Fish feed additive compositions

Abstract

The invention provides a fish feed additive composition comprising one or more probiotics; and one or more nutraceuticals, wherein the one or more probiotics are encapsulated in a plurality of polymeric microparticles. The invention also provides a fish feed composition comprising the fish feed additive composition. In addition, the invention provides a use of the fish feed additive composition or the fish feed composition as medicament for the treatment or prevention of a bacterial infection. The invention also provides the use of the fish feed additive composition or the fish feed composition for aquaculture of fish. In one embodiment, the one or more probiotics are selected from one or more of the group consisting of L. paracasei, L. plantarum, L. helveticus, L. rhamnosus (LGG). In another embodiment, the one or more nutraceuticals is curcumin.

Description

[0001] FISH FEED ADDITIVE COMPOSITIONS

[0002] FIELD OF INVENTION

[0003] The present invention provides fish feed additive compositions and more particularly, fish feed additive compositions comprising one or more probiotics and one or more nutraceuticals.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Global fish production from aquaculture reached approximately 126 million tons in 2023, with an estimated value of USD 296.5 billion. Over the past few decades, while capture fisheries have remained relatively stable, aquaculture has experienced substantial growth. Between 1990 and 2018, global aquaculture production increased by 527%, accompanied by a 122% rise in total fish consumption during the same period. However, despite these advancements, an estimated 35% of total fisheries and aquaculture production is lost or wasted annually. A significant contributor to these losses is the widespread occurrence of bacterial infections and disease outbreaks, which place considerable economic pressure on the industry. The global economic loss due to aquaculture-related diseases is estimated to exceed USD 6 billion annually, with common pathogens such as Streptococcus, Vibrio, and Aeromonas accounting for approximately USD 1 billion of this total.

[0007] For the past five decades, antibiotics have served as the primary method for controlling infectious diseases in aquaculture. However, this practice has contributed to the emergence of antibiotic-resistant bacteria and the spread of transferable resistance genes in both fish pathogens and commensal bacteria within aquatic environments. These resistance genes can be disseminated through horizontal gene transfer, potentially reaching human pathogens and posing significant public health risks. As a result, the use of antibiotics in aquaculture has been banned or heavily restricted in many regions, including the European Union, the United States, and China. These concerns have prompted increased interest in developing sustainable and environmentally friendly alternatives for disease control in aquaculture systems.

[0008] One promising strategy involves the incorporation of probiotics into aquafeeds. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”. Since their initial application in aquaculture in 1986, probiotics have been demonstrated to improve growth performance, enhance immune responses, and increase resistance to infectious diseases. Commonly used probiotics in aquaculture include members of the phylum Firmicutes, particularly lactic acid bacteria (LAB) such as Lactococcus, Lactobacillus, and Bacillus species, which are applied either as single strains or in combination. These probiotics exert their beneficial effects through various mechanisms, including gut colonization, production of antimicrobial compounds, competition with pathogens for nutrients and adhesion sites, and the enhancement of nutrient absorption and host metabolism.

[0009] Specific LAB strains such as Lactococcus lactis CLFP 101 , Lactiplantibacillus plantarum CLFP 238, and Limosilactobacillus fermentum CLFP 242 have demonstrated inhibitory activity against pathogens like Aeromonas hydrophila, Aeromonas salmonicida, Yersinia ruckeri, and Vibrio anguillarum. Moreover, these probiotics contribute to the production of digestive enzymes and bioactive compounds that promote fish growth and health.

[0010] In addition to probiotics, the use of natural bioactive compounds, often referred to as nutraceuticals, has gained traction in the development of sustainable aquaculture practices. Among these, curcumin - a polyphenolic compound derived from turmeric (Curcuma longa), has received significant attention for its diverse biological activities, including antioxidant, antiinflammatory, antimicrobial, immunomodulatory, and antiparasitic effects. Curcumin supplementation has been shown to improve growth performance, hematological parameters, immune function, antioxidant capacity, and disease resistance in aquaculture species. It has demonstrated antibacterial activity against common pathogens such as A. hydrophila and Aeromonas sobria, and has been reported to enhance survival and disease resistance in shrimp infected with Vibrio harveyi, silver catfish exposed to Streptococcus agalactiae, and rainbow trout challenged with A. salmonicida subsp. achromogenes.

[0011] Despite the demonstrated benefits of both probiotics and curcumin, most studies have examined their effects independently. There is a notable lack of research exploring their combined application, which could potentially yield synergistic effects.

[0012] Thus, there is a need for alternative and / or improved fish feed additive compositions for solving at least the above-mentioned problems.

[0013] SUMMARY

[0014] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses. 1 . A fish feed additive composition comprising: one or more probiotics; and one or more nutraceuticals, wherein the one or more probiotics are encapsulated in a plurality of polymeric microparticles.

[0015] 2. The fish feed additive composition according to Clause 1 , wherein the one or more probiotics are selected from one or more of the group consisting of Limosilactobacillus fermentum, Lentilactobacillus kefiri, Lentilactobacillus hilgardii, and more particularly, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lactobacillus helveticus and Lacticaseibacillus rhamnosus.

[0016] 3. The fish feed additive composition according to Clause 2, wherein the one or more probiotics are selected from one or more of the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lentilactobacillus kefiri, Lactobacillus helveticus. Lentilactobacillus hilgardii and Lacticaseibacillus rhamnosus.

[0017] 4. The fish feed additive composition according to Clause 3, wherein the one or more probiotics are selected from one or more of the group consisting of L. paracasei, L. plantarum, L. helveticus, L. rhamnosus (LGG).

[0018] 5. The fish feed additive composition according to Clause 4, wherein the one or more probiotics is L. plantarum.

[0019] 6. The fish feed additive composition according to any one of the preceding clauses, wherein the one or more nutraceuticals is selected from the group consisting of a phytochemical (such as curcumin), a carotenoid (such as beta-carotene, and astaxanthin), a flavonoid (such as quercetin), a catechin, and an essential oil (such as thymol, and limonene).

[0020] 7. The fish feed additive composition according to Clause 6, wherein the one or more nutraceuticals is curcumin.

[0021] 8. The fish feed additive composition according to any one of the preceding clauses, wherein the one or more probiotics are provided in an amount of from 105to 108CFU / g of feed, optionally wherein the one or more probiotics are provided in an amount of from 106to 107CFU / g of feed.

[0022] 9. The fish feed additive composition according to any one of the preceding clauses, wherein the one or more nutraceuticals are provided in an amount of from 0.05% w / w to 5% w / w of feed, such as from 0.1% w / w to 3% w / w, optionally wherein the one or more nutraceuticals are provided in an amount of 0.5% w / w of feed.

[0023] 10. The fish feed additive composition according to any one of the preceding clauses, wherein the mass ratio of the one or more probiotics to the one or more nutraceuticals is from 5:1 to 1 :1 , such as about 4:1 , such as about 3:1 , such as about 2:1 , optionally wherein the mass ratio of the one or more probiotics to the one or more nutraceuticals is about 2:1 .

[0024] 11 . The fish feed additive composition according to any one of the preceding clauses, wherein: the one or more probiotics are provided in an amount of from 105to 108CFU / g of feed, wherein the one or more probiotics is L. plantarum; and the one or more nutraceuticals are provided in an amount of from 0.05% w / w to 5% w / w of feed, wherein the one or more nutraceuticals is curcumin; optionally wherein: the one or more probiotics are provided in an amount of from 106to 107CFU / g of feed, wherein the one or more probiotics is L. plantarum; and the one or more nutraceuticals are provided in an amount of from 0.1% w / w to 3% w / w of feed, wherein the one or more nutraceuticals is curcumin.

[0025] 12. The fish feed additive composition according to any one of the preceding clauses, wherein the microparticles encapsulating the one or more probiotics have a size of from 1 to 10 pm.

[0026] 13. A fish feed composition comprising the fish feed additive composition according to any one of Clauses 1 to 12.

[0027] 14. The fish feed composition according to Clause 13, wherein the fish feed composition comprises: fish meal; wheat flour; wheat gluten; soybean meal; corn starch; sardine oil; vitamin mix; mineral mix; and filler / additives, where the fish feed additive composition forms part of the filler / additives.

[0028] 15. The fish feed composition according to Clause 14, wherein the fish feed composition comprises: fish meal 10 wt%; wheat flour 20 wt%; wheat gluten 32 wt%; soybean meal 20 wt%; corn starch 6 wt%; sardine oil 8 wt%; vitamin mix 0.5 wt%; mineral mix 0.5 wt%; and filler / additives 3 wt%, where the fish feed additive composition forms part of the filler / additives.

[0029] 16. The fish feed composition according to Clause 15, wherein the fish feed additive composition forms from 0.05wt% to 5 wt% of the fish feed composition, such as from 0.1 wt% to 3 wt%, optionally wherein the fish feed additive composition forms about 1.5wt% of the entire fish feed composition.

[0030] 17. The fish feed composition according to Clause 15 or Clause 16, wherein the one or more probiotics are provided in an amount of from 105to 108CFU / g of the fish feed composition, optionally wherein the one or more probiotics are provided in an amount of from 106to 107CFU / g of the fish feed composition.

[0031] 18. The fish feed composition according to any one of Clauses 13 to 17, wherein the fish feed composition is in the form of pellets.

[0032] 19. Use of a fish feed additive composition according to any one of Clauses 1 to 12, or a fish feed composition according to any one of Clauses 13 to 18, in the manufacture of a medicament for the treatment or prevention of a bacterial infection. 20. Use of a fish feed additive composition according to Clauses 1 to 12 or the fish feed composition according to Clauses 13 to 18 for aquaculture of a fish.

[0033] BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1 depicts the agar well diffusion assay showing inhibition zones of selected probiotics against (A) S. iniae and (B) V. parahaemolyticus.

[0035] FIG. 2 depicts the bacteria lawn assay showing the effects of curcumin at different concentrations on A) probiotic strain L. plantarum sp., pathogens B) S. iniae, and C) I / . parahaemolyticus.

[0036] FIG. 3 includes the representative images showing the inhibition zones of probiotics (A) with curcumin and (B) without curcumin.

[0037] FIG. 4 includes the SEM images of (A) blank spray-dried microparticles and (B) probiotics- encapsulated microparticles.

[0038] FIG. 5 includes the graphs showing the survivability of the probiotic strains: (A) following spray drying and 2-h exposure to SGF, and (B) after 12 weeks of encapsulation and storage at 4 °C. *p < 0.05 using one-way ANOVA and post-hoc Tukey test, n = 3.

[0039] FIG. 6 depicts the effects of the functional feeds on the growth performance of Asian seabass fingerlings from week 0 to week 28. A) Survivability, B) Weight gain of fish, C) Specific growth rate (SGR), and D) Feed conversion ratio (FOR). *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 using one-way ANOVA and post-hoc Tukey test, n = 3.

[0040] FIG. 7 depicts Kaplan-Meier survival curves of Asian sea bass from the five experimental groups following challenge with V. parahaemolyticus. Statistical significance was determined using the Log-rank (Mantel-Cox) test (*p < 0.05, **p < 0.01 , *‘*p < 0.001 , **“p < 0.0001).

[0041] FIG. 8 depicts Kaplan-Meier survival curves of Asian sea bass from the five experimental groups following challenge with S. iniae. Statistical significance was determined using the Logrank (Mantel-Cox) test (*p < 0.05, **p < 0.01 , *“p < 0.001 , ”**p < 0.0001).

[0042] DESCRIPTION The present inventors have developed microparticles encapsulating various probiotics for coadministration with nutraceuticals to enhance antimicrobial activity, reduce the required effective dose of the nutraceutical, and improve probiotic performance - resulting in improved disease control and cost efficiency. This encapsulation technology offers several advantages for probiotic delivery, including protection during storage, improved survival through the gastrointestinal tract, and targeted delivery to the gut for enhanced colonization. Moreover, the combination of probiotic with the nutraceutical may produce synergistic effects, thereby improving the overall functional performance of the composition. As demonstrated in the present Examples, the present invention enhances the stability and bio-efficacy of the probiotic component while also harnessing the known health-promoting properties of the nutraceutical (e.g., curcumin). Furthermore, the co-supplementation of encapsulated probiotics and the nutraceuticals presents a promising approach for the development of functional aquafeeds that promote sustainable disease control and reduce reliance on antibiotics in aquaculture.

[0043] Thus, in a first aspect of the invention, there is provided a fish feed additive composition comprising: one or more probiotics; and one or more nutraceuticals, wherein the one or more probiotics are encapsulated in a plurality of polymeric microparticles.

[0044] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0045] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0046] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of two or more such compounds, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0047] As mentioned above and demonstrated in the present Examples, the combination of the probiotic with the nutraceutical may produce synergistic effects. In certain embodiments, the one or more probiotics may be selected from one or more of the group consisting of Limosilactobacillus fermentum, Lentilactobacillus kefiri, Lentilactobacillus hilgardii, and more particularly, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lactobacillus helveticus and Lacticaseibacillus rhamnosus.

[0048] In certain particular embodiments, the one or more probiotics may be selected from one or more of the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lentilactobacillus kefiri, Lactobacillus helveticus. Lentilactobacillus hilgardii and Lacticaseibacillus rhamnosus.

[0049] In certain embodiments, the one or more probiotics may be selected from one or more of the group consisting of L. paracasei, L. plantarum, L. helveticus, L. rhamnosus (LGG). In certain exemplary embodiments, the one or more probiotics may be L. plantarum.

[0050] When used herein, the term “nutraceutical” refers to a food or part of a food that provides medical or health benefits, including the prevention and treatment of disease, beyond basic nutritional value. In certain embodiments, the one or more nutraceuticals may be selected from the group consisting of a phytochemical (such as curcumin), a carotenoid (such as betacarotene, and astaxanthin), a flavonoid (such as quercetin), a catechin, and an essential oil (such as thymol, and limonene). In certain exemplary embodiments, the one or more nutraceuticals may be curcumin.

[0051] For the avoidance of doubt, the fish feed additive composition may comprise one or more probiotics (such as two probiotics or three probiotics) and one or more nutraceuticals (such as two nutraceuticals or three nutraceuticals).

[0052] In the present invention, the one or more probiotics are encapsulated in a plurality of polymeric microparticles (e.g., Ca2+-crosslinked alginate microparticles). Advantageously, encapsulating the one or more probiotics protects them from the environment, such as the acidic gastric juices in the gastrointestinal tract, which may decrease the viability of the probiotics. The one or more nutraceuticals may or may not be encapsulated - the one or more nutraceuticals may be provided in free form.

[0053] The one or more probiotics may be encapsulated in a plurality of polymeric microparticles by methods known to those skilled in the art, for example, L. L. Tan, M. Mahotra, S. Y. Chan, and S. C. J. Loo, In situ alginate crosslinking during spray-drying of lactobacilli probiotics promotes gastrointestinal-targeted delivery, Carbohydrate polymers, vol. 286, p. 1 19279, Jun 15 2022, doi: 10.1016 / j.carbpol .2022.119279. The present invention is not particularly limited by the exact mode of encapsulation.

[0054] The one or more probiotics may be provided in any suitable amount depending on the final amount required in the fish feed composition. In certain embodiments, the one or more probiotics may be provided in an amount such of from 105to 108CFU / g of feed. In certain exemplary embodiments, the one or more probiotics may be provided in an amount of from 106to 107CFU / g of feed.

[0055] The one or more nutraceuticals may be provided in any suitable amount depending on the final amount required in the fish feed composition. In certain embodiments, the one or more nutraceuticals may be provided in an amount of from 0.05% w / w to 5% w / w of feed, such as from 0.1% w / w to 3% w / w. In certain exemplary embodiments, the one or more nutraceuticals may be provided in an amount of about 0.5% w / w of feed.

[0056] In certain embodiments, the mass ratio of the one or more probiotics to the one or more nutraceuticals may be from 5:1 to 1 :1 , such as about 4:1 , such as about 3:1 , such as about 2:1. In certain exemplary embodiments, the mass ratio of the one or more probiotics to the one or more nutraceuticals may be about 2:1 .

[0057] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1 %, within 0.05%, within 0.01 %, within 0.005%, or within 0.001% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0058] In certain exemplary embodiments: the one or more probiotics may be provided in an amount of from 105to 108CFU / g of feed, wherein the one or more probiotics may be L. plantarum and the one or more nutraceuticals may be provided in an amount of from 0.05% w / w to 5% w / w of feed, wherein the one or more nutraceuticals may be curcumin;

[0059] In certain particular exemplary embodiments: the one or more probiotics may be provided in an amount of from 106to 107CFU / g of feed, wherein the one or more probiotics may be L. plantarum; and the one or more nutraceuticals may be provided in an amount of from 0.1% w / w to 3% w / w of feed, wherein the one or more nutraceuticals may be curcumin.

[0060] As mentioned above, the one or more probiotics are encapsulated in a plurality of polymeric microparticles. In certain exemplary embodiments, the microparticles encapsulating the one or more probiotics may have a size of from 1 to 10 pm, as determined by conventional techniques and / or devices known in the art, such as scanning electron microscopy (SEM).

[0061] As will be appreciated, the present fish feed additive composition may be added to a fish feed composition. As such, in another aspect of the invention, there is provided a fish feed composition comprising the fish feed additive composition as disclosed hereinbefore.

[0062] The fish feed composition may comprise ingredients known in the art. In certain embodiments, the fish feed composition may comprise the following: fish meal; wheat flour; wheat gluten; soybean meal; corn starch; sardine oil; vitamin mix; mineral mix; and filler / additives, where the fish feed additive composition forms part of the filler / additives.

[0063] The ingredients may be present in any suitable amounts in the fish feed composition. In certain embodiments, the fish feed composition may comprise the following: fish meal 10 wt%; wheat flour 20 wt%; wheat gluten 32 wt%; soybean meal 20 wt%; corn starch 6 wt%; sardine oil 8 wt%; vitamin mix 0.5 wt%; mineral mix 0.5 wt%; and filler / additives 3 wt%, where the fish feed additive composition forms part of the filler / additives.

[0064] In certain embodiments, the fish feed additive composition may form from 0.05 to 5 wt% of the entire fish feed formulation, such as from 0.1 wt% to 3 wt%. In certain exemplary embodiments, the fish feed additive composition may form about 1 ,5wt% of the entire fish feed composition. In such exemplary embodiments, the fish feed formulation may comprise about 1 wt% of the one or more probiotics and about 0.5 wt% of the one or more nutraceuticals.

[0065] The one or more probiotics may be provided in any suitable amount. In certain embodiments, the one or more probiotics may be provided in an amount of from 105to 108CFU / g of the fish feed composition. In certain exemplary embodiments, the one or more probiotics may be provided in an amount of from 106to 107CFU / g of the fish feed composition.

[0066] As will be appreciated, the present fish feed additive composition may be pelletised together with commonly used fish feed ingredients. In the case of commercial fish feeds that are already manufactured, the present additive composition may be coated onto these fish feeds using oils such as fish oil, soybean oil to form coated functional fish feeds.

[0067] As demonstrated in the present examples, the present fish feed additive composition or the present fish feed composition comprising thereof may find utility as medicaments. As such, in a further aspect of the invention, there is provided a use of a fish feed additive composition or a fish feed composition as disclosed hereinbefore, in the manufacture of a medicament for the treatment or prevention of a bacterial infection.

[0068] The present fish feed additive composition or the present fish feed composition comprising thereof may also find utility in aquaculture. As such, in a further aspect of the invention, there is provided a use of a fish feed additive composition or the fish feed composition as disclosed hereinbefore for aquaculture of a fish.

[0069] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples. EXAMPLES

[0070] Example 1 : Materials and Methods

[0071] Materials

[0072] Probiotic strains used in this study include 12 strains from 12 species, namely Lactobacillus acidophilus (LAS), Lacticaseibacillus paracasei (LPI), Limosilactobacillus reuteri (LRI), Limosilactobacillus amylovorus (LAM), Limosilactobacillus oris (LOS), Lactiplantibacillus plantarum (LPM), Limosilactobacillus fermentum (LFM), Liquorilactobacillus satsumensis (LSS), Lactobacillus helveticus (LHS), Lentilactobacillus kefiri (LKI), Lentilactobacillus hilgardii (LHI), and Lacticaseibacillus rhamnosus (LGG), as detailed in Table 1 . Of which, strains LAS, LRI, LAM, LOS, LPM and LFM, were obtained from PC Biome Pte Ltd, Singapore. Strains LPI, LSS, LHS, LKI and LHI were strains isolated from milk and water kefir, as reported in Tan et al. (Tan, L.L., et al., Potential Probiotic Strains From Milk and Water Kefir Grains in Singapore- Use for Defense Against Enteric Bacterial Pathogens. Front Microbiol, 2022. 13: p. 857720), and L. rhamnosus GG (LGG) was isolated from a purchased Culturelle® probiotic pill (i - Health , Inc., Crownwell, USA). Two aquaculture pathogens used in this study, including Vibrio parahaemolyticus ATCC 17802 and Streptococcus iniae ATCC 29178 were purchased from ATCC, USA.

[0073] De Man, Rogosa and Sharpe (MRS), Nutrient Broth (NB), Wilkins-Chalgren Anaerobe (WC) media were purchased from Thermo Fisher Scientific, USA. Bacto agar was purchased from BD, USA. All other chemicals used in this experiment, including Curcumin from Curcuma longa and Dimethyl Sulfoxide (DMSO), were purchased from Sigma Aldrich, USA.

[0074] Table 1. Growth conditions of probiotic strains tested.

[0075] Statistical analysis

[0076] Each experiment was performed in triplicate, and results were expressed as mean ± standard deviation (SD). Data were analyzed using one-way ANOVA, and post-hoc Tukey-HSD test, with statistical significance set at p < 0.05.

[0077] Example 2: Growth of microorganisms

[0078] Probiotics were inoculated in MRS media and incubated according to respective conditions, under aerobic or anaerobic environments, as indicated in Table 1 . For anaerobic conditions, strains were handled within the Bactron 300 anaerobic chamber (Sheldon Manufacturing, USA). All pathogens were inoculated in WC broth, with exception of V. parahaemolyticus, which was inoculated in NB supplemented with 3% (w / v) NaCI. Pathogen strains were incubated aerobically at 30 °C conditions for 24 h prior to use.

[0079] Example 3: Antimicrobial Susceptibility Testing (AST)

[0080] Example 3.1 : Screening probiotics against S. iniae and V. parahaemolyticus

[0081] The inhibitory potential of probiotic strains against aquaculture pathogens was evaluated using the agar well diffusion assay. Confluent pathogen cultures were diluted in WC medium based on their optical density at 600 nm (OD6oo)- V. parahaemolyticus and S. iniae were adjusted to an OD6OO of 0.5. A 100 pL aliquot of the diluted pathogen suspension was spread onto WC agar plates, and 6 mm-diameter wells were created using the back of a 200 pL pipette tip. Each well was filled with 50 pL of probiotic culture and the plates were incubated at 30°C for 24 h. After incubation, inhibition zones, defined as clear areas devoid of pathogen growth, were recorded as evidence of probiotic-mediated inhibition. The inhibition zone size was measured as the distance from the edge of the well to the outer boundary of the clear zone.

[0082] Results and Discussion: A library of 11 probiotic strains and three control strains was screened for antagonistic activity against S. iniae and V. parahaemolyticus, as summarized in Table 2. Using the agar well diffusion assay (FIG. 1), 15 probiotic strains exhibiting strong antipathogenic activity were selected. In this assay, inhibition zones were classified as strong (+++) for diameters exceeding 4 mm, intermediate (++) for 2-4 mm, and weak (+) for those less than 2 mm.

[0083] Table 2: List of probiotics screened against S. iniae. V. parahaemolyticus and their antipathogenic effects.

[0084] To investigate the potential role of pH in the observed inhibitory effects, the pH of probiotic cultures was measured, ranging from 3.63 to 4.24. Additionally, MRS broth was adjusted to pH values between 3.0 and 6.2 for comparison. The results indicated that pH alone was not the primary determinant of probiotic antagonism. Notably, S. iniae was not inhibited in MRS broth at pH 3.5, despite this being more acidic than all tested probiotic cultures. Similarly, V. parahaemolyticus showed only weak inhibition at pH < 5.0 in MRS broth, whereas most probiotic cultures demonstrated intermediate inhibition. The results suggest that the inhibitory activity of the probiotics was likely mediated by bioactive compounds rather than pH alone. This observation is consistent with findings from earlier studies on lactic acid bacteria (LAB), which have shown that antimicrobial agents, such as hydrogen peroxide and bacteriocins, are involved in pathogen suppression in aquaculture environments (Zapasnik, A., B. Sokolowska, and M. Bryla, Role of Lactic Acid Bacteria in Food Preservation and Safety. Foods, 2022. 11 (9); Ibrahim, S.A., et al., Lactic Acid Bacteria as Antimicrobial Agents: Food Safety and Microbial Food Spoilage Prevention. Foods, 2021. 10(12)). These studies reinforce the potential of probiotic strains to combat aquaculture pathogens through various biochemical mechanisms, including competition for nutrients, production of antimicrobial compounds, and direct inhibition of pathogen growth.

[0085] Example 3.2: Assessing the compatibility of probiotics and pathogens with curcumin

[0086] An agar dilution method was used to assess the compatibility of probiotics and pathogens with curcumin, ensuring that the selected probiotics were not inhibited by the presence of curcumin and evaluating whether curcumin exhibited inhibitory activity against V. parahaemolyticus and S. iniae. Briefly, probiotic and pathogen inoculums were diluted to an OD500of 0.1 and 0.5 in MRS and WC broths, respectively. A 100 pL aliquot of each diluted culture was spread onto MRS or WC agar plates. A range of curcumin concentrations (5 mg / mL to 0.01 mg / mL) was prepared by serial dilution and pipetted onto the MRS and WC agar surfaces. After 24h incubation at 30 °C, plates were examined for the presence of inhibition zones. Complete inhibition was defined as the absence of bacterial colonies beneath the curcumin or DMSO droplet, while partial inhibition was indicated by visible thinning of the bacterial lawn in the affected area. DMSO at corresponding concentrations ranging from 12.5% to 0.025% was also tested as control samples.

[0087] Results and Discussion:

[0088] To assess the compatibility of curcumin with probiotics, its potential inhibitory effects on probiotic growth were examined. Additionally, its antimicrobial activity against S. iniae and V. parahaemolyticus was evaluated. As shown in FIG. 2A, curcumin did not suppress the growth of the selected probiotic strains, indicating its suitability for co-supplementation.

[0089] In contrast, curcumin exhibited dose-dependent inhibitory effects against S. iniae and I / . parahaemolyticus. For S. iniae, a complete growth inhibition was observed at concentrations of >1 .5 mg / mL curcumin on agar, whereas inhibition occurred at concentrations >0.75 mg / mL curcumin for V. parahaemolyticus (FIG. 2B & FIG. 2C). These results suggest that curcumin possesses selective antimicrobial properties, effectively targeting pathogenic bacteria while maintaining probiotic viability. These results align with earlier studies, which indicated that curcumin can selectively target pathogenic bacteria without adversely affecting beneficial microorganisms (Adamczak, A., M. Ozarowski, and T.M. Karpinski, Curcumin, a Natural Antimicrobial Agent with Strain-Specific Activity. Pharmaceuticals (Basel), 2020. 13(7); Mody, D., A.I.M. Athamneh, and M.N. Seleem, Curcumin: A natural derivative with antibacterial activity against Clostridium difficile. J Glob Antimicrob Resist, 2020. 21 : p. 154-161 ). Based on these findings, curcumin concentrations of 0.75 mg / mL and 1.5 mg / mL were selected for subsequent experiments. These concentrations were further evaluated for their potential synergistic effects against pathogens.

[0090] Example 3.3: Evaluating synergistic effects of probiotics and curcumin on pathogens

[0091] In this assay, the synergistic effects of probiotics and curcumin against pathogens were evaluated using the agar well diffusion method. Based on the findings from Example 3.1 , four probiotics - L. paracasei spp., L. helveticus sp., L rhamnosus GG, and L. plantarum sp. - were selected for further testing. These strains were assessed in combination with curcumin at concentrations of 0.75 mg / mL and 1.5 mg / mL. These concentrations were chosen based on Example 3.2, representing the highest level of curcumin compatible with the probiotics and the lowest concentration effective against pathogens, respectively.

[0092] Pathogen cultures were prepared by diluting them to an ODe0o of 0.5. Curcumin stock solution was then added to the pathogen cultures to achieve final curcumin concentrations of 1 .5 mg / mL or 0.75 mg / mL. The pathogen-curcumin mixtures (100 pL) were spread onto WC agar plates. Wells were created in the agar, and 50 pL of the selected probiotic cultures were added to each well. The plates were incubated at 30 °C for 24 h. After incubation, the plates were examined for the presence of inhibition zones. The size of the inhibition zones was compared to results from the individual probiotic assays to assess whether combined supplementation of curcumin and probiotics enhanced the inhibitory effects on the pathogens.

[0093] Results and Discussion

[0094] The potential synergistic antimicrobial effects of curcumin in combination with probiotics were investigated using the agar well diffusion assay. Four probiotic strains - L. paracasei spp., L. plantarum sp., L helveticus sp., and L rhamnosus GG - were selected for co-administration with curcumin based on their demonstrated inhibitory effects against pathogens and compatibility with curcumin.

[0095] As illustrated in FIG. 3 and summarized in Table 3, the co-supplementation of curcumin with probiotics resulted in a clear synergistic inhibition of the pathogens. The combination generally produced greater antimicrobial effects than either curcumin or probiotics alone, as evidenced by larger inhibition zones. Specifically, a dose-dependent bactericidal effect was observed against S. iniae, with curcumin at 1 .5 mg / mL producing a significantly larger zone of inhibition compared to 0.75 mg / mL. In the case of V. parahaemolyticus, the combination treatment resulted in noticeable thinning of the bacterial lawn across the plate, making it challenging to define and measure distinct inhibition zones.

[0096] The enhanced antimicrobial activity of curcumin may be attributed to its known ability to disrupt bacterial quorum sensing pathways, thereby impeding biofilm formation (Zheng, D., et al., Antibacterial Mechanism of Curcumin: A Review. Chemistry & Biodiversity, 2020. 17(8): p. e2000171 ), as well as its capacity to compromise bacterial membrane integrity, leading to cell death (Tyagi, P., et al., Bactericidal activity of curcumin I is associated with damaging of bacterial membrane. PLoS One, 2015. 10(3): p. e0121313). Importantly, curcumin did not exhibit any detrimental effects on the growth of the probiotic strains, consistent with previous findings by Kim et al. (Kim, J., et aL Synergistic Antibacterial Effects of Probiotic Lactic Acid Bacteria with Curcuma longa Rhizome Extract as Synbiotic against Cutibacterium acnes. Applied Sciences, 2020. 10), who reported that Lactobacillus spp. are tolerant to curcumin - possibly due to their natural association with turmeric rhizomes (Pianpumepong, P. and A. Noomhorm, Isolation of probiotic bacteria from turmeric (Curcuma longa Linn.) and its application in enriched beverages. International Journal of Food Science and Technology, 2010. 45(12): p. 2456-2462). These results highlight the potential of curcumin-probiotic co- supplementation as a promising antimicrobial strategy, offering enhanced efficacy against pathogenic bacteria without compromising probiotic viability.

[0097] Table 3: Combinatorial effects of curcumin and the four selected probiotics against S. iniae. An increase in the size of the inhibition zone indicates a concentration-dependent effect.

[0098] Example 4: Encapsulation of probiotics

[0099] Four probiotic strains that showed synergistic effects with curcumin against the pathogens were then encapsulated using a patented technique (Loo, S.C.J., Tan, L. L., Mahotra, M., Encapsulation of live microorganisms for gastrointestinal-targeted delivery, in Nanyang Technological University, Singapore, W.I.P. Organization, Editor. 2022). Briefly, the probiotic cultures were grown in MRS broth at 30 °C for 24 h, followed by washing to remove the residual media. The cells were then suspended in alginate solution containing protectants to prevent loss during spray drying. The alginate cell suspension was then spray dried together with a cross-linking agent. The encapsulated probiotics were characterized using SEM to observe the shape and size of the microparticles. The survivability of the probiotics after spray drying was assessed by dissolving the spray dried microparticles, serially diluting the suspension, and drop-plating onto MRS agar. The probiotics were also subjected to simulated gastric fluid (SGF) (pH~2) to mimic the environment in the Gastrointestinal Tract (GIT). The SGF was prepared with 0.2 M NaCI and 2000 units / mL porcine pepsin, with the pH adjusted to 2 using HCI.

[0100] For the considered end application as supplements in fish feeds, the encapsulated probiotics must display good storage stability to enable the feeds to be stored at the aquaculture farms. The encapsulated probiotics were stored at 4 °C over a period of 12 weeks and the survivability was tested every four weeks to monitor the loss over the storage period. In addition, aliquots of samples during the storage period were also subject to acid exposure tests under SGF conditions to mimic and assess the viability of probiotics when taken up by fishes.

[0101] Results and Discussion

[0102] To enhance the stability, survivability, and functional performance of sensitive probiotic strains, and to ensure their targeted delivery to the gastrointestinal tract (GIT), four strains exhibiting synergistic antimicrobial activity (Example 3.3) were selected for encapsulation. A patented spray-drying method was used to incorporate each strain into an alginate-based matrix, providing protection against environmental and gastrointestinal stresses and facilitating sitespecific release within the GIT (Loo, S.C.J., Tan, L. L., Mahotra, M., Encapsulation of live microorganisms for gastrointestinal-targeted delivery, in Nanyang Technological University, Singapore, W.I.P. Organization, Editor. 2022).

[0103] FIG. 4 presents electron micrographs of the spray-dried alginate particles, both blank and the encapsulated probiotics. All microparticles were uniformly spherical and measured <10 pm in size. This small particle size is ideal for ensuring uniform mixing with fish feed ingredients without compromising the feed's palatability. Next, the survivability of the encapsulated probiotics was tested both after the spray-drying process and upon exposure to gastric conditions (pH~2). The results of these survivability experiments are depicted in FIG. 5A. Some probiotics experienced a reduction of at least 0.5 logio in CFU, with L. plantarum sp. showing comparatively less reduction in CFU. This loss is within the expected range during the encapsulation process. Additionally, the probiotics were exposed to the SGF (pH~2) to simulate the environment of the GIT. L. plantarum sp. exhibited the highest tolerance to the SGF compared to the other strains. No significant difference was observed in log CFU counts after spray drying (SD) and after exposure to the SGF, while the other probiotic strains showed a significant reduction in CFU.

[0104] The encapsulated strains were also subjected to a 12-week storage stability test to assess the shelf life of the probiotics. As shown in FIG. 5B, all strains demonstrated good survivability when stored at 4 °C for up to eight weeks, although some loss was observed after 12 weeks. The survivability of probiotics under acid exposure was also evaluated after storage. L. plantarum sp. had the highest survival after SGF exposure post-storage compared to all other strains. As a result, L. plantarum sp. was selected as the probiotic strain to be used for subsequent feeding trials.

[0105] Example 5: Investigating the impact of encapsulated L. plantarum sp. and curcumin co-supplementation on fingerling growth Example 5.1 : Pelletizing fish feed for fingerlings

[0106] A fish feed pelletizer was used to produce 1.5 - 2 mm feed pellets suitable for fingerlings in a scalable and cost-effective manner. The basal diet was formulated with essential nutrients and supplemented with encapsulated probiotics (1% w / w, ~6.7 x 109CFU / Kg diet) (Soltani, M., et al., Growth performance, immune-physiological variables and disease resistance of common carp (Cyprinus carpio) orally subjected to different concentrations of Lactobacillus plantarum. Aquaculture International, 2017. 25(5): p. 1913-1933), and curcumin (0.5% w / w) (Jiang, J., et al., Effects of dietary curcumin supplementation on growth performance, intestinal digestive enzyme activities and antioxidant capacity of crucian carp Carassius auratus. Aquaculture, 2016. 463: p. 174-180), based on effective dosages reported to support fish health and growth. During the pelletization process, the ingredients were uniformly mixed and extruded under controlled conditions to ensure consistency and nutrient retention. By adjusting the pelletizer settings, different pellet sizes could be produced to accommodate various developmental stages of fish. Standard fish feed was modified to include encapsulated probiotics and curcumin, while a non-supplemented feed served as the control to evaluate their effects on fish growth performance and resistance to pathogen challenges. All diets were subjected to compositional analysis by Eurofins Food Testing Singapore Pte Ltd., a certified analytical laboratory.

[0107] Results and Discussion

[0108] To investigate the combinatorial effects of probiotics and curcumin on the growth performance of Asian seabass fingerlings, a feeding trial was designed using five distinct functional feed groups: EP, FC, FPFC, EPFC, and a control (non-supplemented) group. The unencapsulated probiotics (FP) were produced through spray drying of the probiotic solution to have an effective comparison against the encapsulated probiotic (EP) samples. Spray drying also enables large-scale production of these probiotics for commercial use. The survivability of the spray-dried probiotics was assessed across multiple batches, yielding an average survivability of approximately 1010CFU / g. These probiotic powders were then incorporated into the respective feed formulations and pelletized to the appropriate size. The final concentration of viable probiotics in the diets was approximately ~6.7 x 109CFU / kg of feed.

[0109] All diets were analysed for their composition, and the resulting nutritional profiles of the functional feeds were found to be comparable, ensuring that any observed differences in fish performance could be attributed to the functional additives rather than to nutritional imbalances. The detailed compositional data are presented in Table 4. Table 4: Compositional analysis of the feeds used for the feeding trial.

[0110] Example 5.2: Growth performance

[0111] The growth performance trial was conducted at the fish facility at Pontus Research in Singapore. Fingerlings were fed with control diet for 2-week acclimation period in 500 L recirculation aquaculture systems (RAS) tanks. Thereafter, similar-sized fish were randomly distributed into 5 experimental groups (EP, FC, FPFC, EPFC and control) as shown in Table 5. Each tank contained 100 fish with an initial average weight of approximately 6 grams, and all diets were tested in triplicate. The fish were hand-fed three times daily to apparent satiation for 28 weeks. The water temperature was maintained at around 29± 1 °C for Asian seabass throughout the trial. All procedures adhered to ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC Approval No.: SFA-MAC-2024-01 ). At the end of the feeding trial, fish were starved for 24 h for complete food digestion for accurate weight measurements. Weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and survival rates were evaluated, with the equations from (Amer, S.A., et al. Long-Term Feeding with Curcumin Affects the Growth, Antioxidant Capacity, Immune Status, Tissue Histoarchitecture, Immune Expression of Proinflammatory Cytokines, and Apoptosis Indicators in Nile Tilapia, Oreochromis niloticus. Antioxidants, 2022. 11 , DOI: 10.3390 / antiox1 1050937). Study groups of different diets fed to Asian seabass fingerlings are as follows:

[0112] 1. Encapsulated probiotic (EP)

[0113] 2. Free curcumin (FC)

[0114] 3. A combination of free probiotics and free curcumin (FPFC)

[0115] 4. A combination of encapsulated probiotics and free curcumin (EPFC)

[0116] 5. A control group with no functional additives FBW - 1BW

[0117] WG =

[0118] IBWX W°

[0119] BW InFBW - InlBW SGR (— ) = x 100 day T

[0120] FCR = FI / WG

[0121] Where FBW = final body weight (g), IBW = initial body weight (g), T = duration of the trial in days, WG = wet weight gain (g) and Fl = estimated feed intake (g).

[0122] Table 5: Different study groups for the feeding trial. FP - Free probiotic; EP - Encapsulated probiotic; FC - Free Curcumin.

[0123] Results and Discussion:

[0124] To evaluate the effects of functional feed supplementation on the growth performance of Asian seabass fingerlings, fish were fed with the formulated diets throughout the feeding trial. Survival analysis indicated that the control group, which received no functional additives, exhibited the lowest survivability among all groups (FIG. 6A; Table 6). In contrast, all groups receiving functional feeds showed improved survival rates, suggesting a potential immunomodulatory effect conferred by the dietary supplements.

[0125] Among the functional groups, fish fed with encapsulated probiotics (EP) exhibited the most pronounced growth response, achieving a significant body weight gain of 33.0 ± 6.3% compared to the control group (FIG. 6B). The group supplemented with free curcumin (FC) demonstrated the next highest gain, with a 26.5 ± 10.9% increase. The combination diet groups FPFC and EPFC also promoted growth, resulting in body weight gains of approximately 8.4 ± 1.5% and 15.4 ± 1 1 .0%, respectively. Additionally, all functional feed groups outperformed the control in terms of feed conversion ratio (FCR) and specific growth rate (SGR), indicating improved feed efficiency and overall growth dynamics (FIG. 6C, FIG.

[0126] 6D & Table 6). Despite exhibiting a higher FCR, the EPFC group achieved the highest survival and moderate weight gain, indicating that the combination of encapsulated probiotics (EP) and free curcumin (FC) enhanced immunoprotection. The improved viability and targeted delivery of probiotics, combined with curcumin’s antioxidant, anti-inflammatory, and antimicrobial properties, may have synergistically stimulated immune function. The resulting increase in immune activity likely elevated metabolic demands, diverting energy from growth and contributing to the higher FCR observed. This reflects an eco-immunological trade-off, where greater investment in immune function often leads to reduced growth efficiency. Similar observations have been reported in fish, where immune activation has been associated with metabolic costs (Bonneaud, C., R.S. Wilson, and F. Seebacher, Immune-Challenged Fish Up-Regulate Their Metabolic Scope to Support Locomotion. PLoS One, 2016. 11 (11 ): p. eO166028; Kim, J.H., et al., Effect of growth rate on transcriptomic responses to immune stimulation in wild-type, domesticated, and GH-transgenic coho salmon. BMC Genomics, 2019. 20(1 ): p. 1024). Although the EPFC formulation may not optimize growth performance, it provides added value in aquaculture environments susceptible to disease, where survival is critical.

[0127] These findings highlight the potential of probiotic and curcumin supplementation to promote health and productivity in aquaculture species. These results are consistent with previous studies, which have demonstrated that functional feeds containing Bacillus probiotics or garlicbased plant extracts can improve growth and feed conversion in aquaculture species (Rimoldi, S., et al., Genetically superior European sea bass (Dicentrarchus labrax) and nutritional innovations: Effects of functional feeds on fish immune response, disease resistance, and gut microbiota. Aquaculture Reports, 2023. 33: p. 101747). Probiotics have been widely documented to enhance fish growth and immunity through several mechanisms, including modulating gut microbiota composition, inhibiting pathogenic bacteria, improving digestion, facilitating nutrient absorption, stimulating host immune responses, and producing bioactive antimicrobial compounds (El-Saadony, M.T., et al., The functionality of probiotics in aquaculture: An overview. Fish & Shellfish Immunology, 2021. 117: p. 36-52). Similarly, curcumin, a polyphenolic compound derived from turmeric, promotes growth by stimulating digestive enzymes such as a-amylase, protease, and lipase. The activation of these enzymes improves nutrient assimilation and feed conversion efficiency, ultimately resulting in enhanced growth with reduced feed input (Sruthi, M.V., et al., Dietary curcumin influences leptin, growth hormone and hepatic growth factors in Tilapia (Oreochromis mossambicus). Aquaculture, 2018. 496: p. 105-11 1.). In addition to its digestive benefits, curcumin’s antimicrobial properties have been implicated in its growth-promoting effects, potentially by mitigating the negative impacts of pathogenic bacteria on fish health and intestinal function (Mahmoud, H.K., et al., Dietary curcumin supplement influence on growth, immunity, antioxidant status, and resistance to Aeromonas hydrophila in Oreochromis niloticus. Aquaculture, 2017. 475: p. 16- 23.), as also reflected in the in vitro antipathogen activity observed in Example 3.2. Taken together, these findings support the conclusion that dietary supplementation with encapsulated probiotics and curcumin, whether administered individually or in combination, can significantly improve the health status, growth performance, and feed utilization efficiency of Asian seabass fingerlings. This functional feed strategy offers a promising, sustainable approach to enhancing productivity in aquaculture systems.

[0128] Table 6: Effects of the functional feeds on the growth performance of Asian seabass fingerlings from week 0 to week 28.

[0129] Example 6: Pathogen challenge test

[0130] Example 6.1 : Challenge test against Vibrio

[0131] Following a 28-week feeding period with functional feeds, a subset of fish from each experimental group was transferred to the ABSL-2 facility for a disease challenge experiment. Fish from the control diet group were used to determine the 50% lethal dose (LD50) of the Vibrio challenge strain via intraperitoneal (i.p.) routes. Each test group underwent challenge trials in triplicate using the predetermined LD50dose and was monitored daily for up to 14 days. The experiment was concluded earlier if any experimental group exhibited 50-100% mortality. The control diet group, exposed to varying Vibrio doses, exhibited an LD50of approximately 108CFU / mL via the i.p. route. All procedures adhered to ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC Approval No.: 202303-186).

[0132] Results and Discussion:

[0133] Following the 28-week feeding trial with the respective functional diets, a pathogen challenge test was conducted to evaluate the protective efficacy of the treatments against Vibrio infection. Fish from each dietary group were exposed to a lethal dose 50% (LDS0) concentration of Vibrio, approximately 108CFU / mL, administered via intraperitoneal (i.p.) injection. FIG. 7 showed that the control group exhibited the expected LD50response, with 50% mortality occurring within the observation period. Among the treatment groups, fingerlings fed with the EPFC and FC diets demonstrated complete protection, achieving 100% survival. The FPFC group showed partial protection, with a survival rate of 33%. In contrast, both the control and EP groups exhibited the lowest survival (FIG. 7). In summary, the survival rates of the experimental groups, in descending order, are as follows:

[0134] EPFC = FC (100% survival) > FPFC (33% survival) > Control = EP

[0135] These findings demonstrate the potential of curcumin, particularly when used alone or in combination with encapsulated probiotics, to enhance disease resistance in Asian seabass fingerlings. The improved survival outcomes suggest protective or antimicrobial roles of curcumin against Vibrio infection, making it a promising ingredient for functional feed development in aquaculture.

[0136] Example 6.2: Challenge test against S. iniae

[0137] At the end of the feeding trial, the fingerlings were subjected to a challenge test against S. iniae, conducted in a manner similar to the Vibrio challenge. Briefly, fish from each test group were injected intraperitoneally with 0.1 mL of normal saline solution (NSS) containing the LD50 dose of S. iniae in triplicate. The LD50 was determined using moderate (-3.1 x 106CFU / fish) and high (-1.5 x 108CFU / fish) bacterial doses. The highest i.p. dose of S. iniae ATCC 29178 resulted in -42% cumulative mortality. To facilitate infection, fish were sedated, and a 1 cm2area of skin was scraped using a scalpel blade before i.p. injection of -2.4 x 107CFU / fish. Mortality was recorded daily for 12 days, and the experiment was concluded earlier if any experimental group exhibited 50-100% mortality. This study followed ethical protocols approved by the Animal Ethics Committee (IACUC) of Temasek Polytechnic (Approval No.: 202312-191 A).

[0138] Results and Discussion:

[0139] Pathogen challenge was also conducted to evaluate the efficacy of the functional feeds in protecting Asian seabass fingerlings against S. iniae infection. The challenge protocol was performed similarly to the Vibrio challenge test. The results showed that the FPFC group conferred the highest level of protection against S. iniae, achieving a 100% survival rate (Figure 8). This was followed by the EPFC and FC groups, each demonstrating a survival rate of 66.7%. In contrast, the EP and control groups again exhibited the highest mortality, with survival rates significantly lower than those of the curcumin-supplemented treatments. In summary, the survival rates of the experimental groups, in descending order, are as follows:

[0140] FPFC (100% survival) > EPFC = FC (66.7% survival) > Control = EP

[0141] These findings highlight the promising role of curcumin-containing functional feeds - particularly when combined with probiotics - in enhancing the resistance of Asian seabass fingerlings to S. iniae infection. This observation is consistent with previous studies reporting that dietary curcumin can strengthen fish immune defence against pathogen infections (Mahmoud, H.K., et al., Dietary curcumin supplement influence on growth, immunity, antioxidant status, and resistance to Aeromonas hydrophila in Oreochromis niloticus. Aquaculture, 2017. 475: p. 16-23; Amer, S.A., et al., Long-Term Feeding with Curcumin Affects the Growth, Antioxidant Capacity, Immune Status, Tissue Histoarchitecture, Immune Expression of Proinflammatory Cytokines, and Apoptosis Indicators in Nile Tilapia, Oreochromis niloticus. Antioxidants (Basel), 2022. 1 1 (5)). Indeed, curcumin has been shown to enhance both innate and humoral immune functions in fish, including elevated lysozyme activity and increased immunoglobulin levels. The results further reaffirm the immunomodulatory benefits of curcumin, highlighting its promise as a functional dietary additive for promoting fish health and disease resistance in aquaculture systems.

[0142] Interestingly, between days 5 and 9 post-challenge, the EP group exhibited higher mortality than the control group. A similar trend was reported in a recent study on seabass, where dietary supplementation with a probiotic mixture comprising Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus led to increased mortality following V. anguillarum infection, compared to the control (Rimoldi, S., et al., Genetically superior European sea bass (Dicentrarchus labrax) and nutritional innovations: Effects of functional feeds on fish immune response, disease resistance, and gut microbiota. Aquaculture Reports, 2023. 33: p. 101747). Although the underlying mechanisms behind this negative effect remain unclear, it suggests that the benefits of probiotics are not universally consistent. Indeed, the other studies have demonstrated the positive effects of probiotic supplementation in enhancing fish survival during pathogen challenges (Gupta, A., P. Gupta, and A. Dhawan, Dietary supplementation of probiotics affects growth, immune response and disease resistance of Cyprinus carpio fry. Fish & Shellfish Immunology, 2014. 41 (2): p. 1 13-1 19; Jafarzadeh, F., et al., Harnessing paraprobiotics and postbiotics for enhanced immune function in Asian seabass (Lates calcarifer): Insights into pattern recognition receptor signaling. Fish & Shellfish Immunology, 2024. 151 : p. 109725). Therefore, the effectiveness of probiotics in aquaculture appears to be context-dependent, influenced by factors such as fish species, probiotic strains, pathogen type, and dosage (Mohammed, E.A., et al. The Significance of Probiotics in Aquaculture: A Review of Research Trend and Latest Scientific Findings. Antibiotics, 2025. 14, DOI: 10.3390 / antibi otics 14030242).

[0143] Overall, the results demonstrate that combined supplementation with probiotics and curcumin provides more effective immune protection than either additive alone. Notably, the EPFC and FPFC groups exhibited significantly lower mortality rates compared to the EP and control group. These findings are consistent with observations from the growth trial (Example 5.2), where combinatorial groups also exhibited higher survival than groups fed only probiotics or curcumin. This suggests a synergistic effect of probiotics and curcumin in enhancing fish immunity and disease resistance.

[0144] Conclusion

[0145] In this project, functional nutritional feeds were developed to enhance the growth and immunity of fingerlings, particularly against S. iniae and Vibrio. Through comprehensive screening, probiotics with strong anti-pathogenic properties were identified, and curcumin was optimized to enhance its anti-microbial properties against pathogens. To ensure efficient delivery, innovative encapsulation methods were implemented, using alginate matrices for probiotics. These techniques ensured feed stability during processing and allowed for controlled release. Feeding trials demonstrated the remarkable efficacy of these functional feeds, with encapsulated probiotics (EP) achieving the most significant growth improvements, including a 33% increase in body weight compared to control. All other groups, such as FC, FPFC, and EPFC, also exhibited significant growth and feed efficiency improvements against control. Notably, curcumin-supplement functional feed groups such as EPFC, FPFC and FC exhibited high survival rates during pathogen challenges, highlighting their effectiveness in strengthening resilience to infections. The EPFC and FPFC groups achieved higher survival rates than their individual counterparts (EP or FC) in the growth trial, and similar or higher survival in the challenge tests compared to EP and FC alone. This indicates a synergistic enhancement of immune function. However, the growth performance of EPFC was slightly lower than that of EP and FC, likely due to a metabolic trade-off in which energy was redirected from growth toward immune activation. Despite this, EPFC offers added value in disease- prone aquaculture settings where survival is a critical priority. Overall, these findings demonstrate the strong potential of functional feeds as a sustainable and scalable solution to boost aquaculture productivity while enhancing the health and resilience of farmed fish. By providing a natural alternative to antibiotics, this approach supports the ongoing shift toward more responsible and environmentally friendly aquaculture practices. To build on these promising results, future research should investigate the molecular mechanisms underlying these effects, particularly their interactions with host immunity and the gut microbiome, and focus on optimizing dosing strategies and feed formulations for practical field application.

[0146] Further aspects and embodiments of the invention are described in the following numbered statements.

[0147] 1 . An enhanced fish feed formulation comprising: a) Fish feed (e.g., see composition in Table 1 below); b) Probiotics; and c) A nutraceutical.

[0148] Table 1 . Filler is a mixture of all ingredients in the same proportion except the additives.

[0149] 2. The formulation according to Statement 1 , wherein the probiotics are encapsulated in Ca2+-crossl inked alginate microparticles.

[0150] 3. The formulation according to Statement 1 or 2, wherein the nutraceutical comprises curcumin.

[0151] 4. The process according to Statements 1 to 3, wherein the nutraceutical is encapsulated in microparticles formed from stearic acid (SA) and ethyl cellulose (EC). In addition to the individual advantages of each of the functional ingredients, their encapsulation and combinatorial supplementation in fish feed provides the following advantages.

[0152] 1. Encapsulation helps to protect the functionality of the additives, during storage as well as during digestion. While the combinatorial use of probiotics and curcumin, improves the efficacy synergistically, encapsulation can help to reduce the dosage by maximizing the functionality. This will be translated as reduction in cost of the additives used and marked improvement in fish health.

[0153] 2. Better disease resistance in fish through combinatorial supplementation 3. Increased survivability of the probiotics in the gastro-intestinal tract through encapsulation

[0154] 4. Preserved bioavailability of the nutraceutical and targeted delivery in the gastro-intestinal tract (GIT) of the fish through encapsulation.

[0155] 5. Early intervention with probiotics, along with an immunomodulatory nutraceutical, as a functional fish feed, stimulates growth rates of fingerlings, with sustained effects even in the grow-out phase.

Claims

CLAIMS1 . A fish feed additive composition comprising: one or more probiotics; and one or more nutraceuticals, wherein the one or more probiotics are encapsulated in a plurality of polymeric microparticles.

2. The fish feed additive composition according to Claim 1 , wherein the one or more probiotics are selected from one or more of the group consisting of Limosilactobacillus fermentum, Lentilactobacillus kefiri, Lentilactobacillus hilgardii, and more particularly, Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lactobacillus helveticus and Lacticaseibacillus rhamnosus.

3. The fish feed additive composition according to Claim 2, wherein the one or more probiotics are selected from one or more of the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus amylovorus, Limosilactobacillus oris, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Liquorilactobacillus satsumensis, Lentilactobacillus kefiri, Lactobacillus helveticus. Lentilactobacillus hilgardii and Lacticaseibacillus rhamnosus.

4. The fish feed additive composition according to Claim 3, wherein the one or more probiotics are selected from one or more of the group consisting of L. paracasei, L. plantarum, L. helveticus, L. rhamnosus (LGG).

5. The fish feed additive composition according to Claim 4, wherein the one or more probiotics is L. plantarum.

6. The fish feed additive composition according to any one of the preceding claims, wherein the one or more nutraceuticals is selected from the group consisting of a phytochemical (such as curcumin), a carotenoid (such as beta-carotene, and astaxanthin), a flavonoid (such as quercetin), a catechin, and an essential oil (such as thymol, and limonene).

7. The fish feed additive composition according to Claim 6, wherein the one or more nutraceuticals is curcumin.

8. The fish feed additive composition according to any one of the preceding claims, wherein the one or more probiotics are provided in an amount of from 105to 108CFU / g of feed, optionally wherein the one or more probiotics are provided in an amount of from 106to 107CFU / g of feed.

9. The fish feed additive composition according to any one of the preceding claims, wherein the one or more nutraceuticals are provided in an amount of from 0.05% w / w to 5% w / w of feed, such as from 0.1% w / w to 3% w / w, optionally wherein the one or more nutraceuticals are provided in an amount of 0.5% w / w of feed.

10. The fish feed additive composition according to any one of the preceding claims, wherein the mass ratio of the one or more probiotics to the one or more nutraceuticals is from 5:1 to 1 :1 , such as about 4:1 , such as about 3:1 , such as about 2:1 , optionally wherein the mass ratio of the one or more probiotics to the one or more nutraceuticals is about 2:1 .11 . The fish feed additive composition according to any one of the preceding claims, wherein: the one or more probiotics are provided in an amount of from 105to 108CFU / g of feed, wherein the one or more probiotics is L. plantarum and the one or more nutraceuticals are provided in an amount of from 0.05% w / w to 5% w / w of feed, wherein the one or more nutraceuticals is curcumin; optionally wherein: the one or more probiotics are provided in an amount of from 10® to 107CFU / g of feed, wherein the one or more probiotics is L. plantarum; and the one or more nutraceuticals are provided in an amount of from 0.1% w / w to 3% w / w of feed, wherein the one or more nutraceuticals is curcumin.

12. The fish feed additive composition according to any one of the preceding claims, wherein the microparticles encapsulating the one or more probiotics have a size of from 1 to 10 pm.

13. A fish feed composition comprising the fish feed additive composition according to any one of Claims 1 to 12.

14. The fish feed composition according to Claim 13, wherein the fish feed composition comprises: fish meal;wheat flour; wheat gluten; soybean meal; corn starch; sardine oil; vitamin mix; mineral mix; and filler / additives, where the fish feed additive composition forms part of the filler / additives.

15. The fish feed composition according to Claim 14, wherein the fish feed composition comprises: fish meal 10 wt%; wheat flour 20 wt%; wheat gluten 32 wt%; soybean meal 20 wt%; corn starch 6 wt%; sardine oil 8 wt%; vitamin mix 0.5 wt%; mineral mix 0.5 wt%; and filler / additives 3 wt%, where the fish feed additive composition forms part of the filler / additives.

16. The fish feed composition according to Claim 15, wherein the fish feed additive composition forms from 0.05wt% to 5 wt% of the fish feed composition, such as from 0.1 wt% to 3 wt%, optionally wherein the fish feed additive composition forms about 1 .5wt% of the entire fish feed composition.

17. The fish feed composition according to Claim 15 or Claim 16, wherein the one or more probiotics are provided in an amount of from 105to 108CFU / g of the fish feed composition, optionally wherein the one or more probiotics are provided in an amount of from 106to 107CFU / g of the fish feed composition.

18. The fish feed composition according to any one of Claims 13 to 17, wherein the fish feed composition is in the form of pellets.

19. Use of a fish feed additive composition according to any one of Claims 1 to 12, or a fish feed composition according to any one of Claims 13 to 18, in the manufacture of a medicament for the treatment or prevention of a bacterial infection.

20. Use of a fish feed additive composition according to Claims 1 to 12 or the fish feed composition according to Claim 13 to 18 for aquaculture of a fish.

Images