The Application of Prebiotics, Probiotics, Synbiotics, Postbiotics and Parabiotics in Aquaculture

Abstract

The aquaculture industry has been growing rapidly over the last decades and has become one of the main producers of aquatic organisms. There has been an increasing interest in using biotic supplements, such as prebiotics, probiotics, synbiotics, postbiotics, and parabiotics, as alternatives to antibiotics and chemical treatments in aquaculture. These biotic agents hold promise for improving the gut health, growth performance, reproductive system, immune response, and disease resistance of aquatic organisms. This review delves into the mechanisms, benefits, and applications of biotic supplements in aquaculture systems. It highlights the growing interest in postbiotics and parabiotics, which, unlike probiotics, do not require live bacteria to deliver health benefits. However, despite their potential, postbiotics and parabiotics are still rarely used in aquaculture. Growth enhancement is often linked to an increase in digestive enzyme activity or the stimulation of host appetite-regulating hormones. Additionally, biotic supplementation can also act as ammonia-oxidizing and nitrite-oxidizing agents to maintain appropriate water quality. They also improve reproductive performance in aquatic species, aiding in broodstock maintenance and enhancement. Moreover, biotic supplements boost innate and adaptive immune responses, helping to combat bacterial diseases in the aquatic environment. Furthermore, the review addresses the safety and challenges of integrating.

1. Introduction

Indonesia’s marine and fisheries industry plays an important role in national food production and economic development [1, 2], often referred to as the Blue Economic Sector. This sector is expected to be the driving force for national food security and development. Nevertheless, exponential population growth contributed to increased food needs. Additionally, wild capture fisheries sectors are facing overfishing and are nearing their capacity limit, resulting in declining production [3]. New techniques for farming aquatic organisms are being introduced to address this issue. Aquaculture is a farming technique that grows aquatic organisms under controlled conditions. This farming technique also allows a selective increase of specific species.

According to data by the Food and Agriculture Organization (FAO) [4], Indonesian aquaculture has been experiencing annual growth of up to 13.65% from 2000 to 2021 and ranked first in Southeast Asia. The FAO report in 2024 [4] shows Nile tilapia, catfish, milkfish, carp, and white-leg shrimp dominate the aquaculture industry in Indonesia. In response to growing demand, aquaculture has adopted intensive practices, which involve maintaining a high density of organisms. However, this approach can lead to increased stress and degraded water quality, compromising animal welfare and increasing their susceptibility to diseases and infections, ultimately resulting in elevated mortality rates [5]. Moreover, the large quantities of organic waste produced can cause considerable environmental harm [5, 6, 7].

The fast growth of aquaculture is humbled by the inevitable outbreak of bacterial, viral, fungal, and parasitic diseases that result in economic losses. In Indonesia, it is reported that a total of around $3.3 million was lost due to fish diseases [8]. To prevent the occurrence of disease outbreaks, prevention and control of aquatic organisms have focused on the use of chemicals and antibiotics, which exert a negative impact on public health through the generation of residual waste and antibiotic-resistant strains [9, 10, 11]. Exploring alternative approaches to reduce infection occurrence in aquaculture without antibiotic properties while also maintaining a high output has become a promising and effective solution.

The probiotic term has been widely used to describe live bacteria that have a beneficial effect with appropriate amounts and remain alive when reaching the gastrointestinal tract [5, 12, 13]. Probiotics play an important role in maintaining microbial balance within the host, improving the beneficial intestinal microbial populations, and increasing digestive enzyme activities, which help improve nutrient absorption while protecting the host from pathogenic bacteria and modulating the immune system [14]. Additionally, these approaches are less hazardous compared to conventional chemical treatment [15]. Various probiotic strains, including Lactobacillus, Bacillus, Pseudomonas, and Pediococcus, are commonly used in aquaculture [16, 17]. To further support probiotic survivability and activity, prebiotics, non-digestible food ingredients that stimulate the growth of beneficial microbes, are often included, leading to the development of synbiotics [18]. Surprisingly, there is a report that stated that microbial viability is not necessary to produce benefits in the host [19]. This information has directed attention towards postbiotics and parabiotics, which refer to metabolic products and inactivated microbial cells, respectively. Moreover, these biotic components can reduce the possibility for probiotics to become a reservoir for resistance genes [20]. Although probiotics have been extensively explored, the roles and comparative advantages of other biotics are gaining increasing attention in aquaculture. Therefore, this review aims to examine the use of these five biotic supplements in aquaculture, the actual concept of use, production, and further research prospects. Ultimately, this review seeks to provide insights into how this biotics can be strategically implemented to improve aquatic animal health, enhance sustainability, and guide future research directions in aquaculture.

2. History and definition of prebiotics, probiotics, synbiotics, postbiotics, and parabiotics

The International Scientific Association for Probiotic and Prebiotics (ISAPP) defines the term probiotic as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [21]. The main challenge when administering probiotics to the host is maintaining their viability. Moreover, probiotics are sensitive to heat and acid. To address this challenge, researchers try to incorporate prebiotics together, which develop the concept of synbiotics. In 1995, Gibson and Roberfroid [22] proposed that prebiotics were non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and activity of certain beneficial bacteria in the colon, before being updated to the current definition (Table 1). This early research laid the groundwork for exploring how dietary components could directly influence the composition and function of the gut microbiome. Nowadays, prebiotics have expanded to not only plant-derived oligosaccharides but also several polysaccharides, like resistant starch and pectin [23, 24]. The primary function of prebiotics is to act as a food source for the beneficial bacteria in the gastrointestinal tract, promoting their growth and activity.

Not until recently has it been found that the viability of the microorganism is not necessary to give the beneficial effect [19, 25]. As such, metabolites derived from probiotics, or their non-viable cells, become a favorable and promising alternative to give beneficial effects known as postbiotics or parabiotics. Currently, the term postbiotics is defined by ISAPP as the “preparation of inanimate microorganisms and their components that confers a health benefit to the hosts”. However, others also try to define postbiotics in different terms, which only highlight probiotics’ byproducts and metabolites without the inanimate microorganism [26, 27, 28]. Table 1 summarizes the definition for biotic supplements.

Table 1: Definition of prebiotics, probiotics, synbiotics, postbiotics and parabiotics

Term	Definition	Examples	Ref
Probiotics	Live microorganisms that confer health benefits when consumed in adequate amounts.	Lactobacillus acidophilus, Bifidobacterium bifidum	[21]
Prebiotics	Selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.	Inulin, Fructooligosachharide (FOS), Galatooligosachharide (GOS)	[29]
Synbiotics	A mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host.	Lactobacillus + inulin	[18]
Postbiotics	Non-viable bacterial products or metabolic products from microorganisms that have biological activity in the host.	Short-chain fatty acids (SCFAs), bacteriocins	[28]
Parabiotics	Non-viable microbial cells (either intact or broken) or crude cell extracts which when administered in adequate amounts, confer a benefit on the host.	Heat-killed Lactobacillus	[28]

The use of biotic supplements in animals, particularly as microbial feeds or additives for aquatic species, dates back to 1986, when Kozasa first introduced the concept using the spore of Bacillus toyoi to increase the growth of yellowtail fish (Seriola quinqueradiata) and reduce the mortality of Japanese eel infected with Edwardsiella sp. [30]. Compared to terrestrial and aquatic animals, the presence of probiotics and other microorganisms in aquatic animals is transient due to the constant flow of water. This poses a positive and negative impact; as the water flows, both probiotic and pathogen bacteria can transmit or adhere to other organisms. Additionally, water is an ideal environment for pathogen bacteria growth, thus making the use of probiotics more complex and requiring constant use since the environment suffers periodic changes, from salinity, temperature, and dissolved oxygen (DO) that can alter the dominant microorganism species present [31].

3. Production of probiotics, synbiotics, postbiotics, and parabiotics

The production of commercial probiotics (Figure 1) aims to achieve the highest possible yield, stability, and consistent performance. The process begins with a pure, contaminant-free stock of probiotics, followed by a fermentation process using suitable media. The probiotic cells are then concentrated through centrifugation and may undergo freezing steps to preserve their viability and extend shelf life [32]. In the production of synbiotics, oligosaccharides or other prebiotics are added alongside the probiotic cells [33]. The production of postbiotics is similar to the preparation of probiotics, as both involve a metabolic process by bacteria that results in the production of various metabolites. However, in postbiotics preparation, it is essential to eliminate the remaining bacteria and retain the metabolites only. Microbial cells can undergo several processes to disrupt the structure of the cell membrane to facilitate the release of intracellular metabolites into the extracellular medium [34]. Common methods used include sonication, heat treatment, pulsed-electric field, and high pressure [34]. The choice of method depends on the specific bacterial strain and the sensitivity and stability of the metabolites under different conditions. Additionally, centrifugation and microfiltration are often done to ensure complete removal of living cells. However, for postbiotics that are secreted by live cells and already present in the extracellular medium, centrifugation and microfiltration steps are typically sufficient [35, 36, 20]. Parabiotics, on the other hand, are probiotic microorganisms that have undergone an inactivation process. Various methods can be used for inactivation, including heat treatment, chemical exposure, and irradiation, with heat treatment being the most commonly applied approach [28]. As a result of this process, inactivated probiotics can yield two distinct types of biotic products: postbiotics, which utilize only microbial metabolites, and parabiotics, which make use of the non-viable microbial cells themselves to deliver health benefits.

In a recent study, it is popular to utilize agro-industrial effluents as media for the growth of probiotic bacteria since they are cheaper and more abundant compared to synthetic media. Using cheese whey effluents able to produce Bifidobacterium lactis BB12A postbiotics, including exopolysaccharides and conjugated linolenic acid bacteriocins up to 160 µg/l, 105 µg/ml, respectively, it also shows antimicrobial properties [37]. The use of agro-effluent activity as a medium is also similar to that of synthetic media. A study by Dominguez [38] found that using soybean protein (20 g/L) resulted in the production of cerein 8A (Bacillus cereus bacteriocin) of 1600 AU/ml, which is similar to brain and heart infusion broth (BHI). The need for certain media and nutrients for maximum bacteriocins production depends on the specific type of bacteriocins. Some amino acids can be self-produced by the bacteria, and some require external supplementation [39]. A study by Yi et al. [40] found that the addition of external amino acids, glycine and cysteine, stimulated the production of lac-B23 bacteriocin from Lactobacillus paracasei J23 by 250% and 325%, respectively. From an economic standpoint, by doing so, the commercialization of bioactive compounds can be profitable for the biotechnology industry [41].

Figure 1: Classification and production of several biotic supplements

4. Prebiotics, probiotics, synbiotics, postbiotics and parabiotics selection criteria

Over the past century, since the discovery of Lactobacillus bulgaricus by Stamen Grigorov in 1905 [42], researchers have continuously worked to isolate and identify new probiotic strains. Advances in DNA sequencing help to extend the probiotic taxa, uncovering strains with novel beneficial properties [43, 44, 45]. However, these novel strains are rarely used compared to traditional probiotics, due to their safety concerns and costly procedures [43, 44, 45]. To be classified as probiotic, a microorganism must undergo rigorous evaluation, including stress tolerance, adhesion ability, anti-pathogenic activity, safety assessment, genome sequencing, and clinical trials, which can be costly. Only those with strong functional properties and no adverse effects on the host are selected [46]. Prebiotics, on the other hand, must positively impact gut health, be stable under various conditions, be resistant to digestion in the upper gastrointestinal tract, and be fermented by gastrointestinal microflora. They should be recognized as Generally Recognized As Safe (GRAS), with no significant adverse effects [29, 47]. Selecting synbiotics requires careful selection of prebiotics and probiotics, ensuring both works complementarily or synergistically. The prebiotic should support the chosen probiotic strains’ growth and remain stable during processing and digestion [18, 29]. Ideally, the combination should show superior effectiveness to either component alone.

Postbiotics, which consist of bioactive compounds produced by probiotics, should have a well-characterized composition, including beneficial microbial byproducts like short-chain fatty acids (SCFAs) or peptides, and be free from contaminants, with the progenitor microorganism already molecularly characterized. The mechanisms of inactivation must be confirmed, and their safety and health benefits should be assesed and well-researched [48]. Similary, parabiotics, which are inactivated microorganisms that still confer health benefits, must ensure health benefits. The chosen strain should retain its beneficial properties after inactivation (e.g., through heat or pressure). The inactivation method should be standardized to preserve bioactive components, and the parabiotic should interact effectively with the host’s cells or microbiota. It also must be safe and stable during storage [49].

5. Application of probiotics, prebiotics, synbiotics, postbiotics and parabiotics in aquaculture

The main usage of probiotics in aquaculture started as a growth promoter. However, new findings about its beneficial effects have been found, including as an immune system and host pathogenic bacteria resistance enhancer and reproduction inducer. The application of biotic supplements in aquaculture can be done by several methods, including mixing them directly with their feed or directly add them to the water.

5.1 Promote growth and nutrient digestibility enhancement

Biotic supplements have been used in aquaculture mainly for their properties that enhance aquatic organism growth (Table 1). Researchers show that adding biotic supplements to animal feed as an additive can increase animals' final weight and reduce the feed conversion rate (FCR), indicating a better and more economical feed. Although some studies also found no significant effect on the addition of probiotics to the feed [50], this varying result can depend on the concentrations of biotic supplement and animal stage life. For example, a study conducted by Haque [51] on Pangasianodon hypophthalmus found that feeding with probiotics during the larval to fry stage results in a higher growth rate in the fry growth stage compared to fry to fingerlings. The exact mechanisms through which biotic supplements enhance growth remain unclear. One factor is linked to the host’s appetite, while the other involves the host’s capacity to digest nutrients and produce enzymes.

Vertebrate appetite is controlled by orexigenic (stimulatory) and anorexigenic (inhibitory) factors within the brain, which receive information from the gastrointestinal tract and peripheral nervous system [52]. Research by Gioracchini et al. [53] found that the mixture of encapsulated freeze-dried Lactobacillus rhamnosus IMC 501® in 10⁶ CFU/g can induce tilapia (Oreochromis niloticus) appetite hormones, neuropeptide Y (NPY), agouti-related protein (AgRP), and ghrelin, while suppressing leptin hormone that gives satiety feeling. The probiotic group had a significantly lower secretion of leptin, while NPY, AgRP, and ghrelin were significantly higher in the control group at a p-value of 0.0001 after 30 days of feeding. Besides that, the insulin-like growth factor (IGF), IGF-1, IGF-2, and IGF-1 receptor also showed an increase in the probiotic group. Another study also found growth-promoting properties in using Mandarin fish (Siniperca chuatsi) as hosts. It was found that the supplementation of Lactobacillus plantarum, L. rhamnosus, or Clostridium butyricum at the concentration of 10⁸ CFU/ml in feed pellet increased the NPY and AgRP genes and decreased leptin secretion, with no effect on ghrelin was observed [54]. Similarly, a study using prebiotics only, β-glucan, GOS, and mannanoligosaccharides (MOS) shows increased growth in Channa striata; however, compared to the probiotic group, Saccharomyces cerevisiae and L. acidophilus the growth enhancement is still lower [55]. Additionally, studies on parabiotics showed results similar to those of probiotics, using heat-killed Bacillus sp. SJ-10 with a concentration of 3.34 × 10⁸ CFU/ml in Paralichthys olivaceus induced the secretion of growth hormone and IGF-1 expression [56]. Another factor that is believed to be related to growth enhancement is an increase in digestive enzymes, such as protease, amylase, lipase, and cellulase [57]. Probiotic strains can synthesize extracellular enzymes, thus making nutrient absorption more effective. A study on the use of Lactobacillus pentosus AS13 as a dietary probiotic for Litopenaeus vannamei at the concentration of 10⁷ CFU/g found that they can secrete digestive enzymes, such as protease and cellulase, and increase their activity [58].

Table 2: The effect of potential biotic supplements on aquatic organism growth performance and growth hormone

Biotic Supplement	Dose	Host	Feeding Duration (fed per day)	Result	Ref
Probiotics
Bacillus coagulans	106 CFU/g	Ctenopharyngodon idella	60 days (2)	↑FBW, ADG, RGR, DE ↓FCR	[57]
Lactobacillus acidophilus
Rhodopseudomonas palustris
Bacillus subtillis	4 × 106 CFU/g	Cyprinus carpio	180 days	↑FCE, SGR, FI ↓FCR	[59]
Bacillus velezensis and Lactobacillus plantarum N11	107, 108 CFU/g	Oreochromis niloticus	30 days (2)	↑FBW, WG, SGR ↓FCR	[60]
Bacillus licheniformis	105, 107 CFU/g	Oreochromis mossambicus	28 days (2)	↑FBW, SGR ↓FCR	[61]
Clostridium butyricum	1.2 × 106 CFU/g	Hybrid tilapia (O. niloticus × O. aureus)	56 days (3)	↑FBW, WG, SGR PR, LR ↓FCR	[62]
B. velezensis AP193	4 × 107 CFU/g	Ictalurus punctatus	70 days	↑WG =FCR	[63]
B. subtilis 7k	106, 108, 1010 CFU/g	hybrid Hulong Grouper (Epinephelus fuscoguttatus × E. lanceolatus)	14 days (2)	↑FBW, WG, ADG, FE	[64]
Chromobacterium aquaticum	107 CFU/g	Danio rerio	8 weeks (2)	↑GK, HK1, G6Pase, PK-L, GHr, IGF-1	[65]
Bacillus thuringiensis QQ1	109 CFU/g	Lates calcarifer	5 weeks (3)	↑FBW, FTL, RGR, SGR, PER, FER, FI, DE ↓ FCR	[66]
Bacillus cereus QQ2
B. subtilis (AQUA-GROW®)	0.5, 1, 1.5 g/kg	L. vannamei	56 days (3)	↑FBW, WG, SGR, FI, ADG ↓ FCR	[67]
L. acidophilus powder	0.1 g/kg	Channa striata	16 weeks (3)	↑WG, RG, SGR, FE, PER ↓FCR	[55]
Saccharomyces cerevisiae	10 g/kg
Prebiotics
β-glucan	2 g/kg	C. striata	16 weeks (3)	↑WG, RGR, SGR, FE, PER ↓FCR	[55]
GOS	10 g/kg
MOS	5 g/kg
Xylooligosaccharide (XOS)	50 g/kg	O. niloticus	84 days (2)	↓FCR =BMI	[68]
GOS
XOS + GOS	25 g/kg each
XOS with plant diet	10 g/kg	Dicentrarchus labrax	7 weeks (6 days a week)	↑WG, FBW =FE, PER, FI	[69]
MOS	6 g/kg	Pangasianodon gigas × P. hypophthalmus	10 weeks (2)	↑FR, PER, DE ↓FCR	[70]
Arabinoxylan-oligosaccharide (AXOS) + inulin	40 g/kg each	L. vannamei	8 weeks (4)	↑FBW, ADG, SGR, DE ↓FCR	[71]
GOS	5 g/kg	Acipenser baerii × A. schrenckii	56 days (2)	↑FBW, WG, SGR ↓FCR	[72]
Synbiotics
Corn Cob derived XOS + L. plantarum CR1T5	10 g/kg; 108 CFU/g	O. niloticus	12 weeks (2)	↑FBW, WG, SGR ↓FCR	[73]
GOS + Enterococcus faecalis	10 g/kg; 6.9× 107 CFU/g	Astacus leptodactylus	126 days (3)	↑FBW, SGR , PER, DE ↓FCR	[74]
MOS + E. faecalis
β-glucan + Pediococcus acidilactici	0.5 g/kg; 5×107 CFU/g	L. vannamei	90 days (5)	↑FBW, WG	[75]
β-glucan + B. subtilis				↑=FBW, WG
Alginate oligosaccharides + B. licheniformis WS-2	10g/kg; 109 CFU/g	A. leptodactylus	60 days (1)	↑WG, SGR, DE ↓FCR	[76]
FOS + E. faecium (Biomin IMBO)	0.5, 1, 1.5 g/kg	Oncorhynchus mykiss	60 days (3)	↑FBW, WG, SGR ↓FCR	[77]
Postbiotics
S. cerevisiae cell wall extract (Megamos®) and freeze-dried L. bulgaricus powder	4 and 2 g/ kg	Mugil capito	60 days	↑FBW, WG, SGR, FI, PER, PPV ↓FCR	[78]
L. plantarum cell-free extract	10 ml/ kg	L. vannamei	15 days (4)	↑FBW, WG, SGR, DE ↓FCR	[79]
L. plantarum fermentation supernatant
B. subtilis LCBS1 cell free extract	8 × 107 CFU/ml	Lithobates catesbeianus	8 weeks (2)	↑FBW, WG, PE ↑=DE	[80]
B. subtilis LCBS1 cell free supernatant				↑=FBW, WG, DE =PE
Bacillus, Lactobacillus, Saccharomyces cell lysate	20 g/kg	Macrobrachium nipponense	8 weeks (2)	↑FBW, WG, SGR	[81]
Parabiotics
Heat-killed (HK) Bacillus sp. SJ-10	3.34 × 108 CFU/ml	Paralichthys olivaceus	8 weeks (2)	=FBW, WG, SGR, FCR ↑IGF-1 expression	[56]
HK L. plantarum
HK B. subtilis LCBS1	8 × 107 CFU/ml	L. catesbeianus	8 weeks (2)	↑FBW, WG, PER, DE	[82]
HK P. pentosaceus PP4012	106 CFU/ g	L. vannamei	12 weeks (2)	↑FBW, SGR, FE	[83]
HK B. cereus BR2	1010 CFU/ml	Clarias sp.	30 days (3)	↑FBW, SGR ↓FCR	[84]
HK B. pumilus SE5	108 CFU/g	Epinephelus coioides	60 days (2)	↑FBW, WG, SGR ↓FCR	[85]
HK Leuconostoc mesenteroides	107 CFU/g	C. gariepinus	60 days (2)	↑FBW, WG, SGR, PER ↓FCR	[86]
HK Edwardsiella sp

↑: higher result compared to control; ↓: lower result compared to control; FBW: final body weight; WG: weight gain; ADG: average daily gain; SGR: specific growth rate; RGR: relative growth rate; BMI: body mass index; FI: feed intake; FR: feeding rate; FE: feed efficiency; FCR: feed conversion ratio; FCE: feed conversion efficiency; DE: digestive enzyme; PER: protein efficiency ratio; PPV protein protective value; PR: Protein retention; LR: lipid retention; IGF-1: Insulin-like Growth Factor 1; IGF-2: Insulin-like Growth Factor 2; GH1: Growth Hormone 1; GHr: Growth Hormone Receptor; GK: Glucokinase; HK1: Hexokinase 1; G6pase: Glucose-6-Phosphatase; PK-L : Pyruvate Kinase, Liver Lysozyme

Overall, Table 2 highlights the beneficial effects of various biotic supplements to support the growth performance of aquatic organisms. Among these, probiotics, particularly Bacillus and Lactobacillus strains, remain the most frequently studied and widely applied. However, when comparing biotic types, the effectiveness of prebiotics, synbiotics, postbiotics, and parabiotics is also evident, with all demonstrating consistent improvements in key growth parameters, including FBW, WG, and SGR. The use of postbiotics, especially cell-free supernatants, offers a practical solution to the limitations associated with maintaining live bacterial viability, though the precise composition of these metabolites still requires further characterization. Meanwhile, parabiotics, primarily produced through heat inactivation, have shown promising results in promoting growth without the risks of introducing live microorganisms.

5.2 Enhance reproduction

Biotic supplements become a promising approach to enhance the reproduction in organisms, as they have the potential to improve fertility, fertilization rates, and hatching rates (Table 3). This effect is largely mediated through the regulation of sex hormones and genetic pathways associated with reproduction, mainly via the hypothalamic–pituitary axis. The primary mechanism involves the enhancement of egg and sperm quality and quantity, as well as an increase in fecundity rates [87]. These biotic supplements may influence the expression of genes related to hormone production, gonadal development, and oogenesis or spermatogenesis, thereby improving reproductive outcomes in both males and females [88]. Several studies have found that the addition of biotic supplements in aquatic animals has a positive effect on reproduction. Gioacchini et al. [89] reported that the addition of Lactobacillus rhamnosus IMC50 at 10⁶ CFU/g increase luteinizing hormone receptor (l hr) levels and a decrease in transforming growth factor beta 1 (tgfb1) and growth differentiation factor 9 (gdf9) in female adult zebrafish (Danio rerio) which help in oocyte maturation. A mature oocyte will increase the success of the fertilization process by ensuring proper meiotic progression, cytoplasmic competency, and synchronization with spawning, ultimately leading to improved embryonic development and higher hatchability rates. A similar result was also obtained in a study conducted by Qin et al. [90]. They found that the addition of Lactobacillus casei and Lactobacillus rhamnosus at 10⁸ CFU/g can increase the fertility of 7-month-old Danio rerio and enhance fish’s fecundity through the increased expression of leptin, kisspeptin2 (kiss2), gonadotropin releasing hormone 3 (gnrh3), and luteinizing hormone (lh), luteinizing hormone/choriogonadotropin receptor (lhcgr), progestin and adipoQ receptor family member 8 (paqr8) gene. These genes are responsible for encoding neuropeptide hormone and oocyte maturation, respectively. Simlarly, the addition of prebiotics, particularly chitosan has been shown to influence reproductive development by enhancing gene expression and reproductive cell activity. This occurs through improved gonad development and stimulation of reproductive hormone secretion by hypotalamus. Interestingly, during the stimulation of hypotalamus to secrete reproductive hormone (gonadthropic hormones) by the pituary glands, it also secretes dopamine that can halt the gonad development. The supplementation of chitosan can act as dopamine antagonist and therby allowing continued gonad development [91].

Table 3: The effect of potential biotic supplements on animal reproduction

Biotic Supplements	Dose	Host	Feeding Duration (fed per day)	Results	Ref
Probiotics
Bacillus subtilis, Bacillus licheniformis	0.03 g/m3	Male Oreochromis niloticus × Oreochromis mossambicus	20 days	↑ T, GSI, HSI, VSI	[92]
		Female O. niloticus ×O. mossambicus		↑ LH, FSH, P4, E2, GSI, HSI
Bacillus spp.	0.0013 g/L (1.5 × 1010 CFU)	Female O. niloticus	60 days (1/ week)	↑ AF, RF, FSH, LH ↑= ED, P4	[93]
		Male O. niloticus		↑ LH, E ↑= FSH
Bacillus cereus NP5	106, 108 CFU/g	Female Clarias gariepinus	84 days (2)	↑ AF, HR, LS	[94]
Lactobacillus rhamnosus (IMC 501®)	106 CFU/ml	Fundulus heteroclitus	8 days	↑ AF, GSI,	[95]
Pediococcus acidilactici (Bactocell®)	108 CFU/g	Carassius auratus	180 days	↑ SM, SD, ED, OD, AF, RF, GSI, HSI, FR, HR	[96]
Bacillus sp. NP5	108 CFU/g	Male C. gariepinus	60 days (2)	↑ SpC, SD, SM, SV	[97]
L. rhamnosus GG	108 CFU/g	MCLR induced female Danio rerio	28 days (2)	↑ FR, HR, CEC, E2, T, VTG	[98]
B. subtilis, B. cereus	0.6 g/kg (4 × 1011 CFU/g)	Male Rhamdia quelen	90 days (4)	↑ GSI, HSI, VSI, SCS	[99]
		Female R. quelen		↑ FR, VSI, DVO
Prebiotics
Yucca extract (AQUA-YUCCA)	0.0013 g/L	Female O. niloticus	60 days (1/ week)	↑ AF, ED, FSH, LH, E, P4, T ↑= RF	[93]
		Male O. niloticus		↑FSH, LH, E, P4, T
MOS	1.5 g/kg	Female O. niloticus ×O. mossambicus	40 days (2)	↑FSH, LH, E2, P4, GSI, HSI, VSI, ED	[100]
		Male O. niloticus ×O. mossambicus		↑T, GSI, HSI, VSI
Chitosan	1 g/kg	Barbonymus gonionotus	60 days (2)	↑FSH, LH, HR, FR, GSI, RF ↓ OP	[91]
Synbiotics
Yucca extract (AQUA-YUCCA) + B. subtilis, B. licheniformis, L. acidophilus (UNI ECOTREAT)	0.0013 g/L	Female O. niloticus	60 days (1/ week)	↑ AF, RF, FSH, LH, E, P4, T ↑= ED	[93]
		Male O. niloticus		↑FSH, LH, E, P4, T
acidophilus, B. longum, B. thermophylus, treptococcus faecium, live yeast, oligosaccharides, amylase, protease, cellulose, pectinase, xylanase, phytase (Hydroyeast Aquaculture®)	15 g/kg	Male O. niloticus	56 days (2)	↑ TW, GSI, SC, SM ↓ SA, DS	[101]
	10 g/kg	Female O. niloticus		↑ OW, ED, AF, RF

↑: higher result compared to control; ↓: lower result compared to control; SM: sperm motility; SD: sperm density; SV: sperm volume; sV: semen volume; SpC: spermatocrit; SCS: sperm cell survival; SC: sperm count; SA: sperm abnormalities; DS: dead sperm; TW: testes weight; ED: egg diameter; OD: ovum diameter; OW: ovarium weight; CEC: cumulative egg count; OP: ovulation period; AF: absolute fecundity; RF: relative fecundity; WF: working fecundity; FR fertilization rate; HR hatching rate; LS: larvae survival; GSI gonadosomatic index; HSI hepatosomatic index; VSI: viscerosomatic index; E2: estradiol; E: estrogen; VTG: vitellogenin; T: testosterone ; LH: luteinizing hormone; FSH: follicle-stimulating hormone; DVO: diameter of vitellogenic oocytes; P4: progesterone

Table 3 shows the potential of probiotics, prebiotics, and synbiotics to improve reproductive performance in aquaculture organisms, primarily through the upregulation of reproductive hormones and the development of reproductive cells. However, currently, the application of postbiotics and parabiotics in this context remais unexplored. Given their higher stability and safety profile compared to probiotics, it is interesting to see the potential effect of those biotics alongside commonly used biotics supplements. Additionally, the current body of research is heavily focused on vertebrate fish species, with no studies on commercially important invertebrates, such as shrimp.

5.3 Boost the innate and adaptive immune system and protect against pathogens

Biotic supplements become a promising safer alternative to prevent aquatic diseases (Table 4). The overall mechanism of how probiotics and their derivatives are able to inhibit pathogenic bacteria in aquaculture is due to their metabolites that have bactericidal or bacteriostatic effects, improving the host gut barrier, nutrient or adhesion competition with pathogenic bacteria, and modulation of the host immune system [102]. The increase of antioxidant enzyme activity and immune-related genes can increase an organism’s survival rate when challenged with aquatic pathogenic bacteria [102, 103]. Chen et al. [104] showed that oral administration of Paenibacillus ehimensi isolated from Nile tilapia culture pools with the concentration of 10⁷ CFU/g to Nile tilapia able to increase their survival rate infected with Aeromonas hydrophila. Analyzed by non-specific immune system parameters, the probiotics enhanced TNF-α and IL-β expression. Another strain of probiotics has also been tested on Nile Tilapia, such as Bacillus velezensis, Lactobacillus plantarum, and Enterococcus faecium [61, 105]. Additionally, a study using synbiotic composed of MOS or XOS with Pediococcus acidilactici or E. faecalis showed an increase in lysozyme activity in narrow-clawed crayfish (Astacus leptodactylus). A significant increase in the survival rate was also found after crayfish were infected with Aeromonas hydrophila compared to control and MOS or XOS only [78]. Similar results were also found using heat-killed L. plantarum in Red tilapia at a concentration of 4 mg/kg, which was able to increase lysozyme content compared to the control [106]. This bacterial strain is also used for Claria macrocephalus and showed similar results [107].

Table 4: The effect of potential biotic supplement on aquatic organism innate, adaptive immune system and blood profile

Biotic Supplements	Dose	Host	Pathogen	Dose	Infection Model	Results	Ref
Probiotics
Bacillus velezensis, Lactobacillus plantarum N11	107; 108 CFU/g	Oreochromis niloticus	Streptococcus agalactiae	107 CFU/ml	IP injection (0.1 ml) for 15 days	↑ LYZ, RBA, PA (before challenge) ↑ SR	[60]
Encapsulated Pediococcus acidilactici (Bactocell PA10)	0.1, 0.2, 0.3 g/m3	O. niloticus fingerlings	Aspergillus flavus	4 ×103 conidia/ml	injection (0.2 ml) for 15 days fed with control diet	↑ RBC, Hb, WBC, P, CAT, GPx, SOD, LYZ, IgM, TNF-α, IL-1β, IL-8 (before challenge) ↓MDA ↑ SR	[108]
Paenibacillus ehimensis NPUST1	107 CFU/g	niloticus	Streptococcus iniae	105 CFU/ml	IP injection (0.02 ml) for 7 days fed with control diet	↑ PHG, LYZ, RB SOD ↑ TNF-α, IL-1β ↑ SR	[103]
			Aeromonas hydrophila	106 CFU/ml
Bacillus subtilis L10	108 CFU/ml	Litopenaeus vannamei	Vibrio harveyi	106 CFU	third abdominal segment injection	↑ProPo, PE, LGBP, SP ↑ SR	[109]
B. subtilis G1
B. subtilis, Bacillus licheniformis (Sanolife PRO-W ®)	0.03 g/m3 (5 ×105 CFU/g)	L. vannamei	Fusarium solani	5 × 104. conidia/ml	swab on wounded section for 14 days	↑SOD, CAT, GPx, ProPO, LYZ, RB, Phag ↑ SR ↓ MDA	[110]
L. plantarum Ep-M17	5 × 108 CFU/g	L. vannamei	Vibrio parahaemolyticus E1	107 CFU/ml	immersion for 2 days	↑ SOD, AKP, CAT ↑ SR	[111]
Bacillus cereus	106 CFU/g	Carassius auratus var. Pengze	-	-	-	↑ TSOD, CAT, GSH, GPx, TLR2, IL-10 ↓ MDA, TNF-ɑ, IL-1 $\beta$ , IFN-Ɣ	[112]
Bacillus amyloliquefaciens	105, 107, 109 CFU/g	Labeo rohita	hydrophila	107 CFU/ml	IP injection (0.1 ml) for 28 days	↑ SOD, CAT, LYZ, IgM ↓ MDA ↑ SR, AST, ALT	[113]
B. velezensis	106 CFU/g	Dicentrarchus labrax	Vibrio anguillarum	2 × 106 CFU/ml	IP injection (0.1 ml) for 6 days	↑ LYZ, NO, Phag, IL-1 $\beta$ , TNF-ɑ, COX-2, DIC ↑ SR	[114]
Prebiotics
MOS	6 g/kg	Pangasianodon gigas × P.hypophthalmus	Edwardsiella ictaluri	6 × 107 CFU/ml	IP injection (0.1 ml) for 2 weeks	↑ WBC, Hb, LYM, Ig, LYZ (before challenge) ↑ SR	[74]
AXOS + inulin	4 mg/g each	L. vannamei	Vibrio alginolyticus	2 × 107 CFU/ml	ventral sinus of cephalothorax injection (0.2 ml) for 5 days	↑ PO, MsyD88, ERK ↑ SR	[75]
			WSSV
GOS	5 g/kg	Acipenser baerii × A. schrenckii	hydrophila	1.5 × 106 CFU/ml	injection (0.2 ml) for 2 weeks	↑ACP, AKP, LYZ, MPO, SOD, CAT, GPx ↓ MDA ↑ SR	[76]
Chitosan	1 g/kg	Barbonymus gonionotus	-	-	-	↑ IgM, LYZ	[91]
GOS	20 g/kg	Astacus leptodactylus Eschscholtz, 1823	-	-	air stress	↑ PO, LYZ, SOD	[115]
FOS, $\beta$ -glucan	0.5, 1, 1.5 g/kg	O. niloticus	Fusarium oxysporum	4 ×103 conidia/ml	injection for 15 days	↑ SOD, CAT, GPx, LYZ, IgM ↓ MDA ↑ SR	[116]
$\beta$ -glucan	0.5, 1, 2 g/kg	Oncorhynchus mykiss fingerlings	-	-	-	↑ IgM, LYZ, C3, C4, ACH50.	[117]
Synbiotics
Corn Cob derived XOS + L. plantarum CR1T5	10 g/kg; 108 CFU/g	O. Niloticus	agalactiae	107 CFU/ml	IP injection (0.1 ml) for 15 days	↑ skin mucus LYZ, POD (before challenge) ↑serum LYZ, POD, ACH50, Phag, RB (before challenge) ↑ SR	[77]
GOS + Enterococcus faecalis	10 g/kg; 6.9 × 107 CFU/g	A. leptodactylus	hydrophila	108 CFU/ml	fifth thoracic legs injection (20 ml) for 2 days	↑SOD, LYZ, NO, PO (before challenge) ↑ SR	[78]
MOS + E. faecalis
B. subtilis, S. cerevisiae, yeast cell wall, B. subtilis fermentation, yucca extract (Bioture)	3 g/kg	L. vannamei	V.parahaemolyticus	1.75 × 106 CFU/ml	injection (0.025 ml) for 2 days	↑ TSOD, LYZ, CAT, ACP, ALT ↓ MDA ↑ SR	[118]
Postbiotics
Bacillus, Lactobacillus, Saccharomyces cell-lysate	20 g/kg	Macrobrachium nipponense	-	-	-	↑ POD, PO, LYZ, AKP, MAPK7, MAK14, IRAK4 ↓ MDA	[85]
L. plantarum VAL6 EPS	4 g/kg	L. vannamei	V. parahae-molyticus	105 CFU/ml	immersion for 9 days	↑ PO, RB, SOD	[119]
Bifidobacterium bifidum VAR2 EPS
Staphylococcus lentus bacteriocin BS-SLSZ2	18 mg/ml	Artemia salina	V. harveyi	106 CFU	immersion for 7 days	↑ SR	[120]
			aeruginosa
Clostridium butyricum metabolites		Ctenopharyngodon idella	-	-	high cottonseed and rapeseed diet	↑ SOD, CAT, GSH = C3, IgM, ACP ↓ MDA	[121]
Parabiotics
HK B. pumilus SE5	108 CFU/g	Epinephelus coioides	-	-	-	↑ Phag, SOD, IgM, C3, IL-8, IL-1 $\beta$ , TLR-2	[85]
HK Pseudoalteromonas piscicida 2515	105 CFU/ml	Paralichthys olivaceus	V. anguillarum	2.5 × 107 CFU/ml	immersion for 10 days	↑ SOD, CAT, POD ↑ IL-10, TNF-ɑ (spleen) ↑ LYZ, IL-1 $\beta$ , TNF-ɑ (head-kidney) ↑ SR	[122]
HK L. plantarum (House Wellness Foods Corp)	50 ppm	P. hypophthalmus	Edwardsiella ictaluri	105 CFU/ml	IP injection 0.1 ml) for 14 days	↑ LYZ, WBC, RBC ↑ SR	[123]
HK P. aeruginosa VSG2	30 mg/kg	Cyprinus carpio	hydrophila	107 CFU/ml	IP injection (0.1 ml) for 15 days	↑ mucus and serum LYZ, Ig, ALP, SOD, CAT, GPx, GSH, ↑ SR ↓ MDA	[124]
HK B. subtilis	108 CFU/g	P. hypophthalmus	-	-	-	↑ Phag, SOD, CAT, GPx ↓ MDA	[125]
Formalin-killed B. amyloliquefaciens COFCAU_P1	108 CFU/g	L. rohita fingerlings	hydrophila	104.5 CFU/ml	IP injection (0.1 ml) for 14 days	↑ SOD, GPx ↑ SR	[126]

↑: higher result compared to control; ↓: lower result compared to control; SR: survival rate; RBC: red blood cells; Hb: haemoglobin; WBC: white blood cells; Phag: phagocytosis; RB: respiratory burst; GLB: globulin; IgM: immunoglobulin M; LYZ: lysozyme; CAT: catalase; GPx: glutathione peroxidase; SOD: superoxide dismutase; POD: peroxidase; TSOD: total superoxide dismutase; GSH: glutathione; NO: nitric oxide; ProPo: prophenoloxidase; MDA: malondialdehyde; PE: peroxinectin; LGBP: lipopolysaccharide and β-1,3-glucan-binding protein; SP: serine protein; CR: crustin; lvLEC: lymphatic vessel endothelial cells; PEN-3ɑ: PEN-3 alpha; COX-2: Cyclooxygenase-2; DIC: dicentracin; ACH50: alternative complement activity; Myd88: myeloid differentiation primary response gene 88; ERK: extracellular signal-regulated kinase; C3: complement component 3; C4: complement component 4; MAPK7: mitogen-activated protein kinase 7; MAK14: mitogen-activated protein kinase-activated protein kinase 14; IRAK4: interleukin-1 receptor-associated kinase 4; TLR-2: toll-like receptor 2; TNF-ɑ: tumor necrosis factor-alpha; IL-1ꞵ: interleukin-1 beta; IL-8: interleukin-8; IL-10: interleukin-10; IFN-Ɣ: interferon gamma; AKP: alkaline phosphatase; ALT: alanine aminotransferase; ACP: acid phosphatase

Table 4 shows how biotic supplements can help protect aquaculture species from common bacterial infections during the rearing process. In many studies, fish or shrimp are intentionally exposed to harmful bacteria, usually through injection or immersion, to test how well the biotics work. After exposure, the animals are either kept on a supplemented diet or returned to a regular one. The disease protection conferred by biotic supplements is closely linked to their ability to enhance both the innate and adaptive immune responses of the host. Specifically, biotics have been shown to upregulate anti-inflammatory cytokines while downregulating pro-inflammatory cytokines, thereby improving the host’s resistance to infection, mortality, and overall immune regulation.

5.4 Improve water quality

The water quality is commonly checked on several parameters, including temperature, pH, DO, nitrite, nitrate, ammonium, and salinity. To obtain water quality, a rapid analysis method is usually employed by using a colorimetric test kit [127]. Maintaining water quality is a vital step in aquaculture production. For example, a study conducted by Evans et al. [128] found that Nile Tilapia exposed to sublethal DO levels resulted in higher stress determined via blood glucose and lower their survivability against Streptococcus agalactiae. Other than that, Abbink et al. [129] studied the effect of different pH and temperature on juvenile yellowtail kingfish (Seriola lalandi) in recirculating aquaculture systems (RAS) and found that the pH of 6.58 and temperature of 21°C resulted in the lowest final weight.

The accumulation of aquatic animal waste also poses a danger in aquaculture environments. Unmanaged waste can result in high ammonia content, the final product of protein metabolism. High ammonia content is toxic to the animals, thus required to be broken down into other substances. Several studies found that the incorporation of probiotics in aquaculture can help to improve water quality parameters (Table 2). A study by Thurlow et al. [63] using Bacillius velezensis AP193 during the production of catfish (Ictalurus punctatus) can reduce the amount of phosphorus, nitrogen, and nitrate-nitrogen from 0.136 ± 0.010, 0.344 ± 0.051, 0.051 ± 0.019 to 0.110 ± 0.013, 0.195 ± 0.025, 0.013 ± 0.005, respectively. Another study found that using Pediococcus acidilactici in Nile tilapia (Oreochromis niloticus) farming can reduce the total ammonia nitrogen (TAN) and ammonia (NH₃) proportional to the amount of P. acidilactici added, with the lowest concentration of TAN and NH₃ at 0.43 and 0.04 ppm [108]. A bacterial consortium containing Bacillus cereus, Bacillus amyloliquefaciens, and Pseudomonas stutzeri can become an ammonia-oxidizing and nitrite-oxidizing agent. These bacteria can reduce the ammonia, nitrite, and nitrate content. However, the activity of nitrite oxidation is slow; this may happen because two out of three bacteria in the consortium are ammonia-oxidizing agents [130]. Prebiotics like MOS have shown promising potential to improve water quality in aquaculture systems. For example, in a study by Kishawy et al. [131] with Nile tilapia raised in a biofloc setup, MOS supplementation helped reduce harmful compounds such as TAN, nitrite, and nitrate by around 50% compared to the control group. This effect was seen regardless of whether the fish were fed plant-based or fish-based diets. This study also compared MOS with glycerol as a carbon source, and both showed similar results for the water quality. However, MOS was also found to increase the growth of beneficial microbes, particularly Bacillus species, in the biofloc. This suggests that MOS can help support the natural growth of native bacteria in the system.

In another study by Saleh et al. [93], a plant-based prebiotic, Yucca schidigera, combined with commercial probiotics (Bacillus subtilis, Bacillus licheniformis, and Lactobacillus acidophilus), significantly reduced TAN from 1.25 mg/L to 0.9 mg/L, and total suspended solids (TSS) from 136 mg/L to 73 mg/L. However, ammonia oxidation led to a slight increase in nitrate concentration, from 0.136 mg/L to 0.192 mg/L, indicating the potential need for denitrifying bacteria to further convert nitrate into nitrogen gas. The study also found that synbiotics was more effective than using either probiotics or prebiotics alone.

Table 5: The effect of potential biotic supplements on aquaculture water quality

Biotic Supplements	Dose	Host	Feeding Duration (fed per day)	Results	Ref
Probiotics
Bacillus subtilis powder	4.76 × 108 CFU/g	Oreochromis niloticus	50 days (3)	↓TAN, NO2, NO3	[132]
Pediococcus acidilactici	106 CFU/ml	Litopenaeus vannamei	110 days	↓NH4+, NH33 NO3	[133]
Bacillus cereus				↓NH4+, NO3 NO2 =NH3
Bacillus toyonensis	1 or 2 × 105 CFU/ml	O. niloticus	70 days (3)	=NH3	[134]
Geobacillus stearothermophilus
B. subtilis, Lactobacillus sp., Saccharomyces sp. Entrobacterium spp. (Prozym powder Ultra ®)	0.002 g/m3	O. niloticus fingerlings	56 days (1)	↓NH4+, NO3 NO2	[135]
subtilis, B.cereus, S. cerevisiae (Microban aqua ®)	0.002 g/m3
B. subtilis, Paracoccus sp., P. acidilactici, Enterococcus sp., Thiobacillus sp. (Aquastar®)	0.0015 g/m3
B. subtilis, Bacillus licheniformis, Bacillus pumilus (Sanolife PRO-W ®)	0.0010 g/m3
B. subtilis, B. licheniformis	0.01, 0.02, 0.03 g/m3	Male and female O. niloticus × O. mossambicus	20 days	↓TAN, NH3	[92]
Prebiotics
MOS	Vary, maintaining the C:N ratio of 15:1	O. niloticus	50 days (3)	↓TAN, NO2, NO3	[131]
Synbiotics
de-potash vinasse (DPV) + Enterobacter ludwigii HS1-SOB, Pseudomonas stutzeri B6-SOB, Cytobacillus firmus C8-SOB	1 L mixed ith sand/1.5 ha pond	L. vannamei	80 days	↓NH3, PO43-	[136]
Yucca extract (AQUA-YUCCA) + B. subtilis, B. licheniformis, L. acidophilus (UNI ECOTREAT)	0.0013 g/L	O. niloticus	60 days (1/ week)	↑ NO3 ↓TAN	[93]

TAN: total ammonia nitrogen, NO₂: Nitrite, NO₃: Nitrate, NH₃: Ammonia, NH₄⁺: Ammonium, PO₄^3-: Phosphate

The application of postbiotics and parabiotics for improving water quality remains unexplored. However, there is a study using feed added with pepsin resulted in lower ammonia, nitrate, and nitrite content in the water. This effect is attributed to pepsin’s ability to hydrolyze dietary proteins into simpler compounds, enhancing protein digestibility and thereby decreasing the excretion of nitrogenous waste into the aquatic environment [137]. This finding can be interesting to look for, because microorganisms have the abilities to produce specific enzymes, including pepsin-like proteases that can aid in protein hydrolysis [138].

5.5 Comparative analysis of biotic supplements in aquaculture settings

The biotic application in aquaculture offers numerous benefits, contributing to more sustainable and efficient aquaculture production. Among the various types, probiotics remain the most widely studied and applied. In contrast to newer biotic supplements that are still emerging in the field. The application of these biotic supplements varies, but the most common is done through a feed mix or by directly mixing it into the water. Probiotics have been applied in a wide range of dosages, with beneficial effects observed even at lower concentrations, although some studies use up to 10¹⁰ CFU/g. The feeding duration in probiotic studies ranges from 14 to 180 days. However, it is best to introduce probiotics at an earlier stage of life. This is likely because probiotics can more effectively colonize the gut before a stable and established gut microflora is formed, in both shrimp and fish species [139,140]. With that being said, probiotics as a living microorganism have the ability to acquire resistance genes [20, 141] and require an appropriate storage condition to maintain their viability. Compared to probiotics, prebiotics offer a more practical and potentially easier approach to modulating the gut microbiota in aquatic animals. Prebiotics work by promoting the growth of beneficial microbes already present in the host’s digestive system or environment [142]. However, their effectiveness is highly dependent on the native microbial composition, which varies between the two. This variability requires careful analysis of the host’s gut microbiota to ensure the prebiotic used is appropriate and effective. Commonly used prebiotics in aquaculture include GOS, FOS, XOS, β-glucan, and chitosan, although novel plant-based prebiotics are also being explored. These compounds are typically added in relatively low amounts, around 0.05% to 5% per kg of basal diet, and are administered over feeding periods similar to those used for probiotics.

Synbiotics can help extend the viability and effectiveness of probiotics by providing a prebiotic that supports their growth. For instance, a study by Huynh et al. [142] found that adding 2% white sweet potato extract enhanced the growth of Lactobacillus spp. However, the same extract also promoted the growth of pathogenic bacteria such as Vibrio harveyi and Vibrio parahaemolyticus, as reflected by a low prebiotic score. This highlights the importance of carefully selecting prebiotics that specifically support beneficial bacteria without aiding harmful ones. In addition to supporting growth, synbiotics can also improve the colonization of probiotics in the host’s gut [141]. Boonanuntanasarn et al. [75], for example, reported that shrimp fed a combination of β-glucan and Bacillus subtilis showed higher levels of lactic acid bacteria (LAB) and reduced Vibrio counts in the intestine after 90 days of feeding. However, incorporating synbiotics in aquaculture can become more costly, especially when 60–80% of operational cost is accounted for feed [141]. Although growth improvement increases overall productivity, it can still be challenging for small-scale farmers found in Indonesian aquaculture [145].

Postbiotics and parabiotics are emerging as promising biotic components to support more sustainable aquaculture practices. They are considered safer and more convenient alternatives to probiotics and synbiotics, as they can deliver similar benefits without the risks associated with live microorganisms. Compared to other biotics, research on postbiotics and parabiotics is still limited, particularly in understanding their modes of action [146]. Therefore, laboratory-scale feeding trials are essential before implementing this biotics in real-world aquaculture systems. Additionally, since the composition and concentration of active metabolites can vary depending on microbial growth conditions, metabolomic studies are crucial. These studies help identify and quantify bioactive compounds using advanced analytical techniques such as mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), or nuclear magnetic resonance (NMR) [147].

6. Biotic supplementation in the aquaculture of Indonesia: Current state

In Indonesia, the use of biotic supplements in aquaculture has gained attention as the industry seeks practices that are sustainable and efficient. They are being integrated into the farming of various aquatic species, mostly in shrimp and fish, to enhance growth rates, improve feed conversion ratios, and enhance disease resistance from aquatic pathogenic bacteria. Table 6 summarizes several studies that utilize various biotic supplements. Biotic supplements have been shown to be effective in combating common diseases in Indonesian aquaculture, such as white spot syndrome virus (WSSV) in shrimp [148] and bacterial infections caused by Aeromonas in freshwater fish [147, 148, 149], which are both devastating reasons for economic losses for aquaculture farmers.

Table 6: Current status of studies on aquacultural biotic supplements in Indonesia

Biotic Supplements	Host	Pathogen	Result	Ref
Probiotics
Bacillus megaterium PTB 1.4	Clarias sp.	Aeromonas hydrophila	The supplementation of both B. megaterium PTB 1.4 P. pentosaceus E2211 can increase catfish survival rate and immune response.	[149]
Pediococcus pentosaceis E2211
Bacillus NP5	Clarias gariepinus	A. hydrophila	The supplementation of Bacillus NP5 at 109 CFU/ml can increase fish growth, survival rate, and immune response.	[150]
Bacillus subtilis, Lactobacillus casei	C. gariepinus	A. hydrophila	The supplementation of B. subtilis and L. casei can increase catfish survival rate and immune response.	[151]
B. subtilis	Epinephelus fuscoguttatus	-	The supplementation of B. subtilis significantly affect protein digestibility efficiency of diet utilization, growth, survival rate, and enzyme activities.	[152]
Synbiotic
Pseudoaltermonas piscicida 1Ub + MOS	Litopenaeus vannamei	Vibrio harveyi	Microencapsulated P. piscicida 1Ub enriched with MOS through Artemia sp. increase the survival rate and immune response after V. harveyi infection.	[153]
Bacillus NP5 + honey	L. vannamei	Vibrio parahaemolyticus	The supplementation of Bacillus NP5 and honey can increase the shrimps’ growth performance and immune response.	[154]
Postbiotic
Nodulisporium sp. KT29 metabolites	L. vannamei	V. harveyi WSSV	The supplementation of 20 mg/kg feed of Nodulisporium sp. KT29 can increase the phenoloxidase activity and respiratory burst and increase survival rate.	[148]
Bacillus cereus SN7 and Vagococcus fluvialis CT21 culture supernatant	-	Vibrio alginolyticus A. hydrophila Pseudomonas aeroginosa	Both culture supernatant shows antimicrobial activity to three fish pathogen using disc diffusion method.	[155]
Parabiotic
HK Lactobacillus plantarum	Juvenile Oreochromis sp.	Streptococcus agalactiae	The supplementation of HK L. plantarum at the concentration of 10-20 mg/kg feed can increase tilapia growth and the concentration of 250 mg/kg offers higher survival rate.	[156]
HK Bacillus NP5	Oreochromis niloticus	S. agalactiae	The supplementation of HK Bacillus NP5 at 1010 CFU/ml can increase the average final weight and specific growth rate, increase enzyme activity (lipase, protease, and amylase). The paraprobiotics also induced immune response	[157]

Indonesia has been actively conducting research on the use of various biotic supplements to enhance aquaculture production. This growing body of work suggests that Indonesia is taking important steps toward strengthening its aquaculture sector, in line with the national goal of positioning aquaculture as a competitive and sustainable pillar of economic growth, and the mission to develop a responsible and environmentally friendly aquaculture industry [145]. However, many challenges remain, particularly for small-scale farmers. These challenges can be grouped into internal and external factors. Internal challenges include issues like poor feed and egg quality, low water quality, and limited capacity to manage aquatic diseases and parasites. On the other hand, external challenges involve limited access to government support, lack of training, and insufficient profitability. These barriers often prevent small-scale farmers from achieving sustainable production goals [145]. In this context, the use of biotic supplements offers promising solutions by helping to improve feed utilization, enhance animal health, and reduce dependency on chemical treatments, making them a valuable tool in overcoming these challenges.

7. Challenges and safety concerns

While biotic supplements offer numerous benefits in aquaculture, their use also comes with several challenges and safety concerns that must be carefully addressed. The effectiveness of these supplements heavily depends on factors such as species, dosage, and duration of treatment. Therefore, preliminary studies are essential when introducing a specific biotic to a new host species to assess its effectiveness and safety.

Among the most significant challenges associated with probiotics and synbiotics are maintaining microbial viability, ensuring successful colonization of the host’s gut, and avoiding the risk of antibiotic resistance gene transfer, which may pose a threat to human health [141]. Although no adverse effects of probiotics have been reported in aquaculture to date, gastrointestinal side effects of probiotics have been reported in humans with inflammatory bowel disease, which can depend on the dose and probiotic strain [158]. This underscores the importance of confirming the safety of probiotics in aquaculture species, especially as they can persist in aquatic animals, which are often consumed raw or half-cooked by humans.

Synbiotics present an additional challenge in that their success depends on selecting the right combination of probiotic and prebiotic strains that work synergistically [18, 29]. On the other hand, prebiotics may be considered a safer option since they are non-living and do not carry the same risks as live microbes. However, their effectiveness relies on the presence of specific native microbiota, which can vary widely. In some cases, prebiotics may even support the growth of pathogenic bacteria, making microbial profiling of the host environment a critical step. Moreover, there is currently no standardized regulation for the use of prebiotics in aquaculture, leaving their application and outcomes largely uncontrolled [159].

Compared to these, postbiotics and parabiotics offer a potentially safer alternative, as they consist of either microbial metabolites or inactivated microbial cells, eliminating the risks associated with live organisms. However, their application is still in its early stages, and further research is needed to validate their efficacy. For postbiotics, metabolomic analysis is a crucial component, enabling the identification and quantification of bioactive compounds produced by microbes. Depending on the research goal, either targeted metabolomics or untargeted metabolomics [147, 159]. Targeted metabolomics focuses on analyzing and measuring specific, well-characterized metabolites of interest, while untargeted metabolomics aims to detect and profile all metabolites present in a sample, including unknown or unexpected compounds [147, 159]. The main challenge here lies in the high cost of equipment and the need for trained personnel, especially when using advanced tools like NMR [147]. For parabiotics, the key issue is determining the optimal inactivation method that preserves biological activity while ensuring safety [160]. It is also important to standardize and promote the concepts of postbiotics and parabiotics, particularly among farmers, to avoid confusion regarding the types of biotic compounds being used in aquaculture. Familiarizing farmers with these terms will help ensure proper application and improve acceptance of these emerging technologies in the field.

Currently, commercial products based on postbiotics and parabiotics remain limited, especially when compared to the more widely available probiotic, prebiotic, and synbiotic formulations. Nonetheless, ensuring the safe use of biotics in aquaculture is critical, not only for maintaining animal health but also for protecting human health, due to the direct connection between aquaculture products and the food chain.

8. Concluding remarks and future prospects

This review highlights the potential use of various biotic supplements as growth promoters, disease protection, reproduction enhancement, and water quality improvement in an aquaculture setting. Aquaculture is faced with growing demand, and therefore, the industry is shifting toward intensification to increase aquatic production. However, this practice often led to disease susceptibility and high environmental waste, compromising animal welfare and elevating the mortality rate. In this context, biotic supplements become a promising alternative to antibiotics for improving aquaculture sustainability. Despite this potential, the efficacy of biotic treatment can vary due to factors such as species differences, dosage, species, and treatment duration. Newer biotic supplements, such as postbiotics and parabiotics, are gaining attention for potential implementation in aquaculture. These biotics offer several benefits, including greater stability during storage and the absence of live microorganisms, which minimizes the risk of disrupting the aquatic microbiome. However, research about their functional metabolites and standardized method of inactivation to preserve their bioactivity is still lacking. Additionally, their modes of action are not yet fully understood and need to be explored further. Furthermore, in a practical aquaculture setting, these terms can be unfamiliar or ambiguous for farmers and need to be introduced to ensure shared understanding. Despite these challenges, advancements in biotechnology and metabolomic research are aiming to create more effective and specific biotic formulations. Continued investment in research, education, and regulatory frameworks will be crucial to fully harness the benefits of biotic supplements, transforming aquaculture into a more resilient and productive industry.

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