Effects of Probiotic-Fermented Chinese Herb on Immune Response and Growth Performance in Common Carp (Cyprinus carpio)

Abstract

This study investigated the effects of fermented Chinese herb (FCH) on the growth indices, leukocyte activity, and biochemical indices of carp (Cyprinus carpio). Astragalus membranaceus (AM), Pericarpium Citri Reticulatae (PCR), and Glycyrrhizae Radix et Rhizoma (GRR) as feed additives enhance immune function, promote growth, and exert anti-inflammatory effects, respectively. Therefore, this study investigated the effects of co-fermented blends of these three herbs on growth performance and related parameters in common carp. By adding 2%, 5%, and 10% of the FCH to co-incubate with carp leukocytes, the results show that all three experimental treatments could enhance the respiratory burst activity and phagocytic activity of carp leukocytes. After 28 days of feeding with basal feed supplemented with 2%, 5%, and 10% (w/v) of the FCH, the weight gain rate and specific growth rate of carp were significantly higher than those of the control treatment without additives (ANOVA, p < 0.05), with the 5% treatment showing the highest. The activities of intestinal digestive enzymes were significantly increased (ANOVA, p < 0.05). On the 21st day, the activities of amylase (AMS), lipase (LPS), and chymotrypsin were increased compared to the control treatment. The 5% and 10% treatments showed significantly higher intestinal digestive enzyme activities compared to the 2% treatment. The serum superoxide dismutase (SOD) levels in both the control and experimental treatments initially increased and then decreased, with all three experimental treatments having higher levels than the control treatment. The activities of liver glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) in the experimental treatments showed no significant changes compared to the control treatment (ANOVA, p > 0.05). However, the serum GPT activity in the 5% treatment was significantly lower than that of the control treatment (ANOVA, p < 0.05), while no significant differences were observed in the other treatments. The results indicate that adding 2~10% of FCH to carp feed can improve intestinal digestion, enhance phagocytic activity and the body’s antioxidant defense capabilities, and effectively promote the growth of carp. It can significantly improve farming efficiency and economic benefits, reduce dependence on chemical drugs, and lower environmental pollution, showing good application prospects in production.

1. Introduction

China is rich in Chinese herbal medicine resources, with approximately 12,000 species of medicinal plants, far exceeding those of other countries. Through thousands of years of exploration, the Chinese people developed countless herbal formulas. Due to the complex and diverse pharmacological effects of Chinese herbal medicines (CHMs), which contain active ingredients such as sugars, glycosides, alkaloids, organic acids, and volatile components, they have been found to enhance animal immunity, promote growth, and leave no residues [1], so they are widely used in aquaculture [2]. However, some CHMs can be toxic, which makes their efficacy uncertain. Studies found that FCH can transform their original large-molecule structures into smaller molecules that are more easily absorbed and broken down by the body [3,4]. This process not only reduces the content of toxic substances in CHMs or converts them into other components to eliminate toxicity, but also generates new active compounds, leading to new pharmacological effects [5]. While enhancing immunity [6,7], it also minimizes residue in organisms and the environment and reduces drug resistance. This study aims to develop a fermented CHM formula. By evaluating its impact on carp growth and biochemical indicators, it represents a significant improvement in the application of CHMs for biological immune defense.

The main bioactive components of AM are astragalus polysaccharides, which can boost immunity, promote growth, and scavenge free radicals. Sun et al. (2020) found that astragalus polysaccharides can significantly improve the growth performance and antioxidant activity of Scophthalmus maximus and maintain positive immune responses [8]. Chen et al. (2024) reported that AM can enhance the stress resistance of carp [9]. Pericarpium Citri Reticulatae (PCR), used as a feed additive, can promote the growth of aquaculture animals [10]. When combined with other CHMs, it can significantly enhance the weight gain of farmed animals [11]. Glycyrrhizae Radix et Rhizoma (GRR) has anti-inflammatory, antiviral, and immunity-enhancing effects. Du et al. (2021) found that the total flavonoids of GRR can alleviate gut oxidative stress and lipid metabolism disorders in Nile tilapia induced by a high-fat diet [12]. Wang et al. (2020) suggested that adding GRR to feed can improve the growth performance and disease resistance of yellow catfish [13]. In this study, AM, PCR, and GRR were jointly fermented to leverage their synergistic effects, aiming to enhance feed utilization and boost the disease resistance of carp.

Carp (Cyprinus carpio) is one of the main freshwater fish species in China, commonly affected by diseases such as gill rot, enteritis, and scale protrusion, which are primarily treated with antibiotics [14]. This not only leads to the development of drug-resistant strains, but also damages the ecological environment. The global trend of sustainable aquaculture is moving toward efficiency, eco-friendliness, and technology-driven approaches, aiming to balance food security with ecological protection. Thus, research on alternative medicines to antibiotics is ongoing and advancing. With the deepening of research on alternatives to antibiotics, studies have shown that Chinese herbal medicine has antibacterial [15,16], antiviral [17,18], antiparasitic [19,20], growth-promoting [21,22], and immune-enhancing effects [23] in aquaculture. This study used probiotics to ferment Astragalus mongholicus Bunge, Citrus reticulata Blanco, and Glycyrrhiza uralensis Fisch to prepare an FCH for feeding carp, aiming to investigate its effects on the growth performance of carp and provide a theoretical basis for the application of probiotic-FCH in aquaculture, contributing to the development of new fishery drugs or feed in the aquaculture industry.

2. Materials and Methods

2.1. Experimental Materials, Reagents, and Instruments

2.1.1. Experimental Strains

Bacillus subtilis GDM1.372 and Lactobacillus plantarum GDM1.191 were purchased from the Guangdong Microbial Culture Collection Center, Guangzhou, China.

2.1.2. Chinese Herbal Medicines

Steamed Citrus reticulata Blanco (produced by Kangmei Pharmaceutical Co., Ltd., batch number: 210901902, origin: Puning, China), Glycyrrhiza uralensis Fisch (produced by Anhui Huifeng Traditional Chinese Medicine Co., Ltd., batch number: 21060102, origin: Inner Mongolia, China), and Astragalus mongholicus Bunge (produced by Anhui Huifeng Traditional Chinese Medicine Co., Ltd., batch number: 21050101, origin: Dingxi, China) were ultra-finely ground for use.

2.1.3. Fermentation Process

The three CHMs were added at 0.2% (w/v) to prepare 100 L of FCH (molasses: 45 g/L, yeast extract: 2 g/L, K₂HPO₄·3H₂O: 2 g/L, MnSO₄·H₂O: 0.05 g/L, and MgSO₄·7H₂O: 0.2 g/L). The mixture was fermented in a 200 L fermenter at 121 °C for 10 min, cooled to 37 °C, and inoculated with 5% (v/v) of a composite bacteria mixture (Lactobacillus plantarum and Bacillus subtilis in a 1:1 ratio). The fermentation was carried out at 80 rpm, 37 °C, pH 8.0, and a final microbial load of 70% under anaerobic conditions for 48 h, then stored at 4 °C for experimental use.

2.1.4. Experimental Feed

The basal feed was purchased from a feed company in Xiamen, containing high-quality fish meal, soybean meal, flour, refined fish oil, stabilized vitamins, and organic chelated minerals (

Table 1. Composition of the basal diet.

Items	Content (%)	Items	Content (%)
Shandong fish meal	2.5	Soybean lecithin	0.5
Soybean meal	34.5	CaH2PO4	1.5
Rapeseed meal	20	Vitamin premix	0.4
Flour	21.9	Mineral premix	0.6
DDGS	7.5	Fungicide	0.05
Rice bran	8.0	Antioxidant	0.05
Fish oil	2.5	Total	100

). Based on previous studies, we selected concentrations—0% (control treatment) 2%, 5%, and 10% (w/v)—within the safe usage threshold of Chinese herbal medicines. The 2% concentration allows for an initial assessment of its effects. The 5% concentration is moderate and may represent the optimal effect. The 10% concentration tests whether higher doses enhance effects or cause negative impacts. These three concentrations help systematically evaluate the impact of FCH with probiotics on carp growth and other indicators, providing a scientific basis for practical applications. The FCH was evenly sprayed on the feed, air-dried under ventilation, and then fed to the fish.

2.1.5. Reagents

Percoll cell separation solution (Shanghai Shuoxing Biotechnology Co., Ltd., Shanghai, China), L-15 cell culture medium (Weisente Biotechnology Co., Ltd., Beijing, China), nitroblue tetrazolium (Xiamen Lulong Biotechnology Co., Ltd., Xiamen, China), green fluorescent microsphere suspension (Changsha Meinong Biotechnology Co., Ltd., Changsha, China), ethanol solution (Xilong Scientific Co., Ltd., Shantou, China), methanol solution, and dimethyl sulfoxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

2.1.6. Instruments

Microplate reader (MULTISKAN GO, Thermo Scientific, Singapore), flow cytometer (CytoFLEX, Beckman Coulter, Shanghai, China), ultra-fine grinder (SQW-601, Shandong Sanqing Stainless Steel Equipment Co., Ltd., Shandong, China), biochemical incubator (LRH-250, Shanghai Yiheng Scientific Instruments Co., Ltd., Shanghai, China), etc.

2.2. Experimental Fish and Feeding Management

Carp (Cyprinus carpio) were purchased from a farm in Zhangzhou, Fujian. After two weeks of acclimatization, 120 healthy carp with an average body weight of (42.56 ± 1.50) g were selected and randomly divided into four treatment groups, with 30 fish in each group (divided into three parallel tanks, 10 individuals in each group, the volume of each tank is 100 L). The fish were fed with feed containing different concentrations of the FCH for four weeks. During the experiment, the water temperature was maintained at 25.9 ± 1.53 °C, dissolved oxygen at 6–9.8 mg·L⁻¹, and pH at 6.5–7.5. The fish were fed daily, and the amount of feed, residual feed weight, water changes, feeding activity, and fish health were recorded. Water quality was maintained throughout the experiment.

2.3. Sample Collection

Before sampling, the fish were fasted for 24 h. Samples were collected on the 7th, 14th, 21st, and 28th days of the experiment. Six carp were randomly selected from each group (two from each tank), weighed, and recorded. The sampled fish were anesthetized in a 0.1 mL/L clove oil solution for 2 min [24], and blood was drawn from the tail vein and placed in 2 mL centrifuge tubes. The serum was separated overnight at 4 °C, centrifuged at 3500 rpm for 15 min, and the supernatant was collected for serum biochemical analysis. The liver and midgut (with intestinal contents removed) of two carp from each group were washed with 0.9% sterile saline. The serum, liver, and intestinal tissue samples were mixed and stored in liquid nitrogen at −198 °C for 2 h, then transferred to a −80 °C freezer for storage and used for physiological and biochemical analysis.

2.4. Peripheral Blood Leukocyte Separation

Fresh carp blood was mixed with L-15 cell culture medium at a 1:9 (v/v) ratio to prepare carp plasma. The original Percoll cell separation solution was mixed with 8.5% NaCl at a 9:1 (v/v) ratio to achieve physiological osmotic pressure, resulting in a 100% leukocyte separation solution. This was further diluted with saline to create leukocyte separation mixtures at concentrations of 70%, 60%, 50%, 40%, 30%, and 20%. Each concentration of the leukocyte separation mixture (0.5 mL) was placed in 1.5 mL centrifuge tubes, and 1 mL of carp plasma was added to each tube. The tubes were centrifuged at 800 g for 20 min at room temperature. The upper layer of leukocytes was mixed with L-15 cell culture medium at a 1:9 (v/v) ratio to prepare a leukocyte suspension. The leukocyte count was performed using a flow cytometer to compare the separation efficiency of different concentrations of the leukocyte separation mixture for carp peripheral blood leukocytes.

2.5. Peripheral Blood Leukocyte Respiratory Burst Assay

The carp leukocyte samples were prepared using the most suitable Percoll cell separation mixture. Preparation method: Diluted blood samples were placed in a density gradient medium (Percoll). Centrifugation caused cells of different densities to distribute across various gradient layers. White blood cells, due to their lower density, accumulated in a specific gradient layer. The respiratory burst activity of peripheral blood leukocytes was measured using the nitroblue tetrazolium (NBT) reduction method [25,26] (

Table 2. Respiratory burst activity assay operation sheet.

	Control Group	Experimental Group	Blank Group
Leukocyte suspension (μL)	100	100	100
5% DMSO solution (μL)			100
FCH (μL)	100	100
PBS (μL)	100	100	100
Incubate at room temperature for 2 h, aspirate 100 μL of the supernatant, and wash three times with 100 μL of L-15 cell culture medium.
L-15 cell culture medium (μL)	50	50
0.2% NBT solution (μL)	50
PBS (μL)		50	100
Incubate at room temperature for 1 h, aspirate 100 μL of the supernatant, wash three times with 100 μL of a 70% methanol solution, blow out the liquid from each well, and air-dry at room temperature.
100 μL of 2 mol/L KOH solution and 200 μL of 100% dimethyl sulfoxide.
Absorbance reading at 630 nm using a microplate reader.

). Each sample underwent three technical replicates, with three replicate wells set up on a 96-well plate.

2.6. Peripheral Blood Leukocyte Phagocytosis Assay

Leukocytes were collected as described in Section 2.4. The phagocytic activity of peripheral blood leukocytes was measured using flow cytometry [26] (

Table 3. Phagocytic activity assay operation sheet.

	Control Group	Experimental Group
Leukocyte suspension (μL)	200	200
FCH (μL)		100
5% DMSO solution (μL)	100
Incubated at room temperature for 2 h.
Microsphere suspension (μL)	50	50
Mix thoroughly, room temperature, protect from light, and react for 1 h.
70% methanol solution (μL)	250	250
Flow cytometry analysis.

). Preparation method: Diluted blood samples were placed in a density gradient medium. Centrifugation caused cells of different densities to distribute across various gradient layers. White blood cells, due to their lower density, accumulated in a specific gradient layer.

2.7. Physiological and Biochemical Index Determination

2.7.1. Digestive Enzyme Assay

Before the assay, the midgut was thawed at 4 °C and mixed with 0.9% saline at a 1:9 (v/v) ratio. The mixture was homogenized in an ice bath and centrifuged (4 °C, 2500 rpm, 10 min), and the supernatant was collected for analysis. The activities of amylase (AMS), lipase (LPS), and chymotrypsin were measured using kits from the Nanjing Jiancheng Bioengineering Institute. Protein concentration was determined using the Bradford method [27].

2.7.2. Biochemical Indices

Serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were measured directly after thawing the serum at 4 °C. Before measuring liver GOT and GPT, the liver was thawed at 4 °C and mixed with 0.9% saline at a 1:9 (v/v) ratio. The mixture was homogenized in an ice bath and centrifuged (4 °C, 2500 rpm, 10 min), and the supernatant was collected for analysis. The enzyme activities were measured using microplate kits from the Nanjing Jiancheng Bioengineering Institute, and the enzyme activity units were determined by comparing the color intensity in an alkaline solution to a standard curve.

2.7.3. Antioxidant Indices

Serum superoxide dismutase (SOD) activity was measured directly after thawing the serum at 4 °C. The optimal sample concentration was selected based on an inhibition rate between 40% and 60%, and the SOD activity was measured using a kit from the Nanjing Jiancheng Bioengineering Institute, Nanjing, China.

2.8. Calculation Formulas

Weight gain rate (WGR, %) = (final weight−initial weight)/initial weight × 100%

(1)

Feed coefficient (FCR) = feed intake/(final weight−initial weight)

(2)

Specific growth rate (SGR, %/d) = (ln final weight−ln initial weight)/
feeding days × 100%

(3)

Respiratory burst activity = OD_N−OD_L

(4)

Respiratory burst increase rate =(m₁−m)/m × 100%

(5)

Phagocytosis rate = n₁/n × 100%

(6)

Phagocytosis increase rate = (f₁−f)/f× 100%

(7)

OD_N is the absorbance value of the N well, and OD_L is the absorbance value of the L well.

m₁ is the respiratory burst activity of the experimental group, and m is the respiratory burst activity of the blank control treatment.

n₁ is the number of peripheral blood leukocytes involved in phagocytosis, and n is the total number of peripheral blood leukocytes.

f₁ is the phagocytosis rate of the experimental group, and f is the phagocytosis rate of the control treatment.

2.9. Statistical Analysis

The experimental results are expressed as mean ± standard deviation (mean ± SD). Data were analyzed using SPSS 25.0 software for one-way ANOVA, followed by Tukey’s test and SNK multiple comparisons. Graphs were plotted using GraphPad Prism 8.0 software.

3. Results and Analysis

3.1. Effects of FCH on Carp Leukocyte Activity

3.1.1. Optimization of Peripheral Blood Leukocyte Separation Solution Ratio

By comparing the separation efficiency of Percoll cell separation mixtures at concentrations of 20% to 70%, the results show a positive correlation between the concentration of the Percoll cell separation mixture and the separation efficiency. A 70% Percoll solution could separate 14.25 × 10⁵ cells/mL of leukocytes, which was sufficient for subsequent experiments (

Table 4. Effect of Percoll cell separation mixture on Cyprinus carpio peripheral blood Leukocytes.

Percoll (%) Percoll Concentration	20%	30%	40%	50%	60%	70%
(g/mL) Separation medium density	1.031	1.043	1.056	1.067	1.077	1.090
(105/mL) Leukocyte concentration	2.37	2.42	3.60	6.19	8.79	14.25

).

3.1.2. Effects of FCH on Carp Leukocyte Respiratory Burst Activity

When 2%, 5%, 10%, and 20% of the FCH were added, the respiratory burst activity of carp peripheral blood leukocytes initially increased and then decreased with increasing concentration. The respiratory burst activity increased by 25%, 40%, 77%, and 18.45%, respectively, showing significant differences compared to the control treatment (ANOVA, p < 0.05) (Figure 1). However, when 20% of the FCH was added, the respiratory burst activity decreased.

3.1.3. Effects of FCH on Carp Leukocyte Phagocytic Activity

The effects of FCH on the phagocytic activity of carp peripheral blood leukocytes are shown in Figure 2. The phagocytic activity of carp peripheral blood leukocytes initially increased and then decreased with increasing concentrations of 2%, 5%, and 10% of the FCH. The 5% and 10% treatments showed significantly higher phagocytic activity compared to the control treatment (ANOVA, p < 0.05), with phagocytosis rates increasing by 69.75% and 79.35%, respectively. However, the 20% concentration inhibited phagocytic activity, reducing it by three times.

3.2. Effects of FCH on Carp Growth Performance

The effects of FCH on the growth of carp in each experimental group are shown in

Table 5. Effects of FCH added to feed on the growth of Cyprinus carpio.

Items	Addition Amount of FCH
	Control Treatment	Treatment 2%	Treatment 5%	Treatment 10%
IBW/g	44.61 ± 3.45	40.41 ± 1.08	42.85 ± 0.58	42.38 ± 3.78
FBW/g	48.64 ± 1.67	44.66 ± 1.29	47.42 ± 1.15	46.76 ± 1. 6
WGR/%	9.02 a ± 3.25	10.51 b ± 3.23	10.67 b ± 7.0	10.34 b ± 6.44
SGR/%	0.30 a ± 0.12	0.35 b ± 0.13	0.36 b ± 0.21	0.35 b ± 0.20
FCR/%	1.85	1.75	1.63	1.69

Note: Different data letters in the same row indicate significant difference (ANOVA, p < 0.05).

. The weight gain rate and specific growth rate of carp in all experimental treatments were higher than those in the control treatment, with significant differences (ANOVA, p < 0.05), showing an initial increase followed by a decrease. The Feed coefficient was lower than that of the control treatment, with no significant differences (ANOVA, p > 0.05), showing an initial decrease followed by an increase. The 5% treatment had the highest weight gain rate and specific growth rate, and the lowest Feed coefficient. The weight gain rate and specific growth rate in the 5% treatment increased by 10.67% and 36%, respectively, compared to the control treatment, while the Feed coefficient decreased by 1.63%.

3.3. Effects of FCH on Carp Digestive Enzymes

During the experiment, the activities of digestive enzymes in all treatments initially increased and then decreased, reaching their peak on the 21st day. The enzyme activities in the three experimental treatments were higher than those in the control treatment, showing a gradual increase with increasing drug concentration. On the 7th day, the 5% and 10% treatments significantly increased the activity of amylase (AMS) compared to the control treatment (ANOVA, p < 0.05). On the 14th day, all three experimental treatments significantly increased the activity of AMS, with significant differences between the treatments (ANOVA, p < 0.05) (Figure 3).

On the 7th day, the 10% treatment significantly increased the activity of lipase (LPS) compared to the control treatment (ANOVA, p < 0.05). On the 14th day, the 10% treatment significantly increased the activity of LPS compared to the control treatment and the 2% treatment (ANOVA, p < 0.05). On the 21st day, the 10% treatment significantly increased the activity of LPS compared to the control treatment and the 2% treatment (ANOVA, p < 0.05), and the 5% treatment also significantly increased the activity of LPS compared to the control treatment (ANOVA, p < 0.05). On the 28th day, there were significant differences between the different additive treatments (ANOVA, p < 0.05) (Figure 4).

On the 7th, 14th, and 21st days, the 10% and 5% treatments significantly increased the activity of chymotrypsin compared to the control treatment (ANOVA, p < 0.05), and there were significant differences between the 10% and 2% treatments (ANOVA, p < 0.05). On the 28th day, the 5% and 10% treatments still showed significant differences compared to the control treatment (ANOVA, p < 0.05), but there were no significant differences between the 2% and 10% treatments (ANOVA, p > 0.05) (Figure 5).

3.4. Effects of FCH on Carp Liver Transaminase Activity

During the experiment, the activities of GPT and GOT in all treatments initially increased and then decreased. In the 2%, 5%, and 10% treatments, the activity of GPT in carp liver tissue showed a gradual decrease, with the 10% treatment having the lowest GPT activity, which was lower than that of the control treatment. The 2% treatment showed different differences at different time points, with a significant increase in GPT activity in carp liver tissue on the 21st day compared to the 7th, 14th, and 28th days (ANOVA, p < 0.05). There were no significant differences in GPT activity in the other treatments during the experiment (ANOVA, p > 0.05) (Figure 6). As shown in Figure 7, except for the control treatment, the GOT activity in carp liver tissue in the other treatments initially increased and then decreased, with the 5% treatment showing the fastest increase. On the 28th day, the 5% treatment significantly increased the GOT activity in liver tissue compared to the control treatment.

3.5. Effects of FCH on Carp Serum Transaminase Activity

The results of serum GPT activity in carp fed with FCH are shown in Figure 8. As the experiment progressed, the GPT activity in both the control and experimental treatments decreased, with the control treatment having higher GPT activity than all experimental treatments. The 5% treatment had lower GPT activity than the other experimental treatments, and from the 7th to the 21st day, the 5% treatment significantly reduced serum GPT activity compared to the control treatment.

The results of serum GOT activity in carp fed with FCH are shown in Figure 9. As the experiment progressed, except for the 10% treatment, the GOT activity in the control and other experimental treatments decreased, with the 5% treatment having lower GOT activity than the other experimental treatments. On the 7th day, the 5% and 10% treatments significantly reduced serum GOT activity compared to the control treatment (ANOVA, p < 0.05). On the 28th day, the 5% treatment significantly reduced serum GOT activity compared to the control treatment (ANOVA, p < 0.05), and there were significant differences between the 2% and 5% treatments compared to the 10% treatment (ANOVA, p < 0.05).

3.6. Effects of FCH on Carp Serum Superoxide Dismutase Activity

The results of serum SOD activity in carp fed with FCH are shown in Figure 10. The SOD activity in both the control and experimental treatments initially increased and then decreased. On the 21st day, the SOD activity in all treatments reached its peak, with the control and 5% treatments showing significant differences on the 14th and 7th days (ANOVA, p < 0.05), but no significant differences on the 21st and 28th days (ANOVA, p > 0.05). The 2% treatment showed significant differences on the 21st and 7th days (ANOVA, p < 0.05), but no significant differences on the 14th and 28th days (ANOVA, p > 0.05). The 10% treatment showed no significant differences between different time points (ANOVA, p > 0.05). On the 7th day, the 5% treatment had significantly higher SOD activity than the control and 10% treatments (ANOVA, p < 0.05). On the 21st day, the 10% treatment had significantly higher SOD activity than the control treatment (ANOVA, p < 0.05).

4. Discussion

4.1. Effects of FCH on Carp Peripheral Leukocytes

Different fish species require different densities of separation solutions for leukocyte separation. In this experiment, a 60% and 70% Percoll mixture with a density of 1.085 was used, which is suitable for the separation of carp peripheral blood leukocytes, consistent with the results of Zhu et al. (2022) [28]. The results show that the addition of 2%, 5%, and 10% of FCH significantly enhanced the respiratory burst and phagocytic activities of carp peripheral blood leukocytes, indicating that these concentrations of FCH can effectively enhance these two activities of leukocytes, thereby improving their overall immune function.

4.2. Growth-Promoting Effects of FCH on Carp

With the promotion of green aquaculture concepts, more and more new green FCHs are being applied in aquaculture. Shi et al. (2022) found that adding Astragalus membranaceus fermented by Lactobacillus plantarum to feed significantly increased the weight gain rate and specific growth rate of carp and reduced the Feed coefficient [29]. Zhao et al. (2017) found that FCH made from Bacillus subtilis and Astragalus significantly improved the weight gain rate, specific growth rate, and condition factor of carp, while reducing the Feed coefficient [30]. In previous studies (Zhao et al. (2017)), it was reported that after comparing the effects of Bacillus subtilis and Lactobacillus rhamnosus on the fermentation of CHM, it was concluded that Bacillus subtilis is the best strain for fermenting CHM, significantly increasing the polysaccharide content in CHM [31]. In this study, Lactobacillus plantarum and Bacillus subtilis were used to ferment Glycyrrhiza uralensis, Citrus reticulata, and Astragalus mongholicus. The results show that adding 2%, 5%, and 10% of the FCH to carp feed significantly increased the weight gain rate and specific growth rate of carp and reduced the Feed coefficient. Furthermore, analysis of intestinal digestive enzymes revealed a significant increase in the activities of lipase (LPS), amylase (AMS), and chymotrypsin. This is because the FCH, containing strains such as Bacillus subtilis and Lactobacillus plantarum, produces various digestive enzymes and bioactive substances [32] that enhance nutrient digestion and absorption. Moreover, CHM is rich in nutrients and feeding stimulants that accelerate intestinal absorption, metabolism, and enzyme secretion [33]. The combined effect of probiotics and CHM improves feed digestibility and utilization, thereby promoting growth.

4.3. Effects of FCH on Carp Transaminase Activity

Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) are common indicators of liver function, and changes in their activities can accurately reflect the degree of liver cell damage, as well as the body’s toxic and pathological conditions [34]. Meng et al. (2010) found that CHM has a certain inhibitory and induction effect on the activity levels of GOT and GPT in the serum of mirror carp [35]. In this study, carp were fed with FCH at concentrations of 2%, 5%, and 10%. During the experiment, in the FCH treatments, the GPT and GOT levels in the liver were slightly higher than in the control treatment. This indicates that adding FCH increases liver metabolism and alters metabolic enzyme levels. However, the increase was not significant, suggesting liver metabolism remained within normal limits. In the liver tissue of carp, the GPT and GOT activities in all groups first increased and then decreased. This was because, in the early stages of the experiment, the liver had to metabolize FCH, increasing its burden and thus elevating GPT and GOT activities [36].

If liver tissue is damaged or diseased, increased cell membrane permeability can cause transaminases to flood into the blood [37], significantly raising serum GPT and GOT activities. During the experiment, in the 10% FCH treatment, serum GOT activity increased with time and was higher than the control treatment on day 28, while other treatments showed decreasing GPT and GOT activities, which remained lower than the control treatment. This indicates that the probiotic-FCH additive effectively protects liver cells, prevents damage, and reduces serum GOT and GPT.

4.4. Effects of FCH on Carp Antioxidant Capacity

Superoxide dismutase (SOD), an enzyme with special physiological activity, is a key indicator of the body’s ability to eliminate free radicals and is integral to the antioxidant defense system. It supports and regulates immune function across the entire body. Ma et al. found that FCH significantly boosts SOD levels in crucian carp blood, enhancing their immune response [38]. Zhang et al. noted that adding fermented Moringa to diets improves juvenile crucian carp’s growth, antioxidant capacity, and immunity [39], aligning with the findings of this study. Here, SOD activity was significantly higher in FCH fermentation liquid groups than in the control group, indicating effective SOD induction in carps. Notably, 21 days of feeding with 5% FCH fermentation liquid showed the most pronounced effect on SOD levels in carps, actively supporting normal cellular functions and boosting immunity.

5. Conclusions

In this study, four concentrations of FCH were tested, with the highest at 10%. Results show that adding 2–5% of FCH from Bacillus subtilis and Lactobacillus plantarum to carp feed for 21 days improved intestinal digestion, enhanced immunity, boosted feed efficiency, and promoted growth, with the 5% concentration being most effective. This research offers a new perspective for developing novel fish medications or feeds in aquaculture.

However, the 10% group showed higher serum GOT and GPT activities, suggesting slight liver damage, though no pathological analysis was conducted. Additionally, the study did not assess the effects on intestinal microbiota. Addressing these limitations and testing the FCH in other animals could clarify its sustainable value in aquaculture, indicating promising application potential.