1. Definition and Characteristic Features
The Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) define probiotics as “Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”
[1]. Recently, the term probiotics has been associated with microbial feed additives that, when controlled in enough amounts, confer health and beneficial impacts on a host of aquatic animals
[2].
Probiotics act as a defense system for the host against harmful microbes or foreign substances
[3][4][5][6]. They also produce beneficial bioactive molecules such as enzymes, proteins, lipids, organic acids, and others. Some of these bioactive molecules improve binding to probiotics and reduce, therefore, the activity of pathogens in the gut region through the surface competition mechanism
[7]. Probiotics play a significant role in strengthening the immune system of the host
[8]. While earlier studies have noted the utilization of probiotics in pigs, poultry, cattle, and humans, their application in aquaculture is a relatively new idea
[9][10]. Probiotics can be administered in two ways in aquaculture. They can be supplemented with feed to modulate gut microbes, or they can be directly added to the water, thereby inhibiting the growth of pathogens. These modes of administration are very critical in the utilization of probiotics in aquaculture
[11][12]. Probiotics can have alive, dead, or microbial cell components and provide benefits to the host when added to feed or rearing water. This is achieved at least in part by improving the microbial balance of the host or ambient environment
[12].
Figure 1 summarizes the different entryways of probiotics and their benefits in the aquaculture system.
Figure 1. Illustration of the use and impact of probiotics in aquaculture systems.
Probiotics appear to be a new agent for the development of aquaculture systems, exerting several favorable effects on growth activity, immune systems, digestion, water quality, the inhibition of pathogens, and the regulation of the gut microbes of aquatic animals. The utilization of probiotics in aquaculture is a modern trend, although its effectiveness in the aquatic ecosystem has not been considered comprehensively. Probiotics are ubiquitous, commonly present in aquatic animals, and play an important protective role throughout the digestive system
[13][14]. Mainly represented by Lactobacilli, these beneficial microorganisms are vital to preventing illnesses and improving aquatic animal GIT functions by excreting secondary metabolites such as lactic acid and other bioactive compounds
[15][16]. These biomolecules, synthesized by probiotics, protect against inhibitory molecules from pathogens
[17]. They can also be extracted from probiotics in terrestrial plants and marine life forms and then utilized to enhance disease resistance, develop the immune system, reduce environmental stress, and increase feed quality levels
[18][19]. Advanced studies in this field have reported microbial by-product biomolecules such as enzymes, lipids, proteins, and immune toxins
[20]. Nowadays, some probiotic products are commercially available and are already used in aquaculture as feed additives
[21]. These microbial by-products are beneficial and are mainly helpful in enhancing the health status of aquatic animals.
Potential probiotic strains are assessed based on physiological, functional, and safety criteria such as stress resistance (e.g., acid and bile tolerance), gut epithelial adherence, survival rates, pathogen-inhibiting activities, large-scale cultivability, non-hemolytic activity, non-pathogenicity, the absence of plasmid-encoded antibiotic resistance genes, and beneficial effects on host animals. These include, for instance, their capacity as growth promoters and anti-inflammatory, antimutagenic, and immunostimulatory agents. Each new strain used for probiotic expansion mainly contains all the aforesaid features
[22][23][24]. Current and potential probiotic species for use in aquaculture are listed in
Table 1.
Table 1. A list of current probiotic strains for use in aquaculture.
2. Possible Modes of Action of Probiotics in Aquaculture
The significant effects of probiotics, e.g.,
Bacillus spp. as feed supplements, include the improvement of growth performance, digestive enzyme activity, resistance to pathogens, and immune responses in aquatic animals
[41][42]. Possible action modes of probiotics in aquaculture include the regulation of amino and fatty acid metabolisms, the excretion of digestive enzymes and vitamins or cofactors, the production of antagonistic compounds that inhibit bacteria, the enhancement of immune responses, the disruption of the quorum-sensing processes of pathogenic organisms, stress improvement, and heavy-metal detoxification.
2.1. Probiotics Act as Growth Enhancers in Aquaculture
Probiotics play a crucial role in digesting complex dietary macronutrients. Additionally, they contribute to the host’s nutrient and vitamin supply and provide essential digestive enzymes, thereby enhancing feed utilization and digestion.
One of the mechanisms that regulates the metabolism of amino and fatty acids is the capacity of various probiotic strains to produce vitamin B12, as revealed by a study on carp guts
[43][44]. In addition, this is helpful for enhancing fish growth and eradicating vitamin B12 deficiency in fish
[45]. Also, essential macronutrients are usually supplied through feed. Various micronutrients such as amino acids, vitamins, and fatty acids are very important for physiological functions as nutrients in aquatic animals
[46][47][48]. For instance, diverse fish species such as carp (
Cyprinus carpio), rainbow trout (
Oncorhynchus mykiss), channel catfish (
Ictalurus punctatus), and tilapia (
Oreochromis niloticus) have been found to synthesize vitamin B12
[49][50][51]. The growth and survival rates of juvenile black tiger shrimp (
Penaeus monodon) were enhanced when they were fed for 100 days with a combination of
Lactobacillus spp., previously isolated from the GITs of chickens
[52]. In fact, probiotics improve the digestive function of aquatic animals by producing or inducing the secretion of different kinds of extracellular enzymes such as proteases, amylases, and lipases.
The function of probiotics results in abridged feed cost, which accounts for 60–70% of the contribution cost of fish production
[53][54]. Both the maximum growth performance and best feed conversion ratio were detected when
O. niloticus was fed with the probiotic
Micrococcus luteus [55][56].
Bacillus subtilis improved feed digestibility; enhanced weight gain and feed conversion; and significantly increased the survival rate of bullfrogs (
Lithobates catesbeianus) fed different doses (2.5, 5.0, and 10.0 g/kg)
[57][58].
Bacillus species aid in the digestion of aquatic animals by supplying exoenzymes (proteases, lipases, and amylases) that enhance digestive enzymes
[59]. The addition of probiotics (a mixture of
Streptococcus faecium, Lactobacillus acidophilus, and
Saccharomyces cerevisiae) at a concentration of 0.1% to Nile tilapia fry diets was found to enhance animal growth and intestinal alkaline phosphatase activity
[60].
2.2. Biocontrol of Bacterial Diseases in Aquaculture
In the past few decades, numerous studies have stated that probiotics synthesize different types of inhibitory substances responsible for antagonistic activity against pathogens. Two probiotic strains of LAB (
Lactococcus lactis MM1 and
Enterococcus faecium MM4) isolated from the intestine of the orange-spotted grouper (
E. coioides) can secrete several inhibitory substances such as hydrogen peroxide and bacteriocin-like substances. These can be utilized to induce antimicrobial activity against different pathogens such as
Staphylococcus aureus,
V. harveyi, and
V. metschnikovi, which affect groupers (
E. coioides)
[61][62]. The probiotic
B. pumilus H2 has strong inhibitory activity against
Vibrio spp. through its main mechanism of amicoumacin production, disrupting the cell membrane and cell lysis and thus showing anti-
Vibrio activity
[63][64]. The probiotic
Bacillus velezensis cell-free supernatant contains different types of bioactive molecules that act against
A. salmonicida infection
[65]. The lipopeptide N3, synthesized by the probiotic
Bacillus amyloliquefaciens M1, has strong antibacterial activity in the whole-cell membrane, which can exert significant effects from ion-conducting channels on the whole-cell membrane and membrane-active properties
[66][67]. The probiotic species
Clostridium butyricum, a culture supernatant, includes different types of inhibitory substances, mainly short-chain fatty acids (SCFAs); it can lower the pH of the intestine and thus decrease the growth of pathogens in fish intestinal epithelial cells
[68]. The probiotic
E. faecium was supplemented in the diets of Olive flounders and can enhance the antibacterial activity
[69].
2.3. Biocontrol of Viral Diseases in Aquaculture
Microorganism strains with potential probiotic effects in aquaculture such as
Pseudomonas spp.,
Vibrios spp., and
Aeromonas spp. induce antiviral effects against hematopoietic necrosis virus (IHNV) infection
[70][71]. Similarly, the potential probiotic strain
Pseudoalteromonas undina VKM-124 has been used to improve Yellow Jack (
Carangoides bartholomaei) larval survival and enhance antiviral effects against Neuro Necrosis Virus (SJNNV) infections
[72][73].
2.4. Immunostimulant Agents in Aquaculture
Immunity development and modulation are among the various health benefits of probiotics in aquaculture. The majority of earlier studies have dealt with the health-boosting capabilities of probiotics in aquatic organisms. Currently, probiotics are significantly focused on the immunological development properties of the piscine immune system, including both innate and adaptive immunities
[74]. Different types of probiotics improve various immunological properties, and notably, several fish use the efficiency of probiotics to vitalize teleost immunity in both in situ and ex situ conditions
[75]. Although promising findings have been reported in previous studies, most immunostimulants do not progress to large-scale functions for fish. Since various immunostimulants in aquaculture produce similar effects, researchers have demonstrated the utilization of probiotics to enhance disease resistance and the immune system of carp fish species
[76][77]. Several carp fish have shown an increase in the production of total serum protein, nitric oxide, lysozyme, albumin, and phagocytic activity via blood leucocytes; express IL-1b, superoxide anion, myeloperoxidase content, respiratory burst activity, and globulin levels; and complement C3, TNF-α, and lysozyme-C
[76][78]. Current study reports indicate that probiotics (either single or mixed types) could enhance the immunological development of fish
[79]. These reports have emphasized the immunomodulating properties of beneficial living cell organisms and the factors that facilitate the optimal induction of defense responses in the fish community. The probiotic strain
B. pumilus SE5 has been isolated from the intestine of the fast-growing grouper,
E. coioides [80][81], and subsequent studies have demonstrated that both viable and heat-inactivated
B. pumilus SE5 could shape intestinal immunity and microbiota
[82] and improve the growth performance and systemic immunity of
E. coioides [83]. The dietary supplementation of the cell wall (CW), peptidoglycan (PG), and lipoteichoic acid (LTA) of the probiotic
B. pumilus SE5 and its effect on intestinal immune-related gene expression and microbiota were evaluated in a 60-day feeding trial. The PG and LTA of the probiotic
B. pumilus SE5 were more effective than the CW in shaping the intestinal immunity and microbiota of
E. coioides [84], even though the mechanisms were largely unclear and needed further study.
2.5. Interference of Quorum Sensing in Aquaculture
Quorum sensing (QS) is a communication system among bacterial cells that is very useful in controlling different kinds of biological macromolecule expressions like virulence agents in cell-thickness-dependent comparative performance
[85]. In this process, QS bacteria produce and generate tiny marker molecules called auto-inducers
[86]. The disruption of the QS process in pathogenic organisms is a potential anti-infective strategy, and different types of methods have been used to investigate QS. These include the inhibition of signal molecule biosynthesis, the application of QS antagonists, the chemical inactivation of QS signals with oxidized halogen antimicrobials, signal molecule biodegradation with bacterial lactonases and bacterial and eukaryotic acylases, and the application of QS agonists in aquaculture
[87][88]. N-acyl homoserine lactones (AHLs) are the most important family of QS auto-inducers utilized in Gram-negative bacteria, and their biodegradation is a potential way to interrupt QS
[89].
Bacillus species were among the first bacteria documented to degrade AHLs through the production of lactonase enzymes. Probiotic
Bacillus strains can effectively secrete quorum-quenching enzymes and could reduce the pathogenic activity of
A. hydrophila YJ-1 and control gut microbiota
[90][91]. The dietary supplementation of probiotics with quorum-quenching activity has been shown to increase the intestinal barrier function and enhance the immune system of crucian carp against
A. hydrophila infection. The quorum-quenching bacteria increase the expression of the tight junction (TJ) proteins ZO-1 and Occludin, which control the permeability and absorption of the intestinal mucosal barrier of crucian carp
[92].
Bacillus sp. QSI-1 has been reported to be a quorum quencher in virulence agent production and the biofilm arrangement of the zebrafish pathogen
A. hydrophila. In experimental trials, fish fed with
Bacillus sp. QSI-1 exhibited a relative survival percentage of 80.8%
[93]. In another study, AHL-degrading
Bacillus sp. was shown to protect shrimp (
Penaeus monodon) against
Vibrio harveyi infection
[94]. Furthermore,
Enterobacter sp. f003 and
Staphylococcus sp. sw120, isolated from fish intestines and pond sediment, respectively, have demonstrated the ability to degrade acyl-homoserine lactones (AHLs) and protect against
A. hydrophila infection in the cyprinid
Carassius auratus gibelio [95]. In a biofilm system, bacteria are resistant to high temperatures, phagocytic cells, surfactants, antibiotics, and antibodies and can alter their vital transmissions via quorum-sensing signaling
[96]. These findings suggest that bacteria capable of degrading AHLs should be considered an alternative to antibiotics in aquaculture for effectively controlling bacterial infections in fish.
2.6. Stress Improvement in the Aquaculture System
Stress in a fish’s life cycle disrupts all production. The cultured species may be weakened and averse to taking feed
[97]. In this condition, probiotics in culture farms can decrease stress levels and help to enhance the innate immune system against pathogens and environmental stressors
[98][99]. Probiotic treatments are very helpful in increasing the production of fish within the given time, and they also reduce the stress level in normal aquaculture practices.
Studies have concluded that the use of some probiotic strains increases chronic stress resistance in zebrafish (
D. rerio)
[100][101]. Supplementation with an experimental nutritional probiotic,
Lactobacillus delbrueckii sp.
Delbrueckii, in sea bass led to a decrease in cortisol levels from 25 to 59 days, which, in fish tissue, is a stress indicator since it is directly engaged with the host’s reaction to stress
[102]. One more approach evaluated how fish treated with probiotics exhibited increased flexibility in stress tests when compared with a control group
[54]. The antioxidative properties of the probiotic
Lactobacillus fermentum induce protective action in the intestinal microbial ecosystem and help to overcome exo- and endogenous oxidative stress
[103]. The probiotic strain
Bacillus coagulans SCC-19 alleviates the nonspecific immune damage induced by cadmium in common carp while also relieving oxidative stress induced by cadmium in fish
[104].
2.7. Reducing Heavy Metals in Aquaculture
Heavy metals such as lead (Pb), cadmium (Cd), silver (Ag), chromium (Cr), mercury (Hg), cobalt (Co), zinc (Zn), iron (Fe), and copper (Cu) are present in the soil, water, and atmosphere
[105][106][107]. These metals can have toxic effects on all organisms and pose a huge risk to food quality, crops, and environmental quality. Heavy metals are mainly connected to anthropogenic action in the ecosystem
[108]. Aqueous release from metal industries (steel, mining, and electroplating) contains elevated levels of heavy metals that end up in water bodies, and they are then also utilized for aquacultural action
[109][110]. These heavy metals accumulate in fish tissue, and this is a matter of great concern with regard to humans consuming them via the food chain and breathing
[108][110][111]. Their elimination is very helpful in reducing the toxic effects of the aquatic environment and outflow is, subsequently, imperative
[112]. Among all the recommended methods of eliminating heavy metals is the process of utilizing microbes, which is cost-effective
[113]. The action mechanisms of probiotics in detoxifying heavy metals can be classified into metabolically independent processes that do not require cellular energy, such as biosorption, and cellular-energy-dependent processes, namely, bioaccumulation and bioprecipitation
[114].
Biosorption relies on a physicochemical process wherein cell-surface structures bind heavy metals through physical interactions. For example,
Lactobacillus acidophilus and
Bifidobacterium angulatum are effective in removing Cd, Pb, and As through electrostatic interactions between heavy-metal cations and the anionic functional groups of cell wall membranes
[115]. Some probiotics release exopolysaccharides (EPSs), which can sequester heavy metals and reduce their bioavailability. The mechanisms underlying EPS-metal binding are mainly related to negatively charged acidic groups and steric structures on the surface of EPSs
[116].
In bioaccumulation processes, probiotics accumulate heavy metals within their cells through energy-dependent processes. This can involve the synthesis and use of metal-binding proteins, such as metallothionein. For instance,
Bacillus cereus can produce metallothionein in order to accumulate Pb
[117].
Bioprecipitation involves the conversion of free metals into insoluble complexes, thereby reducing their bioavailability. Bacteria can catalyze oxidative and reductive processes to facilitate the precipitation of heavy metals.
Micrococcus spp. have been demonstrated to be able to sequestrate heavy metals such as Zn, Cd, Pb, and Fe via calcite precipitation
[118]
Generally, heavy metals activate the sporulation development of
Bacillus species and thus decrease heavy metal absorption
[109][119]. In addition, probiotic strains from aquatic farming sediments can be utilized as dietary supplements and help to remove heavy metals and metal-resistant microbes from the intestines of aquatic organisms, particularly fish, to control the progress of heavy metal accumulation
[120].
3. Major Probiotic Genera as Biocontrol Agents in Aquaculture
The major probiotic genera used in aquaculture are
Lactobacillus and
Bacillus [121]. In most cases,
Bacillus,
Lactobacillus,
Lactococcus,
Leuconostoc,
Pediococcus, and
Weissella are isolated from fish and shellfish guts
[122][123][124][125][126]. Supplementation in aquaculture feed is achieved using single-strain probiotics or associations of various bacteria as multi-strain probiotics (MSPs), which have been reported to have more beneficial effects on hosts owing to synergistic effects between various strains
[127].
Table 2 lists some examples of probiotic-based functional feed additives for aquatic animals.
Table 2. Functional feed additives of major probiotics in aquatic animals.
Probiotics Organisms |
Functions |
Aquatic Organisms |
References |
Bacillus |
|
|
|
B. licheniformis HGA8B |
↑ growth performance and ↓ feed conversion ratio Up-regulation of immune genes |
O. niloticus |
[128] |
B. cereus G19 B. cereus BC-01 |
↑ growth and immunity |
Apostichopus japonicus |
[129] |
B. cereus EN25 |
Immunity and resistance against Vibrio splendidus |
A. japonicus |
[130] |
B. pumilus SE5 |
↑ growth and immunity |
L. vannamei |
[131] |
B. subtilis AB1 |
Bactericidal activity against Aeromonas infection |
O. mykiss |
[132] |
Bifidobacterium |
|
|
|
Bifidobacterium animalis PTCC-1631 |
↑ growth performance, digestion, and nutrient utilization |
O. mykiss |
[133] |
B. lactis PTCC-1736 |
↑ growth, nutrient digestibility, and carcass composition |
O. mykiss |
[133] |
Carnobacterium |
|
|
|
C. divergens C. maltaromaticum |
Antagonistic effects against V. anguillarum, V. viscosus, and A. salmonicida |
- |
[134][135] |
Lactobacillus |
|
|
|
L. plantarum CLFP |
↓ mortality against harmful strain L. garvieae |
O. mykiss |
[136] |
L. acidophilus |
Survival against Staphylococcus xylosus, Aeromonas hydrophila gr.2, and Streptococcus agalactiae infection |
Clarias gariepinus |
[137] |
L. pentosus |
↑ growth performance and feed conversion ratio ↑ survival against Vibrio species |
L. vannamei |
[138] |
Lactococcus |
|
|
|
Lactococcus lactis BFE920 |
Activation of nonspecific immune system Bactericidal activity against S. iniae |
Paralichthys olivaceus |
[139] |
Leuconostoc |
|
|
|
Lc. Mesenteroides CLFP 196 |
↑ survival against A. salmonicida infection |
Salmo trutta |
[140] |
Pediococcus |
|
|
|
P. pentosaceus HN10 |
↑ feed utilization, digestive enzyme activity, and anti-Vibrio activity |
L. vannamei |
[141] |
Enterococcus |
|
|
|
E. casseliflavus CGMCC1.2136 |
↑ growth performance, immunity, and digestive enzyme activity |
Rutilus rutilus caspicus |
[142] |
E. casseliflavus |
↑ growth performance and disease resistance against S. iniae |
O. mykiss |
[143] |
E. durans |
↑ growth performance and survival rate |
O. mykiss |
[144] |
Clostridium |
|
|
|
C. butyricum |
↑ antibacterial activity against Vibriosis infection |
O. mykiss |
[145] |
C. butyricum |
↑ immunity; regulation of gut microbiota; antagonistic effects against Aeromonas sp., Vibrio sp., and Pseudomonas sp. |
C. carpio |
[146] |
Weissella |
|
|
|
W. confusa |
↑ growth performance |
O. mykiss |
[147] |
W. confusa |
↑ growth performance and antibacterial activity against A. hydrophila |
Lates calcarifer |
[148] |
Other strains |
|
|
|
A. veronii BA-1 |
↑ immune system and antibacterial activity |
C. carpio |
[149] |
Micrococcus luteus |
↑ growth performance and feed conversion ratio |
O. niloticus |
[150] |
Pseudoalteromonas undina VKM-124 |
↑ survival and antiviral activity |
Carangoides bartholomaei |
[72] |
Yeast |
|
|
|
S.cerevisiae |
↑ growth performance and resistance against waterborne Cu toxicity |
Sarotherodon galilaeus |
[151] |
S. cerevisiae |
↑ immunity and ↓ mortality against P. fluorescens |
Mystus cavasius |
[152] |
Yarrowia lipolytica |
↑ immune response, antioxidant status, and disease resistance against V. parahaemolyticus infection |
Lutjanus peru |
[153] |
Multi-strain |
|
|
|
B. subtilis and Bacillus licheniformis (BioPlus2B) |
↑ resistance against Y. ruckeri |
O. mykiss |
[154] |
Lactobacillus delbrueckii Lactobacillus rhamnosus L. plantarum B. bifidum |
↑ growth performance and immunity |
Acipenser baerii |
[155] |
Lactobacillus plantarum (STBL1), Saccharomyces cerevisiae (STBS1), and Bacillus safensis (SQVG18) |
↑ growth, antioxidant capacity, digestion, and gut microflora |
P. vannamei |
[156] |