1. Introduction
In China, the rapid growth of aquaculture has led to a shortage of protein feed [
1]. The primary protein source in aquaculture is fish meal (FM) [
2]. In recent years, the rising price of FM has led to the rise in aquaculture costs [
3]; consequently, finding alternative protein sources for fish meal is a topic of concern [
4].
Soybean meal (SBM) is a by-product of oil extraction and is widely used as a source of animal protein. In China, the price of soybean meal containing 46% crude protein is lower than 614.5 USD t
−1. The amino acid composition of soybean meal is relatively well-balanced, making it a viable alternative to fish meal [
5]. The original soybean meal contains numerous anti-nutritional factors, such as oligosaccharides, phytic acid, lectins, and trypsin inhibitors [
6]. As expected, its direct application has negative effects on digestion and absorption of nutrients, and thereby fish growth [
7]. The concentration of these anti-nutritional factors can be reduced through microbial fermentation. Refstie et al. [
8], for instance, demonstrated that fermentation with lactic acid bacteria significantly decreased the level of trypsin inhibitors in soybean meal. Similarly, Hassaan et al. showed that fermentation with
Saccharomyces cerevisiae significantly reduced the phytic acid content [
3]. In recent years, replacing fish meals with fermented soybean meal (FSBM) in aquaculture has become a research hotspot. The preliminary effects of replacing fish meal on the growth of catfish, Nile tilapia, rainbow trout, and grouper were investigated [
9].
Perch is the fourth most popular freshwater fish in China, and it is favored by consumers due to its rich nutrition. The cost of raising largemouth bass is approximately 2000–2300 USD t
−1. The main expenses in the process of bass farming originate from labor, electricity, fish medicine, and feed. According to previous studies, the cost of feeding perch accounts for 30–70% of the total cost of aquaculture [
10], whereas the cost of fish meal accounts for over 50% of all feed ingredients. According to data from the U.S. Department of Agriculture, global fish meal production remained between 4.4 and 4.9 million tons from 2012 to 2020. China is the largest fish meal consumption market in the world, and the annual fish meal consumption is around 2 million tons. Some researchers have investigated the viability of substituting FSBM for fish meal in perch cultures [
11]. However, these studies focused primarily on these growth performance indicators for perch. The comprehensive and in-depth evaluation of FSBM as an alternative to fish meal on perch requires additional research. In the current study, we screened three types of microorganisms:
Bacillus amyloliquefaciens,
Cyberlindnera jadinii, and
Streptococcus thermophilus. Then, we mixed
Bacillus amyloliquefaciens,
Cyberlindnera jadinii, and
Streptococcus thermophilus at a ratio of 1:2:1 for the fermentation of soybean meal. Based on the principle of equal nitrogen and energy of feed in the control and experimental treatment groups, fish meal was substituted with FSBM at 0%, 10%, 30%, and 50%. The effect of replacing fish meal with FSBM on largemouth bass was investigated at three levels: macro (growth performance), microbial (bacterial diversity and metabolic), and gene (key gene expression). The results of this experiment lend theoretical support to the substitution of FSBM for the fish meals.
4. Discussion
Two factors can be attributed to increased amino acid and crude protein content. First, due to the growth and metabolism of microorganisms, some carbon with solid forms was converted into CO
2, increasing the relative content of crude protein. Second, microorganisms transformed a portion of inorganic nitrogen into amino acids, polypeptides, and SCP (signal-cell protein). This result was consistent with that of Hong et al. [
15], who fermented soybean meal with
Aspergillus oryzae GB107 and observed a significant increase in crude protein content. After fermentation, the phytic acid content decreased significantly (
p < 0.05), which might be attributed to improved phytase and phosphatase activity [
16,
17]. Lin and Chen. [
7] reported that the phytic acid degradation was pH-dependent and that the optimal pH range for most phytases was between 4.0 and 6.0. The pH of FSBM was decreased by lactic acid produced by
S. thermophilus, which aided in enhancing phytase activity.
In the current study, FE decreased as the proportion of replaced FSBM increased. Lee et al. [
18] discovered that soybean meal had a lower digestibility and utilization rate than animal protein. Numerous studies have demonstrated that fermentation can reduce anti-nutritional factors in SBM, thereby enhancing the growth performance of fish, the digestibility of nutrients, and the intestinal microbiome [
19]. Lee et al. [
1] reported that FSBM could replace 40% of fish meal by breeding rainbow trout and grouper without impairing their growth or feed efficiency. For shrimp, the replacement proportion can reach 75% [
7]. When the replaced FSBM exceeded a certain threshold, the growth performance of the fish was impaired, and the FE decreased. This is attributable to three factors: (1) the content of indigestible carbohydrate (oligosaccharide) in FSBM is higher than that in fish meal, and protein digestibility is lower; (2) FSBM has low palatability, and anti-nutritional factors are not completely eliminated; (3) the amino acids in soybean meal are unbalanced [
1]. This study demonstrated that FSBM could replace 30% of fish meal in the diet of largemouth bass without adversely affecting their growth. A suitable substitution can enhance the apparent digestibility of dry matter and protein. The presence of anti-nutritional factors in soybean meal impairs the utilization of nutrients in animal feed by inhibiting the activity of enzymes and causing adverse intestinal reactions [
7]. In this study, the content of anti-nutritional factors in FSBM decreased significantly (
p < 0.05), which could improve nutrient utilization. In addition, the enhancement of nutrient utilization may be closely related to organic acids such as lactic acid. For instance, previous studies have demonstrated that adding lactic acid to rainbow trout feed can improve nutrient utilization [
7]. In addition, as the concentration of organic acids increased, the solubility of minerals also increased, which enhanced the utilization rate of nutrients [
20].
Steatosis is a metabolic disorder caused by excess fatty acids entering the liver and a high peroxidation level. Steatosis of liver cells is indicative of liver cell damage. Steatosis is associated with an imbalance in the proportion of fatty acids [
21]. Adding FSBM increased the degree of steatosis, indicating that considering the proportion of essential fatty acids in FSBM was worse than that in fish meals. Steatosis will cause dysfunction of liver function, and then affect the absorption and metabolism of nutrients, resulting in an increase in the feed-to-meat ratio. The intestine is an essential component of the digestive system. Based on its morphology and function, the intestine can be divided into the proximal intestine, midgut, and distal intestine. The distal intestine is the primary site for digestion and absorption of nutrients [
22]. The height and width of the villus determine the area of contact between the mucosal epithelial cells and chyme, which is essential for digestion and absorption. This indicates that the substitution of FSBM has negative effects on intestinal morphology and absorption. In addition, the muscular layer is associated with intestinal peristalsis, and its thickness can indicate intestinal peristalsis capacity. Due to the presence of anti-nutritional factors in FSBM, which harmed the fish intestinal tract to a certain extent, FSBM substitution in the current study may have reduced the thickness of the muscle layer [
11]. After intestinal injury, the utilization of nutrients was reduced, resulting in an increase in feed usage and increased costs. Severe changes in intestinal morphology could ultimately lead to small stature.
Serum biochemical indicators are considered the most important health status indicators. ALT is an essential amino acid transaminase in mitochondria that plays a crucial role in protein metabolism; its serum concentration is correlated with liver damage. Soltan et al. [
23] reported that substituting fish meal with plant protein increased ALT and AST in tilapia serum. However, the results of this study demonstrated that substituting FSBM for fish meal increased the concentration of ALT and decreased the content of AST. The concentration of TP in serum can indicate the capacity for protein metabolism and synthesis. In this experiment, replacing fish meals decreased serum TP levels. Similarly, He et al. [
11] asserted that substituting fish meals with SBM and FSBM in breeding largemouth bass decreased serum TP content. The low digestibility and utilization rate of protein caused by the unbalanced amino acid composition of plant protein may account for the decrease in serum TP [
11]. GLB is a vital immune protein whose concentration correlates with disease resistance. Alkaline phosphatase activity can predict organ dysfunction and a high phosphatase concentration in serum indicates severe liver damage. The substitution of fish meals with FSBM increased alkaline phosphatase activity in the current study. This phenomenon was also observed in sturgeon breeding (
Amur sturgeon) [
24].
The main function of pro-inflammatory factors is to aggregate white blood cells into infected or damaged tissues.
IL-1β plays a crucial role during microbial invasion and tissue damage because it can enhance phagocytosis, accelerate macrophage proliferation, lysozyme synthesis, and white cell migration [
25]. Many studies have shown that probiotics regulate the production of anti-inflammatory factors in animals [
26]. The Hepcidin gene plays a crucial role in iron metabolism and innate immunity. It is a regulator of iron homeostasis and an antibacterial peptide. Iron is a necessary component of infectious pathogens. Hepcidin restricts iron circulation by increasing its concentration, lengthening iron retention in phagocytes. Iron restriction is an innate immune response that can combat bacterial and viral infections in multicellular organisms [
27]. For instance, Hu et al. [
28] reported that the iron-limiting ability of
Hepcidin-1 not only has bactericidal activity but also promotes the resistance of grass carp to
Aeromonas hydrophila. Lee et al. [
27] demonstrated that the expression of Hepcidin genes significantly increased in all organs and blood of olive flounder infected with pathogens. This study showed that the expression levels of
Hepcidin-1 and
Hepcidin-2 genes in the 30% FSBM and 50% FSBM groups were lower than those in the control, indicating that FSBM, to some extent, reduced the chance of largemouth bass infection with pathogens. The antioxidant response is a crucial defense mechanism exhibited by organisms. Therefore, any improvement in antioxidant activity would be beneficial for the health of aquatic animals. Superoxide dismutase (SOD) can decompose reactive oxygen species (ROS). SOD catalyzes the conversion of O
2− to H
2O
2 as one of the main antioxidant defense mechanisms of oxidative stress [
29]. In this experiment, the expression levels of
SOD1,
SOD2, and
SOD3a in the liver of the FSBM treatment groups were greater than those of the control group, indicating that the antioxidant capacity of largemouth bass was enhanced to a certain degree (
Figure 2b). This may be because fermentation increases the bioavailability of vitamins and extracellular polysaccharides, increasing antioxidant capacity [
30]. Apoptosis is a highly regulated form of cell death that is essential for tissue homeostasis and organ development. The apoptosis process is separated into external and internal pathways. Caspase-3 initiates cell apoptosis after ligands bind to the death domain and activate external apoptotic pathways, followed by
Caspase-8 activation [
31]. The intrinsic apoptosis process, also called mitochondrial apoptosis, consists of three steps. Initially, oxidative stress causes mitochondrial dysfunction. Mitochondrial dysfunction plays a crucial role in the apoptotic signaling pathway. As ROS levels rise, lipid peroxides accumulate, and cytochrome-c is released from the cytoplasm [
32]. Then, a stress that can activate
Caspase-9 activates mitochondrial apoptosis. Finally,
Caspase-9 induces liver cell apoptosis and fibrosis through
Caspase-3 [
32]. In this experiment, the levels of
Caspase-9 expression were lower in the 10% FSBM, 30% FSBM, and 50% FSBM treatments than in the control.
Caspase-3 and
Caspase-8 expression levels were higher in the 10%, 30%, and 50% FSBM treatments than in the control. Consequently, substituting FSBM enhances the external apoptotic pathway while diminishing the internal apoptotic pathway.
The gut microbiota is a complex symbiotic system that influences the host’s physiology, nutrition, immunity, and metabolism [
33]. Firmicutes consist of numerous types of lactic acid bacteria, such as Streptococcus, lactic acid bacteria, and Leuconostoc, which are regarded as probiotics and play a crucial role in the fish gut [
22]. Dietary components play a crucial role in determining the composition of the gut microbiota [
34]. Intestinal microorganisms and their metabolites play important roles in digestion, mucosal tolerance, immunity, and disease resistance. At the genus level,
Plesiomonas was less abundant in all treatments than in the control.
Plesiomonas is a prominent member of the Proteobacteria.
P. shigellides is the only
Plesiomonas species and a common intestinal pathogenic bacterium. Infecting tilapia and grass carp with
P. shigelloides can result in varying degrees of tissue damage. The experimental results suggest that FSBM could prevent the colonization of the intestine by
P. shigellides [
35]. Certain members of the genus
Cetobacterium can convert proteins and carbohydrates into vitamin B12 [
35].
Mycoplasma is one of the most prevalent microorganisms in the gut of largemouth bass. The relative abundance of
Mycoplasma was greater in the experimental groups than in the control groups. According to previous studies,
Mycoplasma plays an important role in fish health. For instance, Rimoldi et al. [
36] demonstrated that lactic acid and acetic acid produced by
Mycoplasma in the intestine are beneficial to the health of rainbow trout (
Oncorhynchus mykiss).
Pediococcus is a lactic acid bacterial species. The majority of
Pediococcus can produce pediocin bacteriocins, which are lethal to
Listeria monocytogenes [
37].
Aeromonas possesses a vast array of virulence factors that can cause diseases in fish.
Aeromonas consists predominantly of A.
hydrophila, A.
veronii, and A.
caviae [
38]. This study discovered that substituting fish meals with FSBM increased the relative abundance of
Aeromonas, which may increase the risk of infection in largemouth bass.
Diet composition may alter the metabolic pathways associated with the gut microbiome. L−Asparagine, L−histidine, L−tryptophan, and L−lysine, which are involved in the metabolic pathways of biosynthesis of amino acids and biosynthesis of plant secondary metabolites, were downregulated in the 30% and 50% FSBM treatments. This could be due to the imbalanced amino acid composition of FSBM compared to fish meals. Large-molecule carbohydrates, which cannot be directly absorbed by the intestine, can be degraded by intestinal microorganisms into small-molecule substances that are more readily absorbed by the intestine. Consequently, intestinal microorganisms can influence the health status of the host via metabolic products. Numerous substances, including amino acids, fats [
39], fructose, and glucose [
40], can influence the composition of the intestinal microorganisms in the host. Previous studies have demonstrated that microorganisms and metabolites interact. On the one hand, microorganisms are capable of synthesizing and decomposing metabolites. Metabolites, on the other hand, can stimulate or inhibit the growth of intestinal microorganisms [
41].
Figure 5 shows the correlation between intestinal microorganisms and metabolites. Some metabolites showed a significant (
p < 0.01) positive correlation with certain bacteria and a negative correlation with others. This phenomenon suggests that some microbial metabolites may have inhibitory effects on other microorganisms, resulting in a change in the composition of the microbial community [
41].
Large-scale aquaculture increases the risk of diseases in aquatic animals. Feeding perch with FSBM can alter the microbial diversity of the fish intestine, thereby improving its immunity and reducing disease, which will increase its economic value. The price of SBM is cheaper than FM. Replacing part of FM using FSBM can reduce the initial investment and improve its competitiveness in the market. Replacing part of FM using FSBM (lower than 30%) does not have an adverse effect on the weight of the fish. Fish meat is the largest edible portion, and excessive use of FSBM instead of FM can cause severe weight loss and damage in the liver and intestines of bass, which is not conducive to growth, ultimately leading to a serious reduction in the economic benefits.