Promoting Aquatic Animal Health and Water Quality: A Systematic Review on Probiotics, Prebiotics and Synbiotics in Aquaculture

Abstract

Background: Aquaculture, a vital component of global food security, faces sustainability challenges due to intensive farming practices, including water pollution, disease outbreaks, and antibiotic overuse. Probiotics, prebiotics, and synbiotics have emerged as eco-friendly alternatives to antibiotics. However, research results remain heterogeneous across aquatic species and intervention strategies. Methods: Following PRISMA 2020, we searched two databases (up to January 2026) for in vivo trials. Two reviewers screened and extracted data, and 177 eligible studies were ultimately included, covering single-/multi-strain probiotics (SSP/MSP), live/inactivated microbial preparations, and diverse synbiotic formulations. Results: Among 177 studies, Bacillus spp. were the most widely reported and effective probiotic strains. MSP and synbiotics exhibited superior efficacy in boosting aquatic animal growth performance and disease resistance over SSP in 68% of the included trials. Probiotics act through the competitive exclusion of pathogens, immune modulation, and enhanced digestive enzyme activity; prebiotics selectively stimulate beneficial gut microbiota, improving nutrient absorption and immune function through metabolites such as short-chain fatty acids; synbiotics combine the advantages of both, exerting synergistic effects. Furthermore, as water additives or fermented feed ingredients, probiotics reduce nitrogenous waste and organic pollutants, contributing to bioremediation. Conclusions: All three additives are effective. Standardized application protocols and long-term trials are needed for sustainable aquaculture. This review provides a unified evidence-based foundation for the rational use of these additives in aquaculture.

1. Introduction

Aquaculture has become an integral component of the global food production system, boasting abundant resources and immense potential. As one of the world’s fastest-growing food production industries, it continues to expand [1]. The healthy development of fisheries is crucial to global socioeconomic well-being, as aquatic products are rich in high-quality protein, omega-3 fatty acids, minerals, vitamins, and other nutrients essential for human health [2]. With the ongoing growth of the global population and the rising demand for animal protein, the vital contribution of seafood to global food security and nutrition is becoming increasingly prominent [3,4]. According to an FAO report, the total global fishery and aquaculture production is anticipated to reach 197 million tonnes by 2025, representing a 1.7% increase from 2024 [5]. Regionally, Asia has consistently dominated production, while Africa and Latin America have demonstrated growth rates significantly higher than those of other regions, indicating substantial development potential [3].

However, this rapid industrial growth has been accompanied by a series of sustainability challenges. Extensive farming practices lead to the accumulation of organic waste, triggering water eutrophication [6]. High-density farming increases the risk of opportunistic pathogen outbreaks, often resulting in catastrophic disease epidemics [7]. To maintain production levels and control disease, antibiotics are frequently and heavily used in intensive farming systems. This not only exacerbates the emergence and genetic transfer of antibiotic resistance but also poses a serious threat to public health [8,9]. The development and implementation of Good Aquaculture Practices, incorporating preventive approaches, are employed to enhance environmental performance alongside aquatic product yields and quality.

Probiotics are microbial supplements that confer health benefits to the host when administered at adequate levels [10]. In aquaculture practice, the application of probiotics has expanded from single-strain formulations to multi-strain composite preparations, extending beyond gut regulation to aquatic biological remediation [11,12,13,14]. They have become a vital link connecting aquatic animal health with improvements in the farming environment. Prebiotics constitute a class of carbohydrates that are indigestible by the host, serving as carbon sources and growth factors for endogenous beneficial microbial communities [15]. Upon being broken down and utilized, they promote the growth and proliferation of probiotics, thereby enhancing probiotics’ effects [16]. Synbiotics represent composite formulations combining probiotics and prebiotics [17]. Their synergistic action optimizes aquatic animals’ utilization efficiency for carbohydrates, proteins, and energy, improving growth performance and ultimately establishing a health-promoting microecological symbiosis within the host [18]. In large-scale aquaculture, probiotics, prebiotics, and synbiotics are judiciously selected and combined for use as feed supplements or water conditioners [19,20]. This practice has emerged as one of the most promising avenues to replace antibiotics, alleviate aquaculture environmental pressures, and improve industrial sustainability [21]. A growing body of evidence indicates that the rational use of probiotics, prebiotics, and synbiotics exerts positive effects on the health of farmed species [22,23]. These ingredients are believed to help animals to resist the invasion of pathogenic microorganisms and help them to cope with abiotic stressors, thereby reducing the risk of inflammation [24]. In turn, they may improve the growth performance of farmed species, promote intestinal health, enhance immunity, and help to establish more effective defense mechanisms [19,25,26,27].

This review categorizes probiotics, prebiotics, and synbiotics, primarily addressing single-strain and multi-strain probiotics, as well as live/inactivated microorganisms or microbial metabolic byproducts. To highlight the efficacy of diverse probiotic formats in aquatic organisms, their multifunctional applications as feed supplements, water additives, and fermented feed additives are discussed, alongside a comprehensive analysis of their roles in improving aquaculture environments. The comprehensive synthesis and integration of this subject matter has been lacking. This review aims to provide a theoretical and technical foundation for the application of probiotics, prebiotics, and synbiotics in aquaculture.

2. Materials and Methods

This systematic review was conducted in strict accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines [28]. This systematic review was not registered.

2.1. Literature Search and Databases

A systematic literature search of Web of Science (https://www.webofscience.com/) and Scopus (https://www.scopus.com/) for articles published up to January 2026 was accessed between 9 and 10 January 2026. Key search terms were combined using Boolean operators: (‘probiotics’ OR ‘prebiotics’ OR ‘synbiotics’) AND (‘aquaculture’ OR ‘fish’ OR ‘shrimp’ OR ‘crustaceans’ OR ‘echinoderms’) AND (‘growth performance’ OR ‘disease resistance’ OR ‘immune response’ OR ‘gut microbiota’ OR ‘water quality’ OR ‘feed efficiency’). Synonyms and related terms were incorporated to enhance retrieval comprehensiveness.

Table 1. Search keywords based on PICO headings.

S/N	PICO Theme	Keywords	Synonyms
1	Population	Fish, shrimp, crustaceans, Echinoderm	Shellfish, mollusk, aquatic species, marine fish, freshwater fish
2	Intervention	Dietary supplementation, water additive, fermented feed ingredients	Probiotic, prebiotic, synbiotic, paraprobiotics, postbiotics, beneficial microorganism, Lactobacillus, Bacillus, yeast
3	Comparator	Control/basal diet	No probiotic diet, conventional diet
4	Outcome	Growth performance and feed efficiency	Growth performance, feed efficiency, weight gain, specific growth rate, feed conversion ratio
		Disease resistance and immune response	Disease resistance, immune response, immunity, survival rate, challenge test
		Gut microbiota and digestive health	Gut microbiota, intestinal microflora, microbiome, digestive enzyme
		Water quality	Water quality, ammonia, nitrite

summarizes the keywords and corresponding synonyms based on the PICO (population, intervention, control, outcome) framework. Additionally, a snowballing approach [29] was employed to supplement the database searches by screening the reference lists of included studies and highly relevant reviews to identify additional qualifying articles.

2.2. Inclusion and Exclusion Criteria

This study included peer-reviewed empirical papers published in English that described the administration of probiotics, prebiotics, or synbiotics via dietary supplementation, water additives, or fermented feed to aquatic organisms such as fish, crustaceans, mollusks, or echinoderms, reporting outcomes regarding at least one of the following: growth performance, disease resistance, immune responses, gut microbiota, or water quality parameters. Exclusion criteria encompassed review articles; studies lacking controls or with unclear intervention protocols (e.g., unknown strains, dosages, or administration methods); research focusing on terrestrial animals, plants, or microorganisms not utilized in aquaculture; and studies lacking quantitative results or with unavailable data.

2.3. Screening and Selection

Two reviewers independently conducted the initial search. We removed duplicate records from the initial search results, and full-text publications identified through titles and abstracts were downloaded for further review. Any discrepancies arising during the screening process were resolved through discussion with a third reviewer. The Rayyan QCRI online database (https://rayyan.qcri.org) was utilized during the screening phase of this study.

2.4. Included Studies

The initial literature search yielded 12303 records. After removing duplicates via the Rayyan platform, 4621 articles remained. Combined with 28 articles supplemented through the snowballing method, a total of 4649 articles underwent preliminary screening based on the titles/abstracts. Of these 4649 articles, 3688 were excluded for non-compliance, leaving 861 articles for eligibility assessment. Ultimately, 177 articles were deemed eligible for data extraction. These articles covered the use of single-strain probiotics (SSP), multi-strain probiotics (MSP), or synbiotics (combined with prebiotics). Figure 1 shows the PRISMA flow chart for study selection.

3. Probiotics, Prebiotics, and Synbiotics

Through the rigorous screening and selection process outlined above, the 177 eligible studies provided a robust evidence base to systematically explore the roles of probiotics, prebiotics, and synbiotics in aquaculture.

3.1. Probiotics

The application of probiotics represents a significant advancement in industrial aquaculture, garnering substantial scientific and commercial interest. The following will provide a detailed introduction based on the classification of probiotics, which not only helps to understand the different types of probiotics and their applications in health management but also lays the foundation for exploring the relationship between prebiotics and synbiotics.

3.1.1. Monospecies Probiotics or Multi-Strain Probiotics

Traditional probiotics are defined as ‘live microorganisms that, when administered in adequate amounts, confer health benefits to host organism’ [10]. In aquaculture, by supplementing probiotics directly into feed, it is possible to establish a balanced microbiota in the gastrointestinal tract of the host, improving digestive function, immune responses, and host survival [30]. A wide range of probiotic microorganisms are currently used in aquaculture, in which both Gram-positive and Gram-negative bacteria are effectively administered, such as Bacillus spp. [31], Lactococcus spp. [32], Lactobacillus spp. [33], and Bifidobacterium spp. [34].

Other non-bacterial candidates, such as yeasts and their cellular components, unicellular algae, have also been explored for aquaculture applications [35]. For example, Liu et al. added three lactic acid strains to the diet of sea cucumber, namely L. plantarum W2, Escherichia coli LYB, and L. lactis Nj, which all showed growth-promoting effects and improved non-specific immune indicators. In addition, all three LAB diets increased gut microbial abundance and diversity, contributed to the maintenance of microbial community homeostasis, and enhanced several functional pathways of the gut microbiota related to amino acid metabolism [36]. The results of Zheng et al. showed that diets supplemented with 2% yeast extract significantly improved the growth performance and antioxidant capacity of Pacific white shrimp. In addition, the intestinal microecology of shrimp may be optimized by influencing the intestinal microbial structure, such as increasing the relative abundance of beneficial bacteria and decreasing the relative abundance of opportunistic pathogenic bacteria [37]. Despite these beneficial observations, the efficacy of probiotics is subject to a safe dosage threshold. Research indicates that Rhizobium pusense N7 achieves optimal denitrification performance and zebrafish survival rates at a concentration of 10⁶ CFU/mL. Yet high-dose supplementation increases the abundance of opportunistic pathogens, reduces the denitrification efficiency, and elevates mortality rates [14].

Probiotic preparations have both single and compound forms [38]. Although single-strain probiotics (SSP) have beneficial effects on the health of aquaculture organisms, multi-strain probiotics (MSP) can occupy more ecological niches and exert more diverse regulatory functions, and the synergistic and cumulative effects between different strains can jointly affect the microecological balance of the host, so as to exert a wider range of efficacy in aquaculture [39]. Manuela Pillinger et al. investigated the effects of probiotics alone and in combination on the rainbow trout immune response, particularly in preventing pathogen adhesion and enhancing intestinal barrier function. The results indicate that the use of multi-strain formulations can generate broader synergistic effects and immune-regulatory effects in pathogen invasion and inhibition, and MSP showed a stronger ability to trigger immune responses by activating the production of pro-inflammatory and anti-inflammatory cytokines in rainbow trout intestinal epithelial cells [40]. Ghasem Mohammadi et al. investigated the effects of single and multiple strains of probiotics on the cultivation of Nile tilapia under different culture conditions, and they found that multiple strains of probiotics may promote the immune function of Nile tilapia by enhancing the antioxidant defense system and intestinal health. Their research results indicate that multi-strain probiotics are more effective in enhancing immunity than single-strain probiotics [41]. It is worth noting that the fact that different strains applied to the diet show beneficial effects on host health does not mean that the benefits can be enhanced by combining the effects of different strains. For instance, Kong et al. explored the effects of the single and co-administration of L. lactis L19 and Enterococcus faecalis (E. faecalis) W24 in the diet on the health of Channa argus, especially their effects on the intestinal microbiota, digestive enzyme activity, serum biochemical indices and the modulation of antioxidant capacity. The results showed that L. lactis L19 was more effective than E. faecalis W24 and their mixtures in promoting these effects [42]. Therefore, it is critical to understand the unique biological effects of each strain in the development of MSP. In conclusion, although not all MSP provide superior benefits compared to single-strain probiotics, this does not limit the effectiveness of MSP.

3.1.2. Live/Inactivated Microorganisms or Microbial Metabolic Byproducts

In 2010, Merrifield et al. revised the traditional definition of probiotic preparations for aquatic organisms: ‘probiotic biologics can be considered as live, dead, or components of microbial cells that, when administered through feed or feedwater, can benefit the host by improving host growth performance, feed utilization, immune health, pathogen resistance, and stress response. This is achieved, at least in part, by improving the microbial balance in the host or surrounding environment’ [43]. In other words, both live and inactivated bacterial cells can be used as probiotics in aquaculture [44,45]. Live probiotics can colonize the intestinal mucosal epithelial cells of aquatic animals, competitively excluding pathogenic bacteria; when probiotics are more abundant, they can occupy more ecological niches, compete with pathogenic microorganisms for limited nutrients and space [46], and produce antimicrobial substances, thereby reducing the colonization of pathogenic bacteria and maintaining the balance of the intestinal microbiota [47,48]. As research on probiotics intensifies, the limitations of active probiotics are becoming apparent. In practical applications, the viability and efficacy of live probiotics may be limited, especially in the preparation of solid pellet feeds, where heat treatment tends to inactivate the probiotics, and it is difficult to ensure the stability of probiotics during storage and administration [49]. Therefore, many studies have been devoted to investigating how to maintain the viability and populations of probiotics so that they can be utilized more efficiently by farmed animals. In addition, some live probiotics may have potential safety issues related to antibiotic resistance, the acquisition of relevant virulence genes, and the production of harmful metabolites [50]. Therefore, assessing the toxicological safety of probiotics is an important strategy in screening potential probiotics.

Given the limitations of active probiotic administration, the use of inactivated/heat-killed probiotics (inactive microbial cells or crude cell extracts) and probiotic metabolic byproducts has attracted much attention [51]. The former, defined as paraprobiotics, are microbial cells that are inactivated by heat treatment or other means but whose cellular components (e.g., cell walls, proteins, and nucleic acids) remain, and they have a wide range of bioactivity, being capable of influencing the physiological state of the host [52]. In contrast, soluble factors (products or metabolic byproducts), which are secreted by living bacteria (i.e., probiotics or non-probiotics) or released after bacterial lysis and may positively affect the host, are defined as postbiotics [53]. The introduction of the concepts of paraprobiotics and postbiotics has led to the greater diversification of probiotics in terms of applications and research, deepened the understanding of probiotics, and contributed to the development and expansion of their applications.

As shown in

Table 2. Applications of probiotics in aquatic animal health and aquaculture environments.

Probiotic	Dosage/ Method	Period/Condition	Target Aquatic Organism	Effects	Reference
Bacillus velezensis S141	106 CFU/g diet	56 d	Litopenaeus vannamei	Growth performance (WG, ADG, SGR) (↑) * Survival rate post-WSSV infection (↑) Survival rate (↑) and WSSV copy numbers in co-infection with WSSV and VPAHPND 1 (↓) * Toll/IMD and JAK/STAT pathway genes (STAT, PITH, Vago, Vago4, Vago5, Relish, NF-κB, RPX, DOME) in gills (↑)	[54]
Sebastes schlegelii M1	1 × 106 and 1 × 108 CFU/g of inactivated/active M1, dietary	56 d	Liza ramada juveniles	Growth rate, feed efficiency (↑) Intestinal digestive enzyme activity (↑) Intestinal structure improvements (↑) Immune and antioxidant enzyme activity (↑) IL-2, IFN-γ levels (↑); IL-1β, TNF-α, IL-10 levels (↓) Resistance to V. harveyi infection, protection rate up to 73.33% (↑)	[55]
Pavlova maculatum MACC3	10% chitosan-flocculated, freeze-dried algal biomass supplementation	120 d/Aeromonas salmonicida challenge	Guppies (Poecilia reticulata)	Growth, survival, and proteases, amylases, lipases (↑) Resistance to A. salmonicida challenge, survival rate (↑) Immunological markers (total protein, ig, AKP, protease, bactericidal activity) (↑) Skin pigmentation and ornamentation (↑), carotenoid, pteridine, melanin levels (↑), csf1ra gene expression (↑) Intestinal beneficial genera abundance (↑), pathogenic genera abundance (↓) Bacteroidetes to Firmicutes ratio (↓)	[56]
Probiotic consortium (Bacillus aerius, B. altitudinis, B. pumilus)	1 × 107 CFU/g diet	42 d	Juvenile snubnose pompano (Trachinotus blochii)	Growth performance (WG%, SGR, PER, FCR) (↑) Digestive protease activity (↑)	[57]
Indigenous Bacillus subtilis	1, 10, 100 mg/kg diet (optimal at 10 mg/kg)	8 weeks	Common carp (Cyprinus carpio)	Growth performance (WG, SGR, RGR, FCR, FER, PER) (↑) (10 mg/kg) Innate immunity: neutrophilia, neutrophil-to-lymphocyte ratio (↑) Hepatic integrity: ALT, AST levels (↓) Intestinal histology: mucosal fold hypertrophy (↑) (10 mg/kg); epithelial sloughing and inflammation (↓) (100 mg/kg)	[58]
Immunobacteryne (IMB, from two Bacillus spp.)	0, 0.5, 1, 1.5 g/kg diet (optimal at 1–1.5 g/kg)	60 d	Nile tilapia (Oreochromis niloticus)	Growth performance, growth hormone secretion (↑) (1.5 g/kg) Phagocytic activity, innate immune response (↑) (1–1.5 g/kg) Serum total protein, total cholesterol, triglycerides, glucose (↑) (1–1.5 g/kg) Uric acid, creatinine, liver enzymes (AST, ALT), cortisol (↓) (1–1.5 g/kg) IGF-1 gene expression (↑); HSP70 transcription (↓)	[59]
Bacillus velezensis AP193 BioWiSH Feedbuilder Syn3 (BW)	Trial A: AP193 at 1 × 107 CFU/g; BW at 3.6 × 104 CFU/g Trial B: BW×1 at 3.6 × 104 CFU/g; BW×2 at 7.2 × 104 CFU/g (dietary, top-coated)	26-day recirculating biofloc system (Trial A) 42-day biofloc system (Trial B)	Nile tilapia (Oreochromis niloticus)	Growth performance: (→) * (except FCR in Trial B) Survival, water quality, solids management (↑) (BWx1, BWx2) Water and fecal bacterial composition (↑) (BWx1, BWx2)	[60]
Pediococcus acidilactici	106 CFU/g	60 d, 120 d	Male Atlantic salmon (Salmo salar)	Gonad weight, GSI, sperm concentration (↑) (120 d) Bilirubin, ALT levels (↓) (120 d, within normal range) Sperm quality (membrane integrity, motility): (→) Fertility, embryo viability (↑) Embryonic malformation, mortality (↓)	[61]
PFF2 (Rossellomorea marisflavi spp. DAS-SCF02), PFF3 (Agrococcus spp. RKDAS1), PFF4 (DAS-SCF02 + RKDAS1	PFF2 (1 × 104 CFU/g), PFF3 (1 × 106 CFU/g), PFF4 (1 × 104 + 1 × 107 CFU/g);	8 weeks/V. parahaemolyticus challenge	Nile tilapia (Oreochromis niloticus) juveniles	Hematology (WBC, Hb, RBC, Htc, BP) (↑) Hepatic enzymes (AST, ALT) (↓); protein profile (TP, albumin, globulin) (↑) Metabolites (glucose, TC, TG) (↑) Immune enzymes (lysozyme, MPO) and oxidative response (O2−, RNS) (↑) Immune gene expression (HSP70, IL-1β, C3, IFN-α, IFN-γ, GF1, GH, IL-1, Lyz) (↑) Post-challenge survival (↑)	[62]
Pediococcus acidilactici, Enterococcus faecium, B. subtilis, L. acidophilus, L. plantarum, L. casei, L. rhamnosus, B. bifidum, and S. cerevisiae	0 (P0), 1 × 109 (P1), 2 × 109 (P2), 4 × 109 (P4) CFU/kg diet (4 × 109 CFU/kg as optimal)	8 weeks/flow-through water system	Female rainbow trout (Oncorhynchus mykiss) breeders	Complement component 3, complement component 4, immunoglobulin M concentrations (↑) (P4 vs. P0) Plasma enzyme activity (↓) (P2, P1 vs. P0) Stress indicators: cortisol and glucose levels (↓) (P4 vs. P0) Yolk sac resorption defects (↓) and total malformations (↓) in offspring (P2, P4)	[63]
Live Lactiplantibacillus plantarum (LLP) Dead L. plantarum (DLP)	LLP: 1 × 1011 CFU/kg feed; DLP: dead bacteria	45 d/Vibrio campbellii challenge	Whiteleg shrimp (Penaeus vannamei) juveniles	WG, SGR, PER, survival: LLP (→); PPV (↑) Total hemocyte count, differential hemocyte counts (↑) (LLP) PPO (↑) (LLP, DLP) hsp70 (↑) (LLP, DLP) Amylase, LAP (↑) (LLP) Midgut histomorphology (↑) (LLP) Gut-beneficial bacteria (↑) (LLP) Post-challenge cumulative mortality (↓) (LLP); immune gene expression (↑) (LLP)	[64]

* ‘(↑)’, increase; ‘(↓)’, decrease; ‘(→)’, no change. ¹ White Spot Syndrome Virus (WSSV), Vibrio parahaemolyticus (VP), and Enterocytozoon hepatopenaei (EHP).

, most forms of probiotics enhance the growth and immunity of aquatic animals, and certain strains also contribute to water quality bioremediation, which demonstrates the multifunctional application potential of probiotics in aquaculture.

3.2. Prebiotics

Probiotics and prebiotics are of great interest and application value as important dietary supplements in the aquaculture industry. Unlike probiotics, prebiotics are food ingredients that are indigestible but can be selectively fermented by beneficial intestinal flora, resulting in benefits to host well-being and health by regulating the composition and/or activity of the gastrointestinal microbiota [19,65]. The impact on the microbiome varies depending on the prebiotics used by the host. Almost all of the current prebiotics with a high level of evidence and recognition are carbohydrate polymers, including fructo-oligosaccharides (FOS), inulin, galacto-oligosaccharides (GOS)/trans-galacto-oligosaccharides (TOS), xylo-oligosaccharides (XOS), mannanoligosaccharides (MOS), β-glucans (BG), and others [19,65]. In general, the selective fermentation of prebiotics by Lactobacillus or Bifidobacterium contributes to the enrichment of these commensal bacteria, supporting the composition of the gut microbiota in general [66]. The selective utilization of prebiotics by host microorganisms is key to their physiological effects, and the metabolites resulting from this utilization are the primary physiological drivers. For example, these flora utilize prebiotics for fermentation to produce short-chain fatty acids (SCFAs, mainly consisting of acetic acid, propionic acid, and butyric acid), which modulate a range of intestinal functions, and their activity subsequently affects extra-intestinal sites. The production of SCFAs is largely dependent on the composition and abundance of the gut microbial community, as well as the type and physicochemical properties of the substrate, the intestinal transit time, and the host’s metabolic interaction capacity with gut microbes [67]. First, SCFAs serve as an energy source to support the proliferation and differentiation of intestinal mucosal cells, accelerating the restoration of intestinal histological features and thus maintaining the intestinal mucosal mechanical barrier. Second, they influence intestinal immune cells through multiprotein inflammatory vesicle complexes, which play an important role in maintaining the anti-inflammatory/pro-inflammatory balance and help to modulate the host immune response [68,69]. Finally, an appropriate pH reduction brought about by the supplementation of organic acids promotes mineral absorption (e.g., organic acids have been found to promote phosphorus absorption in several studies on a wide range of fish species), increases digestive enzyme activity, and promotes intestinal peristalsis to expel colonized pathogenic bacteria, thereby maintaining the ecological balance of the intestinal flora [70,71]. With the continuous deepening of omics technology research, it has been discovered that a wider range of beneficial microbial community members can utilize certain prebiotic substrates, and these can be directly or indirectly effective in areas outside the intestine [72]. In recent years, an increasing number of substances—including but not limited to other carbohydrates that are beneficial to the gut flora (e.g., resistant starch), flavonoids, polyphenolic compounds, and other non-carbohydrate substrates (e.g., vitamin E, fatty acids)—have demonstrated prebiotic activity and can also be modified to allow for more intact arrival in the colon or translocation to the intestines to be utilized by the resident microorganisms and enhance their potential as prebiotics [73,74,75]. However, as some potential prebiotic substances are secondary metabolites of plants, obtaining the required prebiotic ingredients through the post-processing of cultivated crops not only adds to the final cost but also interferes with food production. Therefore, given the focus on a recyclable economy, research on the development of certain agricultural byproducts and plant wastes as a source of prebiotic ingredients has gained increased attention [76].

Table 3. Applications of probiotics in aquatic animal health and disease resistance.

Prebiotic	Dosage	Period/Condition	Target Aquatic Organism	Effects	Reference
Aspergillus meal prebiotic	0.3% diet	56 d	Asian seabass (Lates calcarifer)	Growth performance, composition of dorsal fish muscle (→) * RBs, SOD, PA, and LYZ activity (↑) * Mx, C3, TNF, TGF-β1 (↑) Survival post-V. alginolyticus challenge (↑)	[77]
Dietary prebiotic—stevioside	300–500 mg/kg	60 d/cold stress	Liza ramada juveniles	Growth performance, feed efficiency, antioxidant enzymes, and immune function (↑) Intestinal villus structure and absorptive area (↑)	[78]
Dietary microencapsulated inulin	0.8% diet	8 weeks	Striped catfish (Pangasianodon hypophthalmus)	Growth performance, feed efficiency (↑) Cellulase activity, SOD, GPx, immunoglobulin, and lysozyme levels (↑)	[79]
Inulin	2% in 100% plant-based diet	12 weeks	Rainbow trout	Intestinal microbiota, expression of plasma immune markers (→) Growth performance (↓)	[80]
FOS, GOS, INU, MOS, SUC, and WSt 1	3% diet, added to culture water	26 d/biofloc technology system	Penaeus vannamei	Growth performance, nutrient composition of biofloc, nutrient composition of shrimp muscle (→) Significant changes in the bacterial composition of biofloc and shrimp gills and hepatopancreas tissue	[81]
Prebiotic compounds (mannan-oligosaccharides, β-glucans, nucleotides, and nucleosides)	2 g/100 g	60 d	Pacific white shrimp (Penaeus vannamei)	FCR, enzyme activity, and protein retention (↑) Mortality after Vibrio infection (↓) *	[82]
Mannanoligosaccharides (MOS), β-glucans (BG), MOS + BG	0.2% MOS, 0.1% MOS + 0.1% BG	90 d/chronic hypothermia stress	Nile tilapia	Growth performance, blood biochemistry (→) Serum lysozyme (MOS: ↑) Mucus lysozyme (MOS, MOS + BG: ↑) Leukocyte respiratory activity (MOS + BG: ↑) Leukocyte phagocytic function, intestinal integrity (↑) Neutrophil/lymphocyte ratio (↑)	[83]
Laminarin	0.8%, diet	56 d	Spotted sea bass (Lateolabrax maculatus)	Serum T-AOC, GSH, SOD, ACP, AKP, LZM, IgM (↑) Spleen T-AOC, CAT, LZM (↑), MDA (↓) Head kidney GSH, LZM, and immune-related gene expression (↑)	[84]
Commercial prebiotics (Saccharomyces cerevisiae-derived β-glucans and one including inulin)	0.02% BS, 0.20% MB, 0.30% CE, 1.00% IN	55 d	Juvenile vimba (Vimba vimba)	Final growth parameters (BS: ↓) Feed conversion ratio (BS: ↑) Respiratory burst activity of head kidney phagocytes (all prebiotic groups: ↓) Proliferative response of head kidney lymphocytes (BS: ↓)	[85]
Inulin	0.25%, 0.50%, 0.75% diet	31 d/pursuit/capture/atmospheric exposure stress + Aeromonas hydrophila challenge	Hybrid catfish (Pseudoplatystoma reticulatum ♀ × Leiarius marmoratus ♂)	0.25% and 0.75% groups: cortisol homeostasis (↑) Stress induced by pursuit/capture/atmospheric exposure (↓) 0.50% group: serum lysozyme activity, innate immune system function (↑)	[86]
Inulin	0.6% diet	42 d	Red swamp crayfish (Procambarus clarkii)	Weight gain, SGR, FCR (↑) SOD/CAT/GSH-Px/GSH (↑), MDA (↓) LZM/ACP/AKP/C3/C4 (↑) Microbiota diversity, intestinal beneficial bacteria (↑) Pathogenic bacteria inhibition (↓)	[87]

* ‘(↑)’, increase; ‘(↓)’, decrease; ‘(→)’, no change. ¹ Short-chain fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin (INU), mannanoligosaccharides (MOS), sucrose (SUC), and wheat starch (WSt).

demonstrates the benefits of probiotics in controlling pathogenic bacteria and promoting host health.

3.3. Synbiotics

Synbiotics, an important concept in the International Scientific Association (ISPP) consensus statement, are defined as a mixture containing live microorganisms and substrates that can be selectively utilized by host microorganisms for the benefit of the host [88]. Various synbiotics combining different probiotic strains and prebiotic substrates have demonstrated efficacy in aquaculture, such as enhancing gut health, regulating the immune system, preventing disease, and improving water quality, as shown in

Table 4. Applications of synbiotics for sustainable aquaculture.

Synbiotic (Probiotic/Prebiotic)	Target Aquatic Organism	Effects	Reference
Bacillus spp./β-gluco-oligosaccharide	Abalone (Haliotis discus hannai)	Growth performance, immunity, antioxidants, digestive function (↑) * Beneficial gut commensal bacteria (↑)	[89]
Lactic acid bacteria + Bifidobacterium spp./inulin	Common carp (Cyprinus carpio)	Growth, survival (↑) Innate immunity and mucosal immunity (↑) Oxidative stress resistance, digestive ability (↑)	[90]
Acinetobacter KU011TH/chitosan	Catfish (Clarias gariepinus × C. macrocephalus)	Growth performance (→) * Immune function, tissue morphology, disease resistance (↑)	[91]
Pediococcus acidilactici/β-glucan	Florida pompano (Trachinotus carolinus)	Growth performance (→) Blood urea nitrogen and carbon dioxide (↓) *	[92]
Bacillus lincheniformis WS-2/alginate oligosaccharides	Apostichopus japonicus	Growth, digestion, non-specific immunity, disease resistance (↑) Beneficial gut commensal bacteria (↑)	[93]
Bacillus circulans PB7/fructo-oligosaccharide	Labeo rohita	Growth performance, digestive enzyme activity, non-specific immune ability, disease resistance (↑) Beneficial gut commensal bacteria (↑)	[94]
Lactobacillus plantarum/cacao pod husk pectin	Litopenaeus vannamei	Growth performance, immunity, disease resistance, and stress resistance ability (↑) Beneficial gut commensal bacteria (↑)	[95]
Lactobacillus casei/garlic	Barramundi (Lates calcarifer)	Growth performance, immunity, disease resistance, and stress resistance ability (↑)	[96]
Aspergillus oryzae/β-glucan	Nile tilapia (Oreochromis niloticus)	Growth performance, immunity, disease resistance, stress resistance ability (↑) Intestinal surface area, villus length (↑)	[97]
Pediococcus acidilactici/pistachio hull-derived polysaccharide	Nile tilapia (Oreochromis niloticus)	Growth performance, digestion, immune response, disease and stress resistance (↑)	[98]
Lactobacillus acidophilus + Bacillus subtilis + Saccharomyces cerevisiae/fructo-oligosaccharide	Chinese mitten crab (Eriocheir sinensis)	Growth performance, liver and pancreas antioxidant capacity (↑) Mortality rate after transportation stress (↓)	[99]
Synergistic Lactobacillus plantarum L20/Sargassum polycystum hydrolysate	Giant freshwater prawn (Macrobrachium rosenbergii)	Growth performance, innate immune response, and resistance of M. rosenbergii against A. veronii-induced necrotizing hepatopancreatitis (↑)	[100]
Bacillus safensis and Bacillus amyloliquifaciens/pectin	Rohu (Labeo rohita)	Weight gain and feed efficiency (↑) Digestive and glycolytic enzymes (↑) Immune parameters, erythrocytes, hemoglobin, serum protein (↑) Post-challenge survival rate against Aeromonas sobria (↑)	[101]

* ‘(↑)’, increase; ‘(↓)’, decrease; ‘(→)’, no change.

.

Tong et al. discovered that the synbiotic formed by β-glucan and Klebsiella sp. E26 alleviated the adverse effects of low-salinity stress on Litopenaeus vannamei by regulating the gut microbiota, energy metabolism, and immune activation, thereby significantly enhancing growth performance and survival rates [24]. Similar trends were observed for the combination of LDB7 (1 × 10⁷ CFU/g L. delbrueckii subsp. bulgaricus) and AR5 (5 g/kg Asparagus officinalis L. root), which effectively improved rainbow trout’s growth performance, feed utilization, and digestive enzyme activity, while significantly mitigating the adverse effects of crowding stress on immune, antioxidant, and serum biochemical parameters [102]. Moreover, dietary supplementation with a synbiotic mixture (10⁸ CFU g⁻¹ B. amyloliquefaciens + 0.5% β-glucan) elicited the most favorable responses in Channa striata. Specifically, this supplementation enhanced skin mucosal immunity, reduced cortisol levels, and conferred significant resistance against Aeromonas hydrophila, boosting survival by 27.7% post-challenge [103].

The addition of a synthetic dietary supplement containing 1 × 10⁷ CFU/g L. acidophilus and 2.5 g/kg M. oleifera enhanced the relative abundance of beneficial microorganisms in the white shrimp gut. This intervention simultaneously modulated immune signaling pathways, promoting growth and delivering significant health benefits in terms of pathogen immunity [104]. Furthermore, dietary supplementation with B. velezensis AAHM-BV2301 (1 × 10⁸ CFU/kg), chitosan (15 g/kg), and SiNPs could modulate the physiology, gut microbiota, and immune responses of Asian seabass, while enhancing resistance to V. vulnificus [105]. Cadangin et al. reported that Bacillus sp. KRF-7, Bacillus sp. PM8318, and β-gluco-oligosaccharide improved somatic growth in abalone, increased digestive enzyme secretion, and improved the intestinal morphology. Furthermore, observations indicated that these probiotics and prebiotics promoted growth and immunomodulation by reshaping the gut microbiota structure. However, combining these probiotics and prebiotics did not demonstrate any significant synergistic effects [89].

It should be noted that, despite these potential benefits, probiotics, prebiotics, and synbiotics are not a universal solution. Their exact effects may vary depending on the type of animal being raised, the environmental conditions, and the specific usage methods.

4. Probiotic Mode of Action

The mechanism of action of probiotics is complex and multifactorial, involving the competitive exclusion of pathogens and immune regulation, which has been reported in aquatic animals (Figure 2) [35].

Although the specific pathways of action and key regulatory mechanisms of probiotics are still under study, several proposed mechanisms are as follows:

• Competition for nutrients and available energy [106];
• Competition for adhesion site colonization [106];
• Inhibition of compound production [106];
• Enzymatic digestion [107];
• Sources of macronutrients and micronutrients [107];
• Enhancement of immune response;
• Interference with quorum sensing (QS) [108,109].

The colonization of symbiotic bacteria in the host is considered an important determinant of the health and pathological conditions of aquaculture organisms [110]. It is believed that probiotics are able to effectively colonize unoccupied ecological niches after entering the host’s digestive tract by producing adhesion molecules that bind to features of the intestinal mucosal layer and prevent the migration of invaders [46,111,112]. For example, the elongation factor Tu (EF-Tu) of L. plantarum HC-2, a key adhesion factor on its surface, mediates HC-2 adhesion and colonization in the shrimp intestine by interacting with fibronectin (Fib) in the gut [113]. Similarly, six moonlighting proteins were identified as adhesins of L. plantarum PO23 (LP-PO23), which were involved in the specific adhesion of LP-PO23 to the intestinal epithelial cells of olive flounder (Paralichthys olivaceus), with the strongest adhesion ability shown by GAPDH [114]. The competitive advantage of probiotics may be further enhanced by the secretion of a series of inhibitory compounds that can directly inhibit the growth of pathogens or interfere with their adhesion mechanisms [115,116]. Antimicrobial substances known to be produced or released by probiotics include bacteriocins, lysozyme, hydrogen peroxide, proteases, and iron carriers [111]. In addition, it has been reported that E. faecium is able to antagonize pathogens such as Vibrio parahaemolyticus, V. harveyi, and V. alginolyticus in L. vannamei through the production of peptidoglycan hydrolases (PGHs) with bacteriolytic activity [117]. Probiotics are also capable of disrupting the attachment of pathogenic bacteria and their biofilm formation by secreting surfactants such as lipopolysaccharides and lipopeptides [118]. Surfactin has been found to be a lipopeptide-type biosurfactant, produced mainly by the secondary metabolism of the genus Bacillus [119]. It weakens the adhesion abilities of pathogenic bacteria and the initial establishment of biofilms by reducing surface tension, disrupting the cell membrane structure, interfering with population-sensing signals, and inhibiting the formation of adhesion substrates. More importantly, it is able to depolymerize or inhibit established biofilms, thus effectively limiting the ability of pathogenic bacteria to colonize and infect [120]. Biofilms are a key factor in the survival and resistance of many pathogenic microorganisms. Therefore, by disrupting this structure, probiotics can significantly reduce pathogen persistence and resistance [121]. Organic acids (e.g., lactic, acetic, butyric, and propionic acids) are important metabolites produced by probiotics in the gut and can create a chemical microenvironment that promotes antagonism by lowering the intestinal pH [122]. In this way, probiotics may inhibit the proliferation of opportunistic pathogenic microorganisms in vivo. Furthermore, probiotics confer a long-term residency advantage in the intestine through adhesion and colonization. Compared to other microorganisms in the same environment, they can more effectively capture and utilize nutrients and energy sources, hindering the growth and activity of pathogenic microorganisms [46,123]. The probiotic properties of LAB strains were determined using shrimp mucus, with E. faecium NRW-2 demonstrating the highest adherence capacity while significantly reducing Vibrio populations in the shrimp gut [124]. The intestinal mucus could provide binding sites and a source of nutrients for microbial growth [125]. This result suggests that the adhesion capacity of E. faecium NRW-2 to mucus may be related to its competitive exclusion or nutrient competition with Vibrio in the shrimp intestine [124].

Probiotics enhance innate and adaptive immunity in aquatic animals [123]. Several studies have shown that probiotics can stimulate the activity of antioxidant enzymes (superoxide dismutase (SOD), phenoloxidase (PO), catalase (CAT), and glutathione peroxidase (GPx)), as well as non-specific immune enzymes such as lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP), which help maintain oxidative and antioxidant homeostasis in aquatic animals, effectively kill and eliminate pathogenic microorganisms or exogenous substances, and play an immune defense role [126,127,128,129].

Moreover, the recognition of probiotic components by pattern recognition receptors (PRRs) can induce an immune response and exert probiotic effects [130]. Specifically, probiotic surface structures contain microbe-associated molecular patterns (MAMPs) of lipopolysaccharide (LPS), flagellin, lipophosphatidic acid (LTA), peptidoglycan (PG), DNA, and other surface (lipo)proteins, which bind to PRRs expressed on the surfaces of immune cells and intestinal epithelial cells to drive innate immunity [131]. The innate immune system involves a variety of cell types, including monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells [132]. Fish and other aquatic species with poorly developed adaptive immunity have a more pronounced reliance on the innate immune system, with PRRs playing a central role in immune defense, consisting mainly of inherited-encoded Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and retinoic acid-inducible gene-I (RIG)-like receptors (RLRs) [133]. PRRs trigger downstream effects by activating classical signaling pathways (e.g., nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), etc.), inducing the production of interferon I (IFN-I), pro-inflammatory cytokines (e.g., interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF)) and other immune mediators, enhancing the recruitment of immune effectors and activating the non-specific immune system, thereby helping the host to respond rapidly to pathogen invasion [134,135]. Through this mechanism, probiotics not only help to enhance the antipathogenic response but also regulate the strength and duration of the immune response, thereby avoiding excessive inflammatory responses triggered by immune overactivation.

5. Probiotics for Sustainable Aquaculture

5.1. Probiotics as Water Additive Agents

To date, most research on the beneficial functions of probiotics has focused on dietary supplements, and their practical application for direct addition in aquatic environments still appears relatively limited. In recent years, interest in the use of water additives as an environmentally friendly practice has rapidly increased, with a major focus on the effects on the physiological characteristics (growth performance, antioxidant and immune responses) of farmed animals and on farmed water quality [136,137]. Probiotics as water additives have shown promising results in aquaculture, as clearly demonstrated by a study on Nile tilapia, where different doses of probiotic bacteria of the genus Bacillus were added to the water of aquaculture ponds in a 56-day experiment. This showed that the addition of probiotic bacteria in a concentration-dependent manner significantly improved fish growth performance and immune indices and, at the same time, enhanced the resistance to common pathogenic bacteria such as Aspergillus flavus [138]. Moreover, water-applied probiotics are suitable for many types of farmed animals and actively promote healthy fish development throughout the growth phase [139,140,141]. In addition, another important function of probiotics as natural water additives is to reduce the concentrations of toxic nitrogenous compounds in the water [142]. For example, two probiotic strains (B. toyonensis and Geobacillus stearothermophilus) administered to Nile tilapia rearing water reduced the ammonia ranges and median levels in the water [136]. The addition of two probiotic bacteria, Salipiger thiooxidans and Exiguobacterium aestuarii, to the culture water of L. vannamei resulted in denitrification, leading to a significant reduction in the total ammonia and nitrite levels in the water [139].

5.2. Probiotics as Fermented Feed Additives

The development of aquaculture nutrition in China has led to a transition from a dependence on natural productivity to systematic research on the nutritional needs of aquatic species, which has spurred innovation and enhancement in non-traditional aquafeeds [143]. In aquaculture, ingredients of plant and animal origin are widely used to meet the nutritional needs of farmed animals [144]. However, this practice faces many challenges, including the presence of anti-nutritional factors (ANFs), the reduced bioavailability of nutrients, indigestible particles, and microbial contamination [145].

In order to address these issues, the use of fermentation technology to adjust the nutritional value of feeds and the quality of cultured products is a cost-effective and environmentally friendly solution that has been incorporated as an ideal choice for aquafeeds [145,146,147,148]. The core aspect of fermentation lies in the biochemical modification of the substrate by probiotic microorganisms and their enzymes, which degrade complex macromolecules into small molecules in the process of ‘pre-digestion’ and, at the same time, produces large amounts of nutrient-rich microbial proteins and useful metabolites, thus realizing value addition in nutrients and improvements in functional properties [149,150,151]. In addition, this transformation process leads to the reduction or even elimination of anti-nutritional factors in the substrate [152], which means that both conventional and non-conventional fermented feeds have the potential to partially or completely replace fishmeal (FM) in order to further reduce production costs and promote the recycling of resources [153,154].

5.2.1. Effects of Probiotic Fermentation on Anti-Nutritional Factors in Feed Ingredients

The challenge with plant-based ingredients as alternative protein sources is that they contain a number of anti-nutritional factors (ANFs). Most ANFs do not directly cause the death of farmed organisms, but they can negatively affect the growth and health statuses of farmed animals and may also lead to the release of waste into the surrounding environment [155,156]. Common anti-nutritional factors in plant-based feeds include the following:

• Protease inhibitors: Interfere with protein digestion [157];
• Phytates: Binds minerals such as Ca, Fe, Zn, and Mg, thereby reducing their bioavailability [158];
• Lectins: Interfere with nutrient catabolism and absorption by reversibly binding to sugars and/or glycoproteins on the surface cells of the intestinal wall [159];
• Tannins: Interfere with the digestive process by binding to enzymes or to feed ingredients such as proteins or minerals [160];
• Oligosaccharides: May lead to indigestion and intestinal gas buildup [161].

Microbial fermentation is an effective means of hydrolyzing anti-nutritional factors present in feeds through the production of specific enzymes (e.g., phytase, xylanase, and protease), thereby increasing the digestibility of feeds for animals and reducing the load of nutrients (N, P) on the environment [162]. For example, wholemeal lupin flour (WL) successfully reduced the content of oligosaccharides, phytic acid, and alkaloids after 24 h semi-solid fermentation with a probiotic blend and two yeast strains [163]. The use of raw materials with low palatability, such as anti-nutritional factors like saponins, with a bitter taste, may affect feed intake and lead to a decrease in the feed efficiency ratio (FER) and apparent digestibility coefficient (ADC) [164]. The use of probiotic fermentation can mitigate this negative impact on feed acceptance in some cases. For example, the solid-state fermentation of sunflower meal by Saccharomyces cerevisiae (YFSFM) was able to reduce the content of phenolic compounds, represented by chlorogenic and caffeic acids, with phytic acid and saponins showing the same decreasing trends. After 84 days of feeding experiments, YFSFM protein was able to replace at least 25% of fishmeal (FM) without affecting the growth performance and feed utilization of Nile tilapia. In addition, the apparent digestibility of dry matter, protein, lipid, and energy from YFSFM-50 was not affected [165]. When soybean meal is used as a primary protein source, a high level of dietary addition may result in decreased feed utilization, reduced digestive enzyme activity, and impaired intestinal health, primarily related to the presence of anti-nutritional factors in soybean meal [166,167]. Lactobacillus spp. are widely used as probiotics, and fermented soybean meal using Lactobacillus spp. was able to replace FM in white shrimp diets, with the 75% FSBM diet exhibiting the highest apparent digestibility for dry matter, protein, and lipids when compared to other dietary treatments. In addition, the shrimp’s total blood cell counts and phenoloxidase (PO) activity were significantly higher at this level of substitution compared to the other dietary groups [153]. This suggests that fermented soybean meal is effective in counteracting immune irritation and chronic inflammation caused by anti-nutritional factors, thereby maintaining the overall health of farmed animals.

5.2.2. Effects of Probiotic Fermentation on Nutrient Utilization

Probiotic microorganisms are a reliable strategy applied in the aquafeed industry to improve feed nutrients and increase feed utilization through fermentation, thereby enhancing the feed’s nutritional value. For example, solid-state fermentation using the probiotic Pediococcus pentosaceus Y64 significantly increased the acid-soluble protein content and improved the quality of soybean meal [168]. Fermentation not only increases the content of protein in its soluble form but also effectively alters the amino acid profile and fatty acid profile of the feed. The free amino acids, such as threonine, serine, methionine, leucine, isoleucine, and tyrosine, were significantly increased in commercial feeds for A. japonicus fermented by Corynebacterium glutamicum. In addition, the total content of polyunsaturated fatty acids in the fermented feeds was higher than that in unfermented feeds, and the fatty acid profiles showed better nutritional value [169]. High-fiber, hemicellulose, and similar polymers often limit the utility of certain dietary feed ingredients, especially macroalgae [145,170]. It has been shown that A. japonicus lacks endogenous amylase, cellulase, and fucoidan enzymes, so these enzymes must be supplied by adding exogenous microorganisms [171]. By fermenting the feed for juvenile A. japonicus with probiotic complexes, the crude fiber proportion in the feed can be effectively reduced, while the content of reducing sugars can be increased [147]. Alginic acid and polysaccharides increase the viscosity of feed and affect its absorption. During the fermentation process, Pseudoalteromonas sp. D11 was able to degrade fucoidan into small-molecule active oligosaccharides, and these active oligosaccharides played an important role in digestion and absorption in sea cucumbers [147]. The improvement in the nutritional potential of the above feed was attributed to the synergistic degradation of complex stored substances by various enzymes secreted by the probiotic strains, which were converted into simple compounds with higher bioavailability [172]. Overall, the biotransformation of various nutrients and the addition of bioactive compounds can improve the quality of feeds.

Generally, the efficiency of fermented feeds is also evaluated through feeding trials. A co-fermented diet of probiotic complexes (L. acidophilus, L. reuteri, and L. plantarum) significantly improved the feed efficiency (FE) and protein efficiency (PER) of juvenile largemouth bass (Micropterius salmoides), promoting the digestion and absorption of dietary protein [173]. In the diet of tilapia, fermented soybean meal performs better than raw soybean meal, and a diet based on fermented soybean meal improved fish growth and feed utilization efficiency [168]. When a fermented feed is ingested by farm animals, it directly interacts with the intestinal tract and other digestive organs, affecting the intestinal flora, digestive enzyme activity, and growth performance [169]. Wang et al. found that the use of probiotic-fermented kelp scraps fed to spiny cucumbers resulted in a significant increase in growth performance in the fermented group and a significant increase in the diversity of the intestinal microbiota compared to the unfermented feed [174]. Zhang et al. developed a synergistic and effective probiotic blend and applied it to the solid-state fermentation of commercial feeds for Penaeus vannamei. The feeding results showed that 1 × 10⁸ CFUg⁻¹ of fermented feed increased the activity of digestive enzymes such as protease, amylase, and lipase and improved the growth performance of P. vannamei, as well as creating a better microecological environment for the shrimp intestinal tract [175].

Microorganisms serve as fermentation agents to enhance feed, but they also possess probiotic properties, such as immune and antioxidant abilities. Dietary fermented wheat bran polysaccharides prepared using S. cerevisiae and B. subtilis fermentation positively regulated intestinal antioxidant-related gene expression and the gut microbiota in zebrafish, thereby improving their growth performance [176]. After 40 days of feeding juvenile A. japonicum with a fermented feed using Corynebacterium glutamicum, transcriptome analysis revealed 4492 differentially expressed genes enriched in the immune system and metabolic pathways. Further gene expression analysis showed that the upregulation of genes such as NFκB1, TLR, TLR3, TRAF6, MyD88, and p38 activated the TLR signaling pathway in sea cucumber, enhancing its immune response and growth performance [169]. Barramundi (Lates calcarifer) fed a probiotic-fermented Sargassum linearifolium diet (termed PF-SL) exhibited higher glutathione peroxidase and total antioxidant activity. The PF-SL diet upregulated the expression of anti-inflammatory genes (occludin, claudin1, and nrf2) and suppressed the expression of pro-inflammatory genes (hsp70 and tnf-α) according to qRT-PCR analysis. In addition, in a 27-day V. harveyi provocation trial, the mortality rate for the PF-SL diet was only 14%, significantly lower than the 46% rate in the unfermented group and 67% rate in the control group [177].

5.2.3. Effects of Probiotic Fermentation on Microbial Contamination

Probiotics involved in fermentation, such as lactobacilli, produce organic acids and bacteriocins through metabolism, which can inhibit the growth of harmful microorganisms via competition (energy and nutrient competition) [178], increase the permeability of the extracellular membrane [179], change the intracellular osmotic pressure [180], and inhibit the synthesis of macromolecules and other metabolic functions [181]. This means that some of the feed ingredients that are easily contaminated (e.g., a variety of byproducts) regain the lost margin of safety, and it effectively extends the shelf life [182]. In evaluating the effects of LAB as inoculants on microbial and mycotoxin contamination in fermented feeds, it was observed that an increase in the lactic acid concentration and the proportion of L. plantarum significantly inhibited the proliferation of undesirable microorganisms. It was found that L. plantarum Q1-2 and L. salivarius Q27-2 reduced aflatoxin B1 by 34.17% and 16.57% and deoxynivalenol by as much as 90.61% and 51.03% as compared to the control group [183]. Vegetable waste typically has a high pathogen load [184,185]. However, after inoculation with exogenous probiotics (S. cerevisiae, B. subtilis, and L. plantarum), significant changes were observed in the phenotypic characteristics and structure of the bacterial community. Specifically, this process led to a significant decrease in the abundance of Proteobacteria (Pantoea, Pseudomonas) and Ascomycota (Alternaria, Epicoccum, Fusarium) associated with decay and pathogenicity, with the enrichment of L. plantarum and S. cerevisiae playing a crucial role [186].

In summary, biofermentation technology has a significant impact on the health and safety of aquatic animals, the quality of aquatic products, resource conservation, and environmental friendliness, as well as the advancement of key processing technologies in the aquafeed industry.

6. Probiotic Effects on the Farming Environment

Intensive aquaculture is threatened by different pollutants, both external and internal, where harmful emissions of organic and inorganic metabolites usually lead to the deterioration of the surrounding environment, especially exacerbating eutrophication—a change that places additional environmental stress on farmed organisms [31,187]. Traditional methods of controlling toxic metabolites typically involve biofilters and water exchange, collectively referred to as recirculating aquaculture systems (RAS). While RAS are effective in maintaining water quality, they require regular testing of multiple water quality parameters—a model that is not only expensive but also difficult to access for specific technologies, which limits its popularity [188]. In this context, the introduction of probiotics (as additives to water or feed) is a promising bioremediation strategy for maintaining water quality and reducing contaminant levels [31]. Studies have shown that ponds with the highest fish stocks tend to exhibit higher nitrogen (N) and carbon (C) nutrient loads in the sediment and water column, which favors the development of ammonia-oxidizing archaeal (AOA) and ammonia-oxidizing bacterial (AOB) communities—microorganisms that play a key role in nitrogen removal through coupled nitrification and denitrification processes [189]. In addition, other types of probiotics, such as L. plantarum and B. subtilis, when used alone or in combination, have been shown to be effective in reducing NH₄⁺ and NO₂⁻ levels in cultured water while improving water transparency [190]. Further studies by Hu et al. showed that Bacillus spp. could reduce ammonia nitrogen, phosphate, and the chemical oxygen demand by up to 36.3%, 28.9%, and 15.2%, respectively, in sterilized aquaculture systems [191]. Meanwhile, probiotics help to reduce the levels of toxic nitrogen compounds by supporting beneficial bacterial populations and reducing pathogen loads, which together improve water quality [192,193]. A metagenomic analysis of the nitrogen cycle revealed that probiotic addition elevated the relative abundance of genes related to ammonia oxidation, nitrification, and denitrification (e.g., amoC_B, hao, nirS, and nosZ), as well as altering the composition of the microbial community. Specifically, it increased the abundance of genera such as Limnohabitans and Sediminibacterium and decreased the abundance of the genera Roseomonas and Rubrivivax [194]. These changes significantly contributed to nitrogen removal and overall water quality improvement in the aquaculture system.

7. Conclusions and Future Perspectives

Recent advances in multi-omics technologies, including metagenomics, transcriptomics, and proteomics, have greatly accelerated research on probiotic interactions within the host microbiome. In the study of composite probiotics, leveraging synergistic interactions among multiple strains has become a key strategy for enhancing functionality and complementarity. The optimization of fermentation processes, particularly through standardized solid-state fermentation, has shown considerable promise in improving feed quality and stability, facilitating broader application within the feed industry.

Beyond their traditional roles in growth promotion and immune enhancement, probiotics have evolved into core components of sustainable aquaculture, serving as eco-friendly bioremediation agents and multifunctional tools. They not only act as alternative immunoprophylactic strategies, complementing or replacing vaccines and drugs, but also mitigate water pollution and promote environmental health.

Several key practical considerations need to be emphasized to ensure the reliable and effective application of probiotics in aquaculture settings.

Administration period: The microbiota is critical for the early development and health of marine vertebrates and invertebrates, and proper feeding affects larval growth, survival, and the quality of breeders and juveniles [195,196]. Most aquatic organisms need 4–8 weeks of continuous probiotic supplementation for stable gut colonization, and early dietary or waterborne application is recommended to establish beneficial microbial communities in early growth stages.

Temperature adaptation: It is necessary to select probiotic strains with thermal adaptability in line with the temperature ranges of aquaculture systems to ensure their efficacy under such conditions [197]. Bacillus spp. and Clostridium butyricum are suitable for warm-water aquaculture (25–35 °C) [198,199], while LAC tolerate cold water (5–19 °C) [200].

Salinity matching: For marine and brackish water aquaculture systems, salt-tolerant strains (e.g., B. velezensis and Cytobacillus firmus isolated from marine aquaculture systems [201]) are preferred. Under low-salinity stress, the probiotic dosage can be increased by 10–20% to improve stress resistance and colonization efficiency [202].

Water quality coupling: When the water quality deteriorates (ammonia nitrogen > 0.5 mg/L, nitrite > 0.1 mg/L), probiotics should be applied as water additives, combined with aeration and moderate water exchange, to enhance the bioremediation of nitrogenous waste and organic pollutants.

Future efforts aimed at harnessing the full potential of probiotics should focus on the following priorities:

• Optimizing strain combinations to maximize synergistic effects;
• Deepening the understanding of probiotic mechanisms, particularly their immunomodulatory and microenvironment-regulating functions;
• Refining and standardizing fermentation processes and production workflows;
• Quantifying the environmental benefits of probiotics in aquaculture and establishing robust environmental footprint assessment systems to support green certification;
• Strengthening safety evaluations to ensure safe and broad application.