 16.65d	4344.44 ± 8.42g
	7	4.60 ± 0.01b	100.00 ± 2.22g	362.88 ± 7.88de	6041.94 ± 17.35b	1072.63 ± 16.65g	3747.22 ± 3.18k
7.14	3	4.66 ± 0.03a	223.70 ± 3.39a	395.62 ± 5.63a	6305.83 ± 30.05a	1587.73 ± 76.30a	5434.72 ± 4.34a
	5	4.66 ± 0.01a	191.85 ± 3.39bc	351.57 ± 39.33e	6316.94 ± 17.35a	1304.93 ± 25.43d	4552.78 ± 3.18e
	7	4.65 ± 0.02a	154.07 ± 3.39d	383.31 ± 6.85b	6316.94 ± 19.25a	1118.08 ± 33.30g	3809.72 ± 3.18j
9.52	3	4.58 ± 0.01bc	199.26 ± 4.63b	355.54 ± 6.52e	5233.50 ± 16.67d	1451.38 ± 16.65b	5143.06 ± 3.18b
	5	4.58 ± 0.01bc	183.70 ± 3.39c	343.63 ± 9.23f	5333.61 ± 25.46c	1264.53 ± 25.43ef	4504.17 ± 2.08f
	7	4.58 ± 0.02bc	135.56 ± 4.44e	336.09 ± 6.18f	5297.50 ± 41.67c	991.83 ± 25.43j	3650.00 ± 5.51l
11.90	3	4.51 ± 0.01e	188.15 ± 3.39c	312.48 ± 7.62g	4925.28 ± 33.68e	1229.18 ± 50.87f	4608.33 ± 2.08d
	5	4.52 ± 0.01e	143.70 ± 3.39e	302.56 ± 6.52h	4861.39 ± 17.35f	1052.43 ± 41.90h	4025.00 ± 4.17i
	7	4.51 ± 0.02e	100.74 ± 3.39g	297.20 ± 6.52h	4700.28 ± 4.81h	774.68 ± 50.87k	3663.89 ± 4.34l

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

). 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 × 10⁶ 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. Effects of the water content on the activities of several enzymes in fermented G. biloba leaves.

Water Content (%, w/w)	Time (d)	Enzymes Activity (U/g)
		Amylase	Lipase	Protease	Pectinase	Cellulase	Hemicellulase
35	3	4.51 ± 0.02c	173.18 ± 2.22b	375.43 ± 6.13a	5804.44 ± 17.35c	1273.27 ± 9.61b	4486.47 ± 2.08c
	5	4.51 ± 0.02c	145.23 ± 2.22d	373.88 ± 5.85a	5712.28 ± 8.33d	877.88 ± 16.65e	3989.29 ± 3.18h
	7	4.52 ± 0.01c	92.77 ± 3.39g	372.19 ± 5.85a	5688.93 ± 12.73e	812.65 ± 16.65f	3398.34 ± 3.18k
45	3	4.60 ± 0.02a	194.07 ± 3.39a	378.55 ± 6.76a	6111.39 ± 20.97a	1431.18 ± 9.61a	4934.72 ± 5.24a
	5	4.60 ± 0.02a	147.41 ± 3.39d	378.15 ± 8.25a	6116.94 ± 12.73a	1289.78 ± 25.43b	4275.00 ± 4.17e
	7	4.60 ± 0.01a	102.22 ± 2.22f	374.98 ± 6.44a	6072.50 ± 16.67b	1077.68 ± 34.66c	3691.67 ± 4.17i
55	3	4.59 ± 0.03ab	176.30 ± 3.39b	378.35 ± 5.28a	5808.61 ± 120.57c	1284.73 ± 16.65b	4518.06 ± 5.24b
	5	4.57 ± 0.02ab	128.15 ± 3.39e	375.77 ± 6.13a	5730.83 ± 22.05d	900.93 ± 25.43e	4031.94 ± 3.18f
	7	4.57 ± 0.01ab	82.96 ± 3.39h	373.59 ± 5.85a	5708.61 ± 17.35d	830.23 ± 16.65f	3608.33 ± 2.08j
65	3	4.51 ± 0.01c	162.96 ± 3.39c	360.30 ± 7.32b	5553.06 ± 133.94f	1269.58 ± 16.65b	4483.33 ± 4.17c
	5	4.52 ± 0.05bc	128.89 ± 2.22e	357.12 ± 6.44b	5355.83 ± 8.33h	1102.93 ± 16.65c	3920.83 ± 2.08g
	7	4.52 ± 0.02c	80.00 ± 2.22h	341.05 ± 5.85c	5403.06 ± 12.73g	850.43 ± 9.61f	3406.94 ± 4.34
75	3	4.50 ± 0.02c	142.96 ± 3.39d	354.74 ± 5.85b	4886.39 ± 4.81j	1077.68 ± 50.87c	4330.56 ± 6.36d
	5	4.51 ± 0.04c	105.19 ± 4.63f	341.65 ± 5.85c	4847.50 ± 44.10jk	966.58 ± 16.65d	3615.28 ± 4.34j
	7	4.50 ± 0.03c	74.07 ± 3.39j	328.15 ± 6.13d	4836.39 ± 29.27k	825.18 ± 34.66f	2955.56 ± 3.18l

Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

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 × 10⁶ 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.

3.4. 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. The main components in the G. biloba leaves before and after fermentation.

	Proteins (mg/g)	FAAs (ug/g)	RS * (ug/g)	PPAs (mg/g)	Gas * (%)	Cellulose (%)	Lignin (%)
Before	69.75 ± 0.001b	2708.33 ± 0.02a	6214.29 ± 0.01a	40.08 ± 0.02b	2.33 ± 0.01a	60.92 ± 0.00a	1.82 ± 0.03a
After	115.03 ± 0.001a	236.11 ± 0.07b	3285.71 ± 0.01b	44.34 ± 0.01a	1.11 ± 0.02b	48.70 ± 0.03b	1.12 ± 0.05b

* RS represents reducing sugars and GAs represent ginkgolic acids. Note: Different lowercase letters in the same column indicate that the values are significantly different (α = 0.05).

. 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. The antioxidant activities of G. biloba leaves before and after fermentation.

Extraction Solvent		ABTS Free Radical Scavenging Capability (%)	DPPH Free Radical Scavenging Capability (%)	Total Reduction Capacity
Water	Before	1.540 ± 0.002	0.739 ± 0.001	0.282 ± 0.004
	After	2.269 ± 0.001	0.898 ± 0.001	0.360 ± 0.002
80% Methanol	Before	3.943 ± 0.001	2.217 ± 0.001	0.263 ± 0.004
	After	4.592 ± 0.001	2.652 ± 0.001	0.332 ± 0.001
80% Ethanol	Before	3.241 ± 0.001	2.304 ± 0.001	0.292 ± 0.007
	After	4.322 ± 0.001	2.579 ± 0.001	0.340 ± 0.002
80% Acetone	Before	2.809 ± 0.001	2.449 ± 0.001	0.275 ± 0.001
	After	3.566 ± 0.001	2.782 ± 0.001	0.373 ± 0.003

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.

4. Conclusions

In this work, solid-state fermentation of G. biloba leaves using E. cristatum was fulfilled successfully. The spore number, main biologically active components, and several digestive enzymes’ activities were chosen as the indexes to optimize the medium components and fermentation conditions. The optimal medium included 10% (w/w) whole G. biloba seeds in the medium. The optimal water content, pH, inoculum size, and fermentation time for the growth of E. cristatum were found to be 45%, 4.5, 4.76 × 10⁷ CFU/100 g wet medium, and eight days, respectively. After fermentation, the spore number increased by about 40 times. The content of the TTLs remained stable. The content of flavonoids was enhanced up to 31.57 ± 0.04 mg/g, which was much higher than the original content of 14.44 ± 0.06 mg/g. The contents of protein and PPAs increased by 64.9% and 10.6%, respectively. The antioxidant activity was also improved. The fermented leaves had more kinds of aroma substances than the unfermented leaves. The ginkgolic acids, lignin, and cellulose decreased by 52.4%, 38.5%, and 20.1% than before, respectively. The hydrolysis of crude fibers by the enzymes was salutary for boosting taste and promoting the digestion of feed in animals. Therefore, the solid-state fermentation of G. biloba leaves using E. cristatum is expected to be a neoteric approach for the production of fermented feed.

5. Patents

A Chinese patent named as “a method of solid-state fermentation of G. biloba leaves using E. cristatum and its application” (CN201710734672.9) has been published.