Dietary supplementations with novel prebiotics, probiotics, and synbiotics for the modulation of growth, cellular and humoral immunity, and disease resistance in olive flounder (Paralichthys olivaceus)

Abstract

Olive flounder (Paralichthys olivaceus) is commercially cultured in China, Japan, and the Republic of Korea. Antibiotics used in flounder aquaculture produce antibiotic-resistant pathogens and residual effects on human. Prebiotics, probiotics, and synbiotics are considered natural functional food ingredients and an alternative feeding strategy for immunomodulation and antibiotic eradication. Prebiotics are non-digestible oligosaccharides, fermented by intestinal microbiota to produce short-chain fatty acids that bind G protein receptors. Probiotics are usually incorporated into the diet at a certain concentration to alter the intestinal microbial population through colonisation or implantation. Synbiotics are combinations of pre- and probiotics that produce synergistically better benefits than separately. β-Glucooligosaccharides (BGO), were fed (0.1%) to olive flounder (14 ± 0.5 g) for 8 weeks to identify the prebiotic potentials. The results demonstrated that the BGO diet produced higher levels (P < 0.05) respiratory burst (RB), lysozyme (LSZ), and superoxide dismutase (SOD) compared to control. Fish challenged with Streptococcus iniae (1×108 CFU mL1), showed a significantly (P < 0.05) lower death rate compared with that of the control. Olive flounder was also dietary supplemented with 1×108 CFU g1 Lactococcus lactis subsp. lactis I2 (LI2) with 0.1% BGO (LI2 + BGO) for 8 weeks, to quantify the synbiotic effects on the growth and immunity. Results showed the synbiotic diet had significantly better (P < 0.05) responses in terms of weight gain (WG) and specific growth rate (SGR), RB, LSZ, and SOD, intestinal LAB viability, and the relative tumor necrosis factor (TNF)-α expression in liver and spleen. Challenged with S. iniae, the synbiotically fed group exhibited higher (P < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Bacillus sp. SJ-10 (BSJ-10) and the previously recognized prebiotic BGO and their combination as a symbiotic were supplemented to olive flounder (10 ± 0.25 g) as neither BSJ-10 nor BGO (control), 1 × 108 CFU g1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 108 CFU g1 BSJ-10 + 0.1% BGO (BSJ-10+BGO) for 8 weeks, to quantify BSJ-10 and BSJ-10+BGO probiotic and synbiotic activities respectively. Fish WG, SGR, feed conversion ratio, and protein efficiency ratio (PER) in BSJ-10, BGO and BSJ-10+BGO diets were modulated (P < 0.05) verses control. Specially, WG and SGR were significantly higher in BSJ-10+BGO than that of BSJ-10 and BGO. The RB, SOD, and LSZ of fish fed BSJ-10 and BSJ-10+BGO were significantly higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10+BGO were statistically higher compared with individual components but microvillus length was increased only in BSJ-10+BGO relative to control. During S. iniae challenge (1 × 108 CFU mL–1), survival rate was significantly higher in all treatment groups versus control. Moreover, in BSJ-10+BGO, protection against S. iniae infection and transcription of TNF-α and interleukin (IL)-6 in gill were significantly higher than the individual component. Heat-killed (HK) 1×108 CFU g–1 BSJ-10 (HKBSJ-10) probiotic was supplemented to olive flounder (~ 9.64 g) for 8 weeks, to identify HK probiotic effects on the growth and immunity. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced WG and PER, 1.17 and 1.11 folds respectively. LZS and SOD in HKBSJ-10 group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1β, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with S. iniae, the HKBSJ-10-fed group exhibited significantly higher protection against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Flounders (~ 14.91 g) were divided into 6 groups and fed with control diet, D-1: [without BSJ-10 and Lactobacillus plantarum (LP)]; positive controls, D-2: (1×108 BSJ-10) and D-3: (1×108 LP); and treatments, D-4: (0.75 BSJ-10 + 0.25 LP) × 108, D-5: (0.50 BSJ-10 + 0.50 LP) × 108, and D-6: (0.25 BSJ-10 + 0.75 LP) × 108 CFU g1 diet for 8 weeks to estimate probiotics mixtures effects on growth and innate immunity. Group D-4, demonstrated better (P < 0.05) growth and feed utilizations parameters (FUP) compared to control and positive controls. Similar modulation was also dipicted in RB in all treatments, and expression of TNF-α, IL-1β, IL-6, and IL-10 in different localized organs in D-4. Moreover, D-4 and D-5 increased RB, SOD, LZS, and MPO verses control, but only D-4 increased microvillus length. Challenged with S. iniae (1×108 CFU mL1), fish in D-4 and D-5 were survived at certain percentages up to 14 days, whereas other group’s 100% mortality reached earlier / at 11.50 days. Therefore, after these dietary supplementation experiments prebiotic potential of BGO; probiotic activities of BSJ-10, HKBSJ-10, and ratio specific BSJ-10 and LP mixtures; and synbiotic actions of BGO + LI2 and BGO + BSJ-10 in olive flounder, positively modulated growth and FUP, increased cellular and humoral immunity, elevated cytokine genes transcriptions in different localized organs, and protected fish against streptococcosis.