Solid state fermentation of Ginkgo biloba leaves and its effects on antioxidant capacity and breast muscle quality of Broilers

doi:10.21203/rs.3.rs-7833307/v1

Ginkgo biloba leaves, abundant in various bioactive ingredients, have promising applications as animal feed additives through microbial fermentation. In this study, we conducted an orthogonal optimization experiment with 7 factors and 3 levels using Ginkgo biloba leaves as the substrate, employing two probiotic strains Bacillus licheniformis and B. natto as the fermentation strains, respectively. The results indicated that the fermented leaves were enriched with probiotics and demonstrated ehanced levels of flavonoids, total amino acids, crude protein and aroma substances. Additionally, the palatability of the Ginkgo biloba leaf fermentation product as a feed additive was significantly improved. Specifically, the flavonoid content increased by 16.7% and 10.6%, total amino acids by 17.22% and 31.05%, total protein by 43.83% and 58.74%, and protease activities reached 1307 U/g and 1510 U/g, for B. licheniformis and B. natto, respectively. Furthermore, supplementation with 0.3% to 0.6% (mass fraction) of the Ginkgo biloba leaf fermentation product in broiler diets significantly improved feed efficiency to a certain extent, with the most significant improvement observed when 0.3% or 0.6% of B. natto fermented product was added (P < 0.05). Through biological solid-state fermentation and conversion, the biological activity, nutritional value, and palatability of Ginkgo biloba leaves were significantly enhanced. When used as a Ginkgo biloba leaf feed additive, it promoted the development of the thymus and spleen, improved the antioxidant capacity of broilers, and enhance muscle quality to a certain extent, thereby establishing a new pathway for the efficient and high-value utilization of Ginkgo biloba leaves.

Ginkgo biloba leaves

solid state fermentation

Bacillus

feed additive

antioxidation

1. Fermentation of leaves significantly increased bioactive compounds.

2. The fermented product demonstrated enhanced palatability and feed efficiency.

3. Supplementation with the fermented product promoted the boosted broiler health.

4. Solid-state fermentation transformed leaves into a high-value feed.

Graph Abstract

For an extended period, the issues of drug residues and food safety caused by the abuse of antibiotics in livestock and poultry growth have become increasingly prominent. To safeguard human food safety, the Ministry of Agriculture and Rural Affairs of China has instituted a complete ban on the use of antibiotics in animal feed, effective from January 1, 2020. In this content, forest-derived feed additive products have received widespread attention [1–3]. Forest-derived plants and their extracts, with their advantages of being purely natural, non-toxic and side-effect-free, and non-inducing antibiotic resistance, have increasingly become research hotspots of research as alternatives to antibiotics [4–5].

Ginkgo biloba, as an esteemed traditional medicinal plant, is abundantly resourced in China. Ginkgo leaves are replete with various active ingredients, including flavonoids, terpenes, polysaccharides, organic acids, sterols, and other substances [6–7]. Ginkgo biloba extract, which is rich in ginkgo flavonoids and ginkgolides, has been widely used in the food, health care, cosmetics, and pharmaceutical industries [8–10]. In recent years, Ginkgo biloba extract has also been utilized as a feed additive in livestock and poultry feeding. Tan et al. investigated the effects of supplementing Ginkgo biloba extract (GBE) in high-fat diets on growth performance, plasma biochemical indices, liver histology, and other aspects of hybrid grouper [11]. The results revealed that incorporating 0.50 to 4.00 g/kg of GBE in the diet effectively increased the plasma high-density lipoprotein (HDL) content of the fish, decreased the low-density lipoprotein (LDL) and triglyceride (TG) contents, and exerted significant effects on the moisture, crude protein, and lipid content in the liver, as well as the protein content of the whole fish. Yang et al. conducted to investigate the potential of GBE in alleviating fatty liver hemorrhage syndrome (FLHS) in laying hens, which indicated that GBE improved the biochemical blood indices in a laying hen model of FLHS induced by a high-fat diet [12].

However, the traditional solvent extraction method for Ginkgo biloba extract often results in the loss of multiple active ingredients and increases both preparation and usage costs. Alternativelu, microbial fermentation of Ginkgo biloba leaves offers a superior approach for better preserving and releasing its active ingredients, enabling the utilization of the entire biomass. Zhang et al. demonstrated that broiler chickens fed with Ginkgo biloba leaf feed additives fermented by Candida utilis (CF group) and Aspergillus niger (AF group) exhibited capacity and a healthier intestinal microbial ecology [13]. Guo et al. utilized Bacillus natto to ferment Ginkgo biloba seeds, yielding a product with a higher total flavonoid content and lower ginkgolic acid content, thereby enhancing the bioactivity of Ginkgo biloba and ensuring food safety [14]. Cao et al. discovered that Ginkgo biloba leaf fermentate, used as a feed additive, significantly promoted the weight gain rate of yellow-feathered chickens and facilitated the development of immune organs such as the spleen and thymus [15]. Furthermore, Cao et al. incorporated a composite fermentate of Ginkgo biloba and Eucommia ulmoides leaves to the diets of broilers, which improved the antioxidant capacity and breast muscle quality [16].

Bacillus represents a crucial class of microbial resources, recognized as a safe microorganism approved for use in the feed industry. This bacterium has the remarkable ability to produce an array of digestive enzymes, including amylase and protease, within the intestinal tract of animals. These enzymes not only aid in digestion but also provide animals with a multitude of vitamins and other essential nutrients [17–19]. Consequently, Bacillus has been officially included in the feed additive list by the Ministry of Agriculture of China, reflecting its importance and safety in animal feed. Notably, Bacillus exists in a resilient spore form, which possesses exceptional tolerance to harsh external conditions such as high temperatures encountered during feed pelletizing. This robust nature ensures its stability and effectiveness, thereby presenting vast potential for widespread development and application [20].

In this study, we conducted microbial solid-state fermentation experiments on Ginkgo biloba leaves using B. licheniformis and B. natto as the fermentation strains, respectively. We optimized the conditions for solid-state fermentation, analyzed the changes in active ingredients of Ginkgo biloba before and after fermentation, and investigated the effects of Ginkgo biloba fermentate on the antioxidant capacity and meat quality of broiler chickens to obtain a non-polluting, high-activity, and multifunctional biological feed additive. The results will lay the foundation for the preparation, promotion, and application of Ginkgo biloba-based biological feed additives.

Strains and Media

B. licheniformis and B. natto were purchased from the Microbial Species Preservation and Management Center of the Institute of Microbiology, Chinese Academy of Sciences (Tianjin, China), and preserved by the Microbial Technology Research Laboratory of the College of Chemical Engineering, Nanjing Forestry University. The Ginkgo biloba leaves were sourced from Ginkgo Gardens in Pizhou, Jiangsu Province, China. Soybean meal and wheat bran were commercially purchased in Nanjing, China.

Culture medium for strains: 0.5% beef extract, 1.0% peptone, and 0.5% sodium chloride by mass. For solid culture medium, 2.0% agar by mass was added.

Fermentation medium: consisting of Ginkgo biloba leaf powder, soybean meal, and wheat bran in a mass ratio of 14:3:3. After thorough mixing, nutrient salts were added (1.50% glucose, 0.30% dipotassium phosphate, 0.05% magnesium sulfate heptahydrate, and 1.50% peptone by mass). The Ginkgo biloba leaves were naturally air-dried, crushed, and sieved through a 60-mesh sieve for use.

Experimental animals

300 AA broiler chickens (1-day-old healthy) of the same batch and genetic background were selected and purchased from Shandong Yantai Sujiali Poultry Industry Co., Ltd. (Shandong, China) with an average body weight of 50.00 ± 3.56 g, and the difference was not significant (P > 0.05).

Experimental instruments and equipment

D-1 Auto Clave automatic high-temperature and high-pressure steam sterilizer (Beijing Fanke Trade Co., Ltd., China); 5415R benchtop high-speed refrigerated centrifuge (Eppendorf, Germany); ZHWY-2102C shaker (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., China); MH-350 constant temperature and humidity light incubator (Panasonic, Japan); SpectraMax190 full-wavelength microplate reader (Molecular Devices, USA); 65 µm PDMS/DVB solid-phase microextraction (SPME) fiber and 15 mL SPME vial (Nanjing Dahu Science and Technology Co., Ltd., China); Salter shear force tester (GR, USA); CHROMA METER CR-400 colorimeter (Konica Minolta, Japan).

Experimental Methods

Orthogonal experiment for optimizing fermentation medium

A loop of Bacillus was picked from the slant culture medium and inoculated into 50 mL of sterile seed culture medium, and cultured at 37°C with shaking at 180 r/min for 24 h. Using viable cell count and protease activity as indicators, and with B. licheniformis and B. natto as the strains, the fermentation conditions such as carbon source, nitrogen source, inoculum size, solid-to-liquid ratio, initial pH, fermentation time, and fermentation temperature were studied.

Viable cell and spore counting

One gram of fermented sample was weighed and placed in 20 mL of sterile physiological saline. It was oscillated at 37°C and 180 r/min for 30 min to disperse it uniformly. The dispersed sample was then serially diluted, and 0.2 mL of an appropriate dilution was spread onto sterile beef extract-peptone agar plates. 3 parallel samples were set up and cultured upside down at 37°C for 24 h, followed by colony counting. The dispersed sample was placed in an oven at 80°C for 15 min. Then, 0.2 mL of appropriately diluted solution was spread onto sterile beef extract-peptone solid culture medium. 3 parallel samples were set up and cultured upside down at 37°C for 24 h, followed by spore counting.

Enzyme extraction and protease activity assay

One gram of air-dried fermented sample was weighed and placed in 20 mL of sterile physiological saline. It was oscillated at 37°C and 200 r/min for 30 min. The suspension was then filtered to obtain the crude enzyme extract. The protease activity was assayed according to the Chinese national professional standard SB/T 10317 − 1999 "Method for Determination of Protease Activity".

Detection of aroma compound content

A certain amount of air-dried Ginkgo biloba leaf fermentate was placed in a 15 mL SPME vial. A 65µm PDMS/DVB SPME fiber was inserted from the top of the vial, and after extraction for a certain period, gas chromatography-mass spectrometry (GC-MS) analysis was performed. Chromatographic conditions were as follows: injector temperature of 250°C; oven temperature program: held at 40°C for 1 min, increased to 64°C at a rate of 2°C/min, then increased to 160°C at a rate of 3°C/min, further increased to 250°C at a rate of 10°C/min, and held for 5 min; carrier gas: 99.99% high-purity helium, carrier gas flow rate: 1 mL/min; chromatographic column: TR-5MS capillary column. Mass spectrometry conditions: EI ion source, temperature of 250°C, ionization voltage of 70 eV, interface temperature of 250°C, quadrupole temperature of 150°C [21].

Extraction and detection method of Ginkgo flavone content

5 grams of fermented sample were taken, and 70% volume fraction ethanol was used as the extraction solvent. Under the condition of a solid-to-liquid ratio of 1:30 (g:mL), ultrasonic extraction was performed for 30 min. The extract was filtered to obtain the sample solution for analysis. A 5 mL aliquot of the sample solution was accurately pipetted into a 10 mL stoppered test tube, with distilled water used as a blank control. To each tube, 0.3 mL of 5% sodium nitrite solution was added, allowed to stand for 6 min, then 0.3 mL of 10% aluminum nitrate solution was added, allowed to stand for another 6 min, followed by the addition of 4 mL of 4% sodium hydroxide, and the volume was adjusted to 10 mL with distilled water. After standing for 15–20 min, the absorbance at A₅₁₀ was measured. The flavonoid content was calculated using the standard curve equation: A = 8.0581C − 0.0012 (R² = 0.9995), where A is the absorbance at 510 nm and C is the rutin mass concentration.

Detection of crude protein and amino acid content

The crude protein and amino acid content in the fermented samples were determined according to GB/T 6432 − 1994 "Method for Determination of Crude Protein in Feeds" and GB/T 18246 − 2000 "Method for Determination of Amino Acids in Feeds".

Experimental animal handling and feed design

The 300 1-day-old healthy AA broiler chickens were randomly divided into 5 treatment groups: a control group fed a basic diet, a 0.3% B. licheniformis-fermented Ginkgo biloba leaf group (0.3% BL group), a 0.6% B. licheniformis-fermented Ginkgo biloba leaf group (0.6% BL group), a 0.3% B. natto-fermented Ginkgo biloba leaf group (0.3% BN group), and a 0.6% B. natto-fermented Ginkgo biloba leaf group (0.6% BN group). The experiment lasted for 42 days, with each treatment group comprising 6 replicates of 10 chickens each. A corn-soybean meal-based basic diet was used, and the experiment employed a weight substitution method. In the treatment groups, Ginkgo biloba leaf fermentate was added by replacing an equivalent amount of wheat bran. The diet composition and nutritional levels are shown in Table 1. The chickens were housed in cages with ad libitum access to feed and water. The room temperature was initially maintained at 34–36°C for the first 14 days and then gradually lowered to around 26°C. The lighting schedule was initially 24 h and gradually reduced to 12 h per day.

Measurement indicators and methods for experimental animals

Production performance: Daily feed consumption was recorded. Chickens were weighed on days 1, 21, and 42 after overnight fasting. Average daily feed intake (FI), average daily gain (BWG), and feed conversion ratio (F/G) were calculated.

Serum biochemical indicators: On day 42, six chickens (3 males and 3 females) were randomly selected from each treatment group. Blood samples were collected from the jugular vein and centrifuged at 4500 r/min for 15 min at 4°C to obtain serum. Serum samples were stored at -80°C until analysis. Total protein and albumin were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute). α-tocopherol (α-TOH) content was determined using an enzyme method with a standard curve [22].

Antioxidant indicators in breast muscle: A 0.5 g sample of breast muscle tissue, free of fat and connective tissue, was homogenized with 4.5 mL of pre-cooled physiological saline in an ice bath for 1–2 min. The homogenate was centrifuged at 4500 r/min for 15 min at 4°C, and the supernatant was stored at -20°C for subsequent analysis of muscle antioxidant function and fatty acid composition. Malondialdehyde (MDA) content in serum and liver was measured using the thiobarbituric acid method [23]. Glutathione peroxidase (GSH-Px) activity was determined according to Noguchi et al [24]. Total superoxide dismutase (T-SOD) activity was assessed using the xanthine oxidase method, and total antioxidant capacity (T-AOC) was evaluated using the Fe³⁺ reduction method..

Breast muscle pH and water-holding capacity: One side of the breast muscle was quickly excised and placed on ice. The muscle pH was measured at 45 min and 24 h post-mortem after homogenizing in a mixture of 5 mmol/L iodoacetic acid and 150 mmol/L potassium chloride. For drip loss measurement, a 10 g sample of breast muscle was suspended in a 4°C refrigerator, weighed (W₁), sealed to prevent evaporation, and reweighed after 24 h (W₂) and 48 h (W₃). Drip loss was calculated as follows:

24-h drip loss rate = (W₁ - W₂) / W₁ × 100% (1)

48-h drip loss rate = (W₁ - W₃) / W₁ × 100% (2)

Cooking loss and shear force: Ten-gram samples of breast and leg muscle were wrapped in aluminum foil and cooked in a 75°C water bath for 15 min. The samples were then weighed to determine cooking loss. For shear force measurement, breast muscle samples were sealed in plastic bags and heated in an 80°C water bath until the core temperature reached 74°C. After cooling to room temperature, each sample was cut into 3–5 strips (1 cm × 1 cm × 3 cm, length × width × thickness) parallel to the muscle fibers. Shear force was measured three times using a shear force tester, and the average value was recorded.

Color measurement: Samples of breast muscle (2 cm × 2 cm × 2 cm, length × width × thickness) were prepared, and a colorimeter was used to record the lightness (L), redness (a), and yellowness (b*) values of both breast and leg muscles.

Fatty acid composition of breast muscle: A 0.2 g sample of freeze-dried breast muscle was combined with 1 mL of internal standard and 4 mL of NaOCH₃/methanol. The mixture was incubated at 50°C for 20 min, followed by the addition of 2 mL of hydrochloric acid/methanol solution and incubation at 80°C for 1 h. After cooling to room temperature, 2 mL of water and 6 mL of hexane were added, and the mixture was vigorously shaken and allowed to separate. A 200 µL aliquot of the upper layer was combined with 600 µL of hexane for GC analysis. GC conditions were as follows: column: 0.22 mm inner diameter, 30 m length; column temperature: 195°C; carrier gas: nitrogen at 9 mL/min; injector temperature: 250°C; hydrogen flow rate: 22.5 mL/min; air flow rate: 80 mL/min; split ratio: 60:1; injection volume: 0.5-1 µL.

Total fatty acids (mg/g) = (total peak volume - internal standard volume) / internal standard volume × (mass of internal standard) (3)

Statistical Analysis

Data were organized using Excel and subjected to one-way Analysis of Variance (ANOVA) using SPSS 6.0. When significant differences were observed, multiple comparisons were performed using Duncan’s method. The significance level was set at P < 0.05. All results are presented as mean values.

Results of orthogonal experiment for Bacillus

Orthogonal experimental design is an efficient and systematic approach for conducting multi-factor experiments, which systematically considers multiple factors and their interactions to ensure the comprehensiveness and reliability of experimental results. In this study, an orthogonal experimental design was employed to optimize the cultivation conditions for two Bacillus species, with the results presented in Table 2. This approach systematically considers multiple factors and their interactions to ensure comprehensive and reliable experimental results. The order of significance in influencing the fermentation of Ginkgo biloba leaves was: nitrogen source>carbon source>inoculum size>fermentation temperature>solid-to-liquid ratio>initial pH>fermentation time. The optimal fermentation conditions were: 1.5%(mass fraction) glucose, 1.5% urea, 10% inoculum size, solid-to-liquid ratio of 1.2:1 (g:mL), initial pH of 7, fermentation time of 48 h, and fermentation temperature of 37°C. After fermentation with B. licheniformis, the viable bacterial count in the sample reached 2.189×10¹⁰ CFU/g, and the protease activity was 1307 U/g. While for B. natto, the viable bacterial count in the sample reached 2.840×10¹⁰ CFU/g, and the protease activity was 1510 U/g.

Results of aroma compounds

The release of aroma compounds is crucial for enhancing product palatability in food, feed additives, and related items [25].These compounds improve sensory properties, making products more appealing. Whether it's through natural fermentation processes, the addition of specific aroma extracts, or other methods, the manipulation and enhancement of aroma release is an important aspect in product development and optimization. The results of the aroma compound content in Ginkgo biloba leaf samples fermented by Bacillus were shown in Table 3 and Fig.1. B. licheniformis fermentation increased ketone (e.g., 3-hydroxy-2-butanone) and alcohol (e.g., benzyl alcohol, phenylethanol) aroma compounds, with total aroma compounds increasing by 98.68%. B. natto fermentation increased alcohol and ester (e.g., methyl 2-amino-6-methylbenzoate) aroma compounds, with a 95.26% total increase. These results suggest Bacillus fermentation significantly improves the palatability of Ginkgo biloba leaf bio-feed additives.

Measurement of indicators in Ginkgo biloba leaf fermentates by Bacillus

The results of flavonoid content, sporulation rate, amino acid content, and crude protein content in Ginkgo biloba leaf fermentates before and after fermentation by Bacillus were shown in Table 4. As shown in Table 4, the flavonoid content in the Ginkgo biloba leaf fermentates of B. licheniformis and B. natto increased by 16.7% and 10.6% respectively, compared to before fermentation. The sporulation rates were 82.7% and 84.2%, respectively. The total amino acid content increased by 17.22% and 31.05%, respectively, and the crude protein content increased by 43.83% and 58.74%, respectively. Bacillus fermentation not only enriched the Ginkgo biloba leaf fermentates with probiotics but also significantly increased flavonoid extraction yield and enhanced amino acid and crude protein content.

Effect of fermentates on the production performance of AA broiler chickens

The effect of Ginkgo biloba leaf bio-feed additives on the production performance of broiler chickens was shown in Fig.2. Dietary inclusion of 0.3% or 0.6% fermentates from either B. licheniformis or B. natto significantly improved feed efficiency from days 21 to 42 and overall (days 1 to 42). B. natto fermentates at 0.3% or 0.6% showed the most significant improvement (P<0.05).

The immune organ index is commonly used as an indirect indicator to reflect the immune function of animal organisms, which including the thymus, spleen, and bursa of Fabricius, are responsible for executing immune functions, and their developmental status directly influences the level of immune competence in the organism [26].As shown in Fig. 3, at day 21, the dietary inclusion of 0.3% or 0.6% of B. natto had the most significant effect on increasing the spleen index of broiler chickens. At day 42, the thymus index significantly increased in the groups with the addition of 0.6% B. licheniformis and 0.3% to 0.6% of B. natto Ginkgo biloba leaf fermentates. Additionally, at day 21, the bursa of Fabricius index significantly increased in the groups with the addition of 0.3% B. licheniformis and 0.6% B. natto Ginkgo biloba leaf fermentates.

The impact of fermentation products on abdominal fat deposition and biochemical metabolism

The beneficial microorganisms and their metabolites in microbial fermented feed can regulate animal fat metabolism, reducing abdominal fat deposition. These beneficial microorganisms inhibit the growth of harmful microorganisms through competition, improving the intestinal microecological environment and enhancing the absorption and utilization rate of nutrients by animals. The addition of fermentation products had no effect on the serum total protein content (Table 5), while the serum total cholesterol (TC) content and abdominal fat rate were significantly decreased in the 0.6% BL group and the 0.3% and 0.6% BN groups (P<0.05). Both the 0.6% BL group and the 0.6% BN group significantly reduced the serum triglyceride (TG) content in broilers (P<0.05), with the 0.6% BN group showing a significantly higher reduction compared to the control group and the 0.3% BL group. Both the 0.3% and 0.6% BL groups, as well as the BN groups, significantly increased the serum high-density lipoprotein cholesterol (HDL-C) levels and decreased the serum low-density lipoprotein cholesterol (LDL-C) concentrations in broilers (P<0.05).

The impact of fermentation products on antioxidant capacity and lipid peroxidation levels in AA broiler chickens

The fermented Ginkgo biloba leaves are rich in various antioxidant components, such as Ginkgo flavonoids and ginkgolides, which may undergo better release and transformation during the fermentation process, thereby enhancing their antioxidant effects [27-28].When consumed by broilers, these antioxidants can improve overall antioxidant capacity by: 1. Increasing serum antioxidant enzyme activity (T-SOD, CAT, GSH-Px) 2. Reducing serum lipid peroxidation products (e.g., MDA). It was presented in Table 6 that the effects of Ginkgo biloba fermentation products from BL and BN groups on antioxidant function and lipid peroxidation in the serum and liver of 42-day-old broiler chickens. Ginkgo biloba fermentation products in the 0.6% BL or BN groups significantly increased the activity of T-SOD and the concentration of GSH-Px (P<0.05). The content of MDA was significantly reduced in both the 0.6% BL and BN groups with Ginkgo biloba fermentation products (P<0.05). There was no significant difference in GSH-Px activity (P>0.05). Dietary supplementation with 0.3% and 0.6% Ginkgo biloba fermentation products from either BL or BN groups significantly increased the total antioxidant capacity (T-AOC) levels. Dietary treatments had significant effects on liver T-SOD, T-AOC, and MDA content (P<0.05). Specifically, adding 0.6% Ginkgo biloba fermentation products from the BL group or 0.3% and 0.6% from the BN groups to broiler diets significantly increased liver T-SOD activity and T-AOC levels (P<0.05), while the content of lipid peroxide MDA was significantly decreased (P<0.05).

The impact of fermentation products on breast muscle quality

Drip loss is an important indicator of muscle water-holding capacity [29].Good water-holding capacity preserves muscle moisture and texture during processing. Lower cooking loss indicates better moisture retention during cooking, preserving taste and nutritional value [30].As shown in Table 7, dietary treatments had significant effects on breast muscle quality. Dietary supplementation with 0.6% Ginkgo biloba fermentation products from either the BL or BN group significantly increased the pH_24h of the breast muscle and reduced shear force. The drip loss at 24 h and 48 h in the 0.6% BN group with Ginkgo biloba fermentation products was significantly lower compared to the control group. Additionally, dietary supplementation with 0.3% and 0.6% Ginkgo biloba fermentation products from either the BL or BN groups significantly decreased the cooking loss of the breast muscle.

Impact of Bacillus fermentation on the active components of Ginkgo biloba leaves

Ginkgo biloba resources are widely distributed in China, with its main medicinal value found in its leaves and fruits. To date, researchers have isolated over 160 bioactive components from Ginkgo biloba leaves, including flavonoids, terpene lactones, alcohols, and sterols [31]. Probiotic Bacillus species possess important stress resistance properties and can effectively regulate the intestinal flora balance of animals when used in animal farming, improving feed utilization rates. Their application in microecological preparations, due to their non-toxic and non-polluting characteristics, holds broad prospects for development in the feed industry [32]. In this study, we investigated the fermentation effects of Ginkgo biloba leaves using B. licheniformis and B. natto as fermentation strains. Carbon and nitrogen sources are crucial components of the fermentation medium and important nutrients for microbial metabolites. We employed an Orthogonal experimental design to optimize the fermentation medium for the two Bacillus strains, aiming to increase the viable cell count and protease activity in the fermentation products. Zheng et al. optimized the Bacillus NTGB-178 high-yield spore fermentation process, achieving a spore yield of 6.16×10⁹ CFU/mL in the fermented broth [33]. In this sudy, through Orthogonal experimental method optimization, the Ginkgo biloba leaves fermented by B. licheniformis reached a viable cell count of 2.189×10¹⁰ cells/g and a protease activity of 1307 U/g, while those fermented by B. natto achieved a viable cell count of 2.84×10¹⁰ cells/g and a protease activity of 1510 U/g. After fermentation, the Bacillus strains grew abundantly and decomposed the fibers and flavonoids in Ginkgo biloba leaves, significantly increasing the crude protein and amino acid content in the fermentation products. Specifically, the results showed that the flavonoid content of the B. licheniformis and B. natto fermentation products increased by 16.7% and 10.6%, respectively; the total amino acid content increased by 17.22% and 31.05%, respectively; and the crude protein content increased by 43.83% and 58.74%, respectively. These improvements significantly enhanced the probiotic content, palatability, and nutritional value of the fermented products.

Furthermore, the release of aroma compounds can improve the palatability of food and feed additives. After fermentation by B. licheniformis and B. natto, a total of 18 and 15 aroma compounds, including alcohols, ketones, esters, and others, were detected. The contents of ketone and alcohol aroma compounds in the Ginkgo biloba leaf fermentation products of B. licheniformis were significantly higher than those in the pre-fermentation samples. The contents of alcohol and ester aroma compounds in the Ginkgo biloba leaf fermentation products of B. natto were significantly higher, either. For instance, limonene, a common top-note fragrance, increased by 65.5% after fermentation by B. licheniformis. Phenylethanol, which produced a sweet rose aroma. It was not detected in the pre-fermentation samples but significantly increased (6.23%) after fermentation by B. natto. These results demonstrate that Bacillus-fermented Ginkgo biloba leaf fermentation products have a significant effect on improving the palatability of Ginkgo biloba bio-feed additives.

Impact of Bacillus-fermented Ginkgo biloba leaf products on the production performance and antioxidant function of AA broilers

Ginkgo biloba leaves treated with Bacillus fermentation are rich in enzymes, growth factors, and active components. Moreover, the macromolecules such as cellulose, starch, and protein in Ginkgo biloba leaves are degraded to some extent into small molecules that are easily digested by animals. When added to broiler feed, these fermented Ginkgo biloba leaves improve the digestibility and absorption rate of the feed. Additionally, the nutrient-rich microbial biomass protein and metabolites produced during the Ginkgo biloba leaf fermentation process can alter the physicochemical properties of the feed, enhancing its digestibility, absorption rate, and nutritional value. Our findings are consistent with previous research. Cao et al. reported that adding Ginkgo biloba leaf-eucommia ulmoides leaf fermentation products to the diet significantly improved broiler feed efficiency, digestive enzyme activity, and growth performance [34]. Tanaka et al. found that adding Bacillus subtilis fermentation products to the diet significantly increased broiler body weight, feed intake, and feed efficiency [35]. This experiment found that adding 0.3% or 0.6% of B. licheniformis or B. natto Ginkgo biloba leaf fermentation products to the diet significantly improved broiler feed efficiency, thymus index, spleen index, and bursa of fabricius index. We attribute these improvements to two main factors: first, the fermented Ginkgo biloba leaves eliminate certain anti-nutritive substances in the feed and produce beneficial metabolites, thereby improving the palatability of the Ginkgo biloba leaf fermentation products. Second, the flavonoids and lactone active components in the Ginkgo biloba leaf fermentation products have antioxidant and immune-enhancing effects, suggesting great potential for development and lasting competitiveness in livestock and poultry farming.

In addition, the results showed that adding Ginkgo biloba leaf fermentation products to broiler diets could increase the activity of T-SOD and T-AOC in breast muscle and reduce the concentration of LDL-C in broiler serum. This may be related to the regulation of cellular free radicals and antioxidant balance by the total flavonoids in Ginkgo biloba leaf fermentation products. As active substances with a polyphenolic structure, flavonoid compounds exhibit high antioxidant activity, stabilizing the free radicals generated within cells and exerting an antioxidant effect.

Impact of Bacillus-fermented Ginkgo biloba leaf products on the meat quality of AA broilers

The pH of muscle can affect meat quality characteristics. A rapid decrease in pH after slaughter can lead to protein denaturation, resulting in pale meat. In this experiment, the pH of breast muscle 24 h after slaughter increased with the addition of Ginkgo biloba leaf fermentation products to the diet, and there was a linear relationship between the increase in fermentation products in the diet and the pH. Furthermore, we evaluated meat color, an important indicator for assessing the appearance of muscle in meat products. Meat color is an important indicator for evaluating the appearance of muscle in meat products, generally represented by L*, a*, and b* values, where a smaller L* value, larger a* value, and smaller b* value indicate better meat color [36]. We also assessed water-holding capacity, as indicated by drip loss and cooking loss, and meat tenderness, as measured by shear force. The smaller the drip loss and cooking loss of muscle, the stronger the water-holding capacity of the muscle, and the better the edible quality of the meat. Shear force is an indicator used to evaluate muscle tenderness, with lower shear force indicating more tender meat. Ye et al. added fermented feed to broiler diets, resulting in varying degrees of reduction in cooking loss and shear force, and a significant decrease in muscle b* value [37]. Ashayerizadeh et al. found that adding fermented rapeseed meal to the diet significantly improved the water-holding capacity of broiler breast muscle [38]. In this experiment, adding 0.6% of BL or BN Ginkgo biloba leaf fermentation products to the diet significantly reduced muscle shear force and increased water-holding capacity of breast muscle, thereby enhancing muscle tenderness.

To sum up, this study demonstrates the potential of Bacillus-fermented Ginkgo biloba leaves as a high-value feed additive. Using Ginkgo biloba leaves as the main fermentation substrate, B. licheniformis and B. natto were used as the fermentation strains, and the fermentation conditions were optimized through orthogonal experiments. The results showed that with the addition of 1.5% glucose and 1.5% urea, an inoculum size of 10%, a solid-to-liquid ratio of 1:1.2, an initial pH of 7.0, a fermentation temperature of 37°C, and a fermentation time of 48 h, the flavonoid content of the fermentation products of B. licheniformis and B. natto increased by 16.7% and 10.6% respectively. The total amino acid content increased by 17.22% and 31.05% respectively, with viable cell counts reaching 2.189×10¹⁰ cells/g, spore formation rates of 82.7% and 84.2%, protease activities of 1307 and 1510 U/g, crude protein content increased by 43.83% and 58.74%, and aroma substance content increased by 98.68% and 95.26%, respectively. The fermented products demonstrated multiple benefits as a feed additive. They are rich in probiotics, effectively increase the extraction yield of flavonoids and the content of crude protein, and significantly improve the palatability of Ginkgo biloba leaves. When used as feed additives, these fermented products improved broiler performance in several ways: they enhanced feed efficiency, promoted the development of the thymus and spleen, improved antioxidant function, and enhanced the absorption capacity and tenderness of broiler breast muscle. Notably, B. natto showed a slight advantage over B. licheniformis in these effects. This approach not only enhances the nutritional and functional properties of Ginkgo biloba leaves but also improves their palatability and effectiveness as a feed additive. These findings have important implications for the feed industry and offer a promising strategy for upgrading agricultural by-products.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors’ contributions

Q. Li and L. G. Zhao conceived and designed the experiments. F. Q. Zhang and R. Chen performed the experiments. Q. Li and S. F. Gao analyzed the data. L. G. Zhao and Q. Li contributed reagents/materials/analysis tools. Q. Li wrote the manuscript. X. H. Zhang and L. G. Zhao revised and approved the final version of the paper.

Fundings

This work was supported by the Forestry Achievements of Science and Technology to Promote Projects ([2017] 10) and Nanjing Forestry University Student Practice Innovation Training Program Project (2024NFUSPITP0175) .

Data availability

The datasets generated and/or analyzed during the current work are available from the corresponding author upon reasonable request.

Acknowledgements

The authors gratefully acknowledge State Key Laboratory for Development and Utilization of Forest Food Resource, Nanjing Forestry University for its assistance in technical support. We also thank the anonymous reviewers for their constructive feedback.

Conflict Interest-The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval-This article does not contain any studies with human participants or animals performed by any of the authors.

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Table 1 Composition and nutrient levels of basal diets

Dietary composition/%	0-3 week	4-6 week
Corn	60.74	64.08
Soybean meal	30.82	24.03
Corn protein powder	2.0	4.0
Bran	0.5	1.0
Pork fat	2.03	2.83
Stone powder	1.1	1.27
Calcium hydrogen phosphate	1.39	1.27
Salt	0.2	0.25
L-lysine	0.07	0.16
methionine	0.15	0.1
1% premix	1.5	1
Metabolic energy (MJ/kg)	12.27	12.77
Crude protein/%	21.2	19.3
Calcium/%	1.0	0.91
Effective phosphorus/%	0.43	0.38
L-lysine/%	1.08	0.95
Methionine/%	0.50	0.44
Methionine+Cysteine/%	0.82	0.74

Table 2 Results of orthogonal test on fermentation conditions of Ginkgo biloba leaves by Bacillus

Test		Influence factor								Inspection index
		Carbon source		Nitrogen source	Inoculation amount /%	Initial pH	Solid liquid ratio /(g:mL)	Fermentation time /h	Fermentation temperature /℃	Viable count /(×10⁹/g)		Protease activity /(U·g^-1)
		Carbon source		Nitrogen source	Inoculation amount /%	Initial pH	Solid liquid ratio /(g:mL)	Fermentation time /h	Fermentation temperature /℃	B. licheniformis	B. natto	B. licheniformis	B. natto
1		Glucose		Ammonium sulphate	5	6	1.0:1.0	24	34	3.65	0.0954	865	946
2		Glucose		Urea	10	7	1.0:1.2	48	37	21.89	28.4	1307	1510
3		Glucose		Peptone	15	8	1.0:1.4	72	40	2.41	0.502	927	1010
4		Sucrose		Ammonium sulphate	5	7	1.0:1.2	72	40	1.521	0.1774	773	964
5		Sucrose		Urea	10	8	1.0:1.4	24	34	8.16	9.03	1277	1408
6		Sucrose		Peptone	15	6	1.0:1.0	48	37	1.18	0.328	651	1003
7		Starch		Ammonium sulphate	10	6	1.0:1.4	48	40	0.849	0.534	657	1017
8		Starch		Urea	15	7	1.0:1.0	72	34	7.62	8.06	1052	1373
9		Starch		Peptone	10	8	1.0:1.2	24	37	0.269	0.655	361	1031
10		Glucose		Ammonium sulphate	15	8	1.0:1.2	48	34	2.32	1.60	812	1021
11		Glucose		Urea	5	6	1.0:1.4	72	37	6.68	7.12	1183	1356
12		Glucose		Peptone	10	7	1.0:1.0	24	40	0.362	0.0528	377	879
13		Sucrose		Ammonium sulphate	10	8	1.0:1.0	72	37	0.435	0.126	426	949
14		Sucrose		Urea	15	6	1.0:1.2	24	40	4.47	6.08	1008	1218
15		Sucrose		Peptone	5	7	1.0:1.4	48	34	0.399	1.09	494	1124
16		Starch		Ammonium sulphate	15	7	1.0:1.4	24	37	0.206	2.35	380	1117
17		Starch		Urea	5	8	1.0:1.0	48	40	3.71	5.36	888	1177
18		Starch		Peptone	10	6	1.0:1.2	72	34	2.36	0.187	824	993
Mean value	1	B. licheniformis	6.219	1.497	2.705	3.198	2.826	2.853	4.085
	1	B. natto	6.295	0.813	2.416	2.391	2.337	3.044	3.344
	2	B. licheniformis	2.694	8.755	5.676	5.333	5.472	5.058	5.110
	2	B. natto	2.805	10.675	6.388	6.688	6.183	6.219	6.497
	3	B. licheniformis	2.502	1.163	3.034	2.884	3.117	3.504	2.220
	3	B. natto	2.858	0.469	3.153	2.879	3.438	2.695	2.117
Range		B. licheniformis	3.717	7.592	2.971	2.449	2.646	2.205	2.890
Range		B. natto	3.490	10.206	3.972	4.297	3.846	3.524	4.380

Table 3 Composition and content of aroma compounds in samples before and after fermentation

	Pre fermentation sample	Culture of B.licheniformis	Culture of B.natto
3-hydroxy-2-butanone	--	10.42	--
Cis-3-hexen-1-ol	0.63	2.23	2.70
1-octen-3-ol	--	0.73	1.69
Methyl heptenone	5.59	7.21	7.31
Methyl heptene	--	--	0.51
Methyl 2-amino-6-methylbenzoate	--	1.05	3.05
Methyl phenylacetate	--	--	0.86
Methyl salicylate	--	--	1.25
Limonene	2.58	4.27	--
2,5-dimethylpyrazine	--	--	3.69
Trimethyl pyrazine	--	--	3.06
2,4,4-trimethyl-2-cyclohexen-1-ol	--	0.96	--
Terpinene	--	1.02	--
Isophorone	1.42	1.39	--
Benzyl alcohol	2.02	0.52	6.64
Linalool oxide	2.87	3.84	--
Linalool	0.51	1.22	--
Phenylethanol	--	1.51	6.23
Cyclocitral	1.64	0.54	--
Safranin aldehyde	--	--	1.31
Theophyllone	0.85	1.64	--
Terpenol	0.81	2.38	1.52
Geranyl acetone	3.05	2.33	1.73
Benzofuranone	--	0.39	0.51
Total aroma compounds（R/100）	21.97	43.65	42.06

Table 4 Flavonoid content, spore formation rate, amino acid content and crude protein content of Ginkgo biloba leaves fermented by Bacillus

Index	Pre fermentation sample	Culture of B.licheniformis	Culture of B.natto
Viable count /（×10¹⁰个/g）	--	2.189	2.84
Spore number /（×10¹⁰个/g）	--	1.81	2.39
Spore formation rate /%	--	82.7	84.2
Flavonoid content /%	0.66	0.77	0.73
Total amino acid content/%	15.653	18.348	20.513
Crude protein content /（g/100 g）	21.06	30.29	33.43

Table 5 Effects of Ginkgo biloba extract on serum lipid metabolism and abdominal fat deposition in Broilers

Items	Control	0.3%BL	0.6%BL	0.3%BN	0.6%BN	Error	P value
Items	Control	0.3%BL	0.6%BL	0.3%BN	0.6%BN	Error	linear	Quadratic power
Total protein /(g·dL^-1)	4.201	4.368	4.294	4.322	4.34	0.042	0.14	0.319
Albumin /(g·dL^-1)	1.823	1.832	1.68	1.758	1.814	0.055	0.524	0.754
TC/(mg·dL^-1)	107.96	100.25	92.11	98.46	92.61	2.066	0.042	0.031
TG/(mg·dL^-1)	119.94	112.66	107.67	113.28	104.55	1.399	0.031	0.001
HDL-C/(mg·dL^-1)	63.84	64.81	68.94	66.9	72.54	0.937	0.02	0.006
LDL-C/(mg·dL^-1)	39.5	35.96	34.9	33.63	32.81	0.437	0.027	0.004
Percentage of abdominal fat /(g·kg^-1)	2.86	2.63	2.51	2.44	2.39	0.047	0.041	0.001

Table 6 Effects of Ginkgo biloba leaf fermentation on serum antioxidant function and lipid peroxidation

	Items	Control	0.3%BL	0.6%BL	0.3%BN	0.6%BN	Error	P value
Serum	T-SOD	8.45	8.93	10.09	9.42	10.35	0.645	0.001
	GSH-Px	7.38	7.54	7.89	7.48	7.87	0.089	0.64
	T-AOC	3.45	4.32	4.85	4.39	4.82	0.471	0.034
	GSH	7.7	8.09	8.34	8.27	8.33	0.562	0.038
	MDA	1.05	0.94	0.75	0.9	0.75	0.031	0.001
Liver	T-SOD	10.69	11.2	12.76	12.92	13.09	1.23	0.024
	GSH-Px	9.52	9.46	9.98	9.54	9.96	0.659	0.64
	T-AOC	4.91	5.51	6.91	6.25	6.86	0.356	0.037
	GSH	9.47	10.03	10.25	10.46	10.54	0.98	0.054
	MDA	1.53	1.29	0.95	1.24	0.95	0.136	0.002

Table 7 Effect of Ginkgo biloba leaf fermentation on breast muscle quality of 42 day broilers

		Control	0.3%BL	0.6%BL	0.3%BN	0.6%BN	Error	P value
pH_45min		6.64	6.66	6.9	6.76	6.7	0.041	0.521
pH_24h		6.06	6.09	6.45	6.26	6.46	0.035	0.002
24 h meat color	L*	50.22	50.32	49.94	49.91	51.5	0.057	0.512
	a*	4.32	4.56	4.13	3.79	5.11	0.354	0.056
	b*	17.8	16.57	15.87	16.46	17.07	0.394	0.126
24 h drip loss /%		1.73	1.57	1.21	1.58	1.34	0.026	0.003
48 h drip loss /%		2.44	2.41	2.14	2.22	2.06	0.039	0.026
Cooking loss /%		15.11	12.31	12.69	13.47	12.64	0.512	0.038
Shear force /kg		2.29	2.16	1.97	2.09	1.9	0.026	0.017

No competing interests reported.

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Graph Abstract

Solid state fermentation of Ginkgo biloba leaves and its effects on antioxidant capacity and breast muscle quality of Broilers

Status:

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Abstract

Figures

Highlights

Introduction

Experiments and Methods

Strains and Media

Experimental animals

Experimental instruments and equipment

Experimental Methods

Orthogonal experiment for optimizing fermentation medium

Viable cell and spore counting

Enzyme extraction and protease activity assay

Detection of aroma compound content

Detection of crude protein and amino acid content

Experimental animal handling and feed design

Measurement indicators and methods for experimental animals

Statistical Analysis

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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