Macrobrachium nipponense is a freshwater shrimp with a large breeding area in China and is steadily expanding worldwide (Jiang et al., 2019). The aquaculture industry has suffered significant economic losses in recent years due to recurrent outbreaks of prawn diseases (Hou et al., 2018; Joshi et al., 2014). Traditionally, antibiotics and chemical drugs were used to prevent and treat prawn diseases. However, their use is restricted due to increasing bacteria drug resistance and food safety concerns. While plant extracts offer a promising alternative to antibiotics, their cost-effectiveness remains challenging. Therefore, the search for safe, effective, and economically feasible plant extracts for treating prawn diseases remains a priority (Bulfon et al., 2015). Plant polysaccharides from plant extracts are considered natural alternatives to chemical drugs and antibiotics and are widely used in aquaculture (Tzianabos, 2000).

Prebiotics have been demonstrated to improve host health, which in turn facilitates the growth and development of prawns (Kiernan et al., 2023). Related studies have shown that prebiotics have the characteristics of improving the structure of intestinal flora and stimulating the proliferation of probiotics (Li and Gatlin, 2005; Munir et al., 2016). The main physiological functions of prebiotics include enhancing the body’s immunity (Sredkova et al., 2020), promoting the absorption of minerals (Karakan et al., 2021) and reducing inflammatory response (McLoughlin et al., 2017) and other efficacy. Prebiotics are polysaccharides extracted from plants and can be digested by the bacteria to produce short-chain fatty acids, which have anti-inflammatory and immune-enhancing effects (Jenab et al., 2020). Currently, Atractylodes rhizoma (Sun et al., 2023), mannooligosaccharides (Forsatkar et al., 2018), Yupingfeng polysaccharide (Su et al., 2020), and other prebiotics have been added to aquafeed. Atrhizoma polysaccharide plays a vital role in folk medicine, treating a variety of diseases (Pan et al., 2018) and improving immunity (Sun et al., 2015), which may be due to its ability to alleviate immunosuppression induced by cyclophosphamide (Li et al., 2019a, b). At the same time, Atractylodes polysaccharides can induce TLR4 to activate NF-κB, which in turn activates macrophages to synthesize cytokines (Wei et al., 2016). Previous studies have shown that Atractylodes polysaccharide also plays an important role in heat stress and immune function improvement (Xu et al., 2017; Xu and Tian, 2015). Adding mannooligosaccharides during fish breeding can promote the growth and reproduction (Forsatkar et al., 2018). Yupingfeng polysaccharide is an Astragalus membranaceus based ancient Chinese herbal medicine that regulates humoral and cellular immunity and inhibits inflammation (Fabrizio et al., 2013). Related studies have shown that Yupingfeng polysaccharide can improve the foregut microbiota and intestinal barrier of weanling rabbits and enhance the immunity of rabbits (Sun et al., 2016). Moreover, Yupingfeng polysaccharide could effectively improve the immune response, disease resistance and growth performance of Litopenia vannamen (Su et al., 2020). Astragalus polysaccharide has a variety of biological activities, such as immunomodulatory (Liu et al., 2017), antioxidant (Li et al., 2010), and antibacterial effects (Ma et al., 2017). In aquaculture, Astragalus polysaccharide has been shown to promote growth performance, improve physiological and biochemical indicators and increase the expression of genes related to lipid metabolism (Huang et al., 2023).Parsnip polysaccharide can modulate immune activity, significantly increase immune cell density and macrophage number, and reduce the expression of NO, TNF-α, IL-1β and IL-6 (Fan et al., 2023).

Bacillus coagulans is a lactic-producing bacillus that can produce short-chain fatty acids such as acetic acid and propionic acid, promote gastrointestinal peristalsis, and regulate the structure of microenvironment flora (Zhu et al., 2019). B.coagulans utilize polysaccharides for growth and secrete digestive enzymes and bacteriocins that promote digestion and absorption (Mazkour et al., 2022). B.coagulans can significantly improve shrimp growth performance and serum antioxidant capacity in shrimp culture (Sadat Hoseini Madani et al., 2018). B.coagulans can also up-regulate the transcription level and related enzyme activities of SOD and CAT genes in zebrafish, and protect zebrafish larvae against oxidative stress induced by copper sulfate (Ai et al., 2023). In livestock and poultry breeding, B.coagulans can improve the growth performance of C. vermilmilium, enhance intestinal innate immunity and improve intestinal microbial community (Fu et al., 2019). This study addresses the disease issues faced in the cultivation of M. nipponense by exploring the potential of B.coagulans-fermented plant polysaccharides as a green additive. Plant polysaccharides have garnered attention in aquaculture due to their diverse bioactivities, while B.coagulans is favored for its ability to promote growth, enhance antioxidant capacity, boost immunity and improve gut microbiota. The study focuses on the combined effects of B.coagulans and plant polysaccharides on M.nipponense, aiming to provide a new green additive application scheme for shrimp farming, which can help improve the breeding environment and increase breeding efficiency.

Polysaccharides in aquaculture have attracted considerable attention and commercial interest over the past few decades. A continual search for novel, eco-friendly additives remains essential. Therefore, prebiotic polysaccharides have become a hotspot in research. Prebiotics, which contain polysaccharides and other substances, can promote animal health Plant polysaccharides have been widely studied for their various biological activities. These include antiviral activity, antioxidant activity, anti-fatigue and liver-protective effects (Liu et al., 2015). Five plant polysaccharides (Atractylodes polysaccharide, Paraspinatus polysaccharide, Mannose, Astragalus polysaccharide and Yulingfeng polysaccharide) can improve growth performance, antioxidant capacity and immune function in animals (Wu, 2020; Su et al., 2020; Cui et al., 2023; Erdenebileg et al., 2023). In addition, B.coagulans can promote animal growth, improve antioxidant capacity, enhance immunity and improve intestinal microbial flora (Zhang et al., 2021). Meanwhile, B.coagulans can break down plant polysaccharides and promote their uptake in animals (Shinde et al., 2020). However, there are few studies on the use of plant polysaccharides in combination with B.coagulans. Therefore, the effect of polysaccharide fermentation with B.coagulans for M.nipponense was investigated in this experiment.

It was found that the survival rate of the group fed with the polysaccharide B.coagulans was the highest. This may be due to the anti-inflammatory, antibacterial and immune-enhancing effects of paraspinals (Javadi and Sahebkar, 2017). Meanwhile, Paraspinatus polysaccharides also possessed antioxidant and anti-allergic properties (Guang et al., 2023). In addition, B.coagulans was found to fully restore the immunosuppression induced by cyclophosphamide, thereby improving its immunity (Bomko et al., 2017), which may also be an essential reason for the improved survival rate. Therefore, it was shown that feeding M.nipponense with Paraspinatus polysaccharide fermented by B.coagulans could improve the survival rate of A.hydrophila infection.

B.coagulans can produce enzymes such as protease, lipase and amylase (Gupta et al., 2016), which promote the absorption of nutrients in the intestine of animals. Studies have found that feeding compound bacterial agents such as Bacillus subtilis for Pengze crucian carassius improved the activities of protease, amylase and lipase (Luo et al., 2021). In addition, studies on Clostridium butyricum and B.coagulans have found that they can improve the gut microbiota structure and promote the growth of steelhead trout (Fan et al., 2019). The results of this study showed that the hepatopancreatic digestive enzyme activities of M.nipponense were increased after Atractylodes polysaccharide, mannose and Paratractylodes polysaccharide, which was consistent with the results of previous studies. Therefore, adding polysaccharides of Atractylodes japonicum, Paraspinatus, Astragalus and Yupingfeng to in the diet can improve the activities of non-digestible enzymes of M.nipponense. This result is mainly caused by B.coagulans, but there are few studies on the effect of the polysaccharides (Atractylodes rhizoma polysaccharide, Paraspinatus polysaccharide, Astragalus polysaccharide, Yupingfeng polysaccharide) on digestive enzymes. The ability was limited to the study of the effects of polysaccharides of Atractylodes rhizoma, Paratractylodes and Astragalus on digestive enzymes of M.nipponense. Therefore, the molecular mechanism underlying the effect of polysaccharides on the digestive enzymes of M.nipponense could not be determined.

Serum biochemistry reflects homeostatic changes in the body’s internal environment. ALT is mainly distributed in hepatocyte plasma and AST is primarily present in hepatocyte mitochondria and plasma. Hepatocyte necrosis causes ALT and AST to enter the blood circulation, thereby increasing the levels of ALT and AST in the body (Iweala et al., 2019). Oxidative stress in the liver can cause lipid peroxidation, thereby changing the permeability of the liver cell membrane and increasing ALP levels in vivo (Albasher et al., 2019). At the same time, liver injury can cause the rapid diffusion of fatty acids in the liver, resulting in the increase of TG and TC content in the liver. TG and TC also directly reflect the degree of lipid peroxidation in the liver (Wang et al., 2019b). Reduced liver function can further impair renal function, affecting the synthesis, transport, and release of ALB in the body, thereby reducing the level of ALB in the blood (Dongzhe et al., 2023). In the present study, the N, NF, NG, NH and NB significantly reduced the content of ALT. However, the N, NF and NG significantly reduced AST content. It has been previously found that dietary supplementation of Bacillus coagulans significantly reduced serum ALT and AST levels induced by high cholesterol in rats (Aminlari et al., 2018). The elevation of ALT and AST induced by cadmium poisoning was alleviated in rats fed with symbiotics B.coagulans with Lactobacillus plantarum and inulin (Jafarpour et al., 2017). In other words, B.coagulans mixed with polysaccharide can reduce ALT and AST content and protect the liver. Mannose and Atractylodes polysaccharide can effectively protect the liver injury in mice by significantly decreasing the activities of AST and ALT in serum (Han et al., 2016). Dietary Astragalus polysaccharide supplementation can improve the growth performance, alleviate liver dysfunction, and reduce ALT and AST content in the liver of piglets (Wang et al., 2019a). In addition, relevant studies have shown that dietary Astragalus polysaccharide can significantly reduce the content of glucose, triglyceride, cholesterol and nitric oxide in fish serum (Wu et al., 2019). In addition, it was found that replacing fish meal with Atractylodes Macrocephala Polysaccharide decreased the albumin and globulin concentrations and increased the feed coefficient and total bile acid activity of catfish (Zhuo et al., 2022). In summary, this is consistent with the present study and confirms the authenticity of the data in this study.

Oxidative stress is a primary biochemical reaction of organisms to resist external environmental stimulation and adapt to the living environment. The oxidative defense system is the basis of resistance to external stimuli, which is a balanced system composed of antioxidant enzymes and oxidative stress products. SOD is an important antioxidant enzyme in the oxidative defense system. SASC and T-AOC reflect the body’s overall antioxidant capacity. MDA is critical for oxidative stress and demonstrates the degree of oxidative damage in the body. Oxidative stress can also lead to decreased immunity and damage the immune system. ACP and AKP, as immune enzyme indicators, reflect the body’s immune ability. In this study, it was found that diet made from the fermentation broth of B.coagulans with Yupingfeng polysaccharide significantly increased the levels of ACP, AKP, SASC and T-AOC in M.nipponense. These results suggested that the fermentation broth of B.coagulans with Yupingfeng polysaccharide could increase the antioxidant capacity and immune capacity of M.nipponense. This may be due to the improvement of the antioxidant capacity of M.nipponense by regulating serum immunity through the fermentation broth of B.coagulans of Yupingping polysaccharide. Previous studies have shown that Yupingfeng polysaccharide could down-regulate the mRNA expression levels of pro-inflammatory cytokines, NF-kB, TLR-4 and iNOs, enhancing the antioxidant capacity (Sun et al., 2017). At the same time, Yupingfeng polysaccharide could significantly increase the density of immune cells and the number of macrophages and reduce the levels of NO, TNF-α, IL-1β and IL-6. Adding 1.6 g/kg Yupingfeng polysaccharide to the diet of grass carp can improve the immunity of grass carp (Wang et al., 2016). The above results indicated that Paratricans polysaccharide had some immunomodulatory activity (Fan et al., 2023). In addition, it was found that the fermentation broth of Atractylodes rhizoma polysaccharide increased the contents of ACP and AKP in M.nipponense. Atractylodes polysaccharide and Atractylodes lactone have been found to have anti-inflammatory and immunomodulatory effects in previous studies (Gu et al., 2019; Tang et al., 2017). However, the defect of this study is that the cause of the high MDA in the fermentation broth of B.coagulans with polysaccharide was not explored.

Invertebrates rely on innate immunity to defend against disease invading their bodies (Reboul and Ewbank, 2016) and shrimp are crustaceans and rely mainly on innate immunity (Patnaik et al., 2023). These include physical defense, cellular immunity, and humoral immunity (Riera Romo et al., 2016; Thaiss et al., 2016). In the model organism Drosophila antimicrobial peptide gene pathway, Toll and IMD play an important role in mediating G^- and G⁺ infections, respectively (Huang et al., 2009) and shrimp immunity (Su et al., 2020). Toll receptor, Myd88 and Dorsal are key signaling molecules in the Toll pathway (Dong et al., 2022) and Myd88 is a receptor protein that connects Toll (Arts et al., 2007). Relish, a downstream transcription factor of the IMD pathway, belongs to the NF-kB family and participates in the body’s humoral immune response (Luo et al., 2022). IMD immunodeficiency is involved in antimicrobial responses in vivo. Previous studies found that Yupingfeng polysaccharide could enhance serum immunity and Toll gene expression in Litopenia vannamen (Su et al., 2020). At the same time, researchers showed that Yupingfeng could regulate the release of cytokines in mouse macrophages, mainly due to the enhanced degradation of IkBα, which in turn activated NF-kB, causing pro-inflammatory cytokine-related protein and mRNA expression (Fabrizio et al., 2013). In addition, Yupingfeng also inhibited the expression of pro-inflammatory cytokines (Fabrizio et al., 2013). The expression levels of Toll, IMD and Relish in the hepatopancreas of Litopenaeus vannaeus can be significantly increased by adding Marine Rhodotorula to the diet (Jin et al., 2022). Increased expression of Relish was observed in the hepatopancreas after artificial infection with A. hydrophila (Liang, 2022). In addition, Yupingfeng polysaccharide was found to increase the expression of Toll receptors in the serum of Litopenia vannamen and promote the body health (Su et al., 2020). Astragaloside has also been found to exert anti-inflammatory effects through the Myd88/NF-kB pathway (Shi et al., 2021). Astragalus polysaccharides can improve juvenile crucian carp’s growth performance and innate immunity (Wu, 2020). This is consistent with the results of the present study, which showed that the addition of Astragalus and Yupingfeng polysaccharides to the diet of M. nipponense increased the expression of Myd88 and Toll, respectively, and thus increased the expression of related immune genes in M.nipponense.

In general, these fermented polysaccharides’ primary mechanisms of action involve absorption in the intestines, entry into the bloodstream, and effects on various tissues and organs throughout the body. Specifically, Yupingfeng polysaccharides enhance liver function and the liver’s antioxidant capacity by increasing the serum levels of TP, ALB, AKP and T-AOC Saposhnikovia divaricata polysaccharides can increase the content of TP in the serum, improve liver function, and regulate the liver’s antioxidant function by adjusting the content of AKP and TP. Additionally, Saposhnikovia divaricata polysaccharides can increase the content of alpha-amylase content, enhancing metabolic function. Astragalus polysaccharides can reduce the content of ALT in the serum and increase the content of TP, thereby enhancing liver function. Moreover, Astragalus polysaccharides enhance the body’s antioxidant function by increasing the content of T-AOC, TP, and AKP, thereby enhancing the body’s immune capacity ( Figure 5 ). In summary, among the five types of fermented polysaccharides screened, Yupingfeng polysaccharides, Saposhnikovia divaricata polysaccharides and Astragalus polysaccharides have a significant effect on improving the antioxidant capacity and immunity of M.nipponense.

M.nipponense was co-influenced by polysaccharides and B.coagulans and the adding of fermented polysaccharides to the diet increased the weight gain of the prawns. The combined use of polysaccharide fermentation with B.coagulans can improve the survival rate of M.nipponense infected with A.hydrophila; the survival rate of the group with B.coagulans and Saposhnikovia divaricata polysaccharides (NF) was 66.67% at 96 hours. Adding polysaccharides fermented with Bacillus coagulans to the diet significantly impacted the prawns’ immune capacity and digestive enzyme activity. The addition of fermented Astragalus and Yupingfeng polysaccharides with B. coagulans to the diet increased the expression of Myd88 and Toll, respectively. The addition of fermented Atractylodes macrocephala polysaccharides with Saposhnikovia divaricata polysaccharides and Mannose, as well as Yupingfeng polysaccharides with B.coagulans, significantly reduced the expression of Relish and promoted the growth of the M.nipponense.