Evaluation of the synbiotic effects of Saccharomyces cerevisiae and mushroom extract on the growth performance, digestive enzyme activity, and immune status of zebrafish danio rerio

Abstract

Background: The quest for candidate probiotics and prebiotics to develop novel synbiotics for sustainable and profitable fish farming remains a major focus for various stakeholders. In this study, we examined the effects of combining two fungal probiotics, Saccharomyces cerevisiae and Aspergillus niger with extracts of Jerusalem artichoke and white button mushroom to develop a synbiotic formulation to improve the growth and health status of zebrafish (Danio rerio). An initial in vitro study determined the most effective synbiotic combination, which was then tested in a 60-day in vivo nutritional trial using zebrafish (80 ± 1.0 mg) as a model animal. Four experimental diets were prepared: a control diet (basal diet), a prebiotic diet with 100% selected mushroom extract, a probiotic diet with 10⁷ CFU of S. cerevisiae/g of diet, and a synbiotic diet with 10⁷ CFU of S. cerevisiae/g of diet and 100% mushroom extract. As readouts, growth performance, survival, digestive enzyme activity and innate immune responses were evaluated.

Results: In vitro results showed that the S. cerevisiae cultured in a medium containing 100% mushroom extract exhibited the maximum specific growth rate and shortest doubling time. In the in vivo test with zebrafish, feeding them with a synbiotic diet, developed with S. cerevisiae and mushroom extract, led to a significant improvement in the growth performance of zebrafish (P < 0.05). The group of zebrafish fed with the synbiotic diet showed significantly higher levels of digestive enzyme activity and immune responses compared to the control group (P < 0.05).

Conclusion: Taken together, these results indicated that the combination of S. cerevisiae and mushroom extract forms an effective synbiotic, capable of enhancing growth performance and immune response in zebrafish.

Keywords: Funal probiotics; Growth; Immunity; Natural extract; Probiotic; Zebrafish.

Fig. 1

Growth of Saccharomyces cerevisiae and Aspergillus niger in presence of mushroom or artichoke extract. Growth curve of A. niger and S. cerevisiae in medium containing mushroom or artichoke extract (2, 6.25, 12.5, 25, 50, 75 and 100%) as the only carbon source. A and B: growth of the two fungal strains in presence of artichoke extract. C and D: growth of the two fungal strains in presence of mushroom extract. Data are the mean of three replicates (n = 3). Positive control: Sabouraud dextrose broth medium with the standard amount of glucose; negative control 1: Free glucose Sabouraud dextrose broth medium containing a concentration of 100% of plant extract without inoculation fungi; negative control 2: Free glucose and plant extract (as a prebiotic) Sabouraud dextrose broth with fungi

Fig. 2

Comparison of (A) µ max (h^− 1) and (B) doubling time (h) and (C) final pH of the synbionts in presence of plant extract. A. niger and S. cerevisiae were culture in 100% of mushroom and artichoke extracts. Data are the mean of three replicates ± SE. Different letters display significant difference in each column (P < 0.05)

Fig. 3

Growth curve of synbionts in presence of artichoke or mushroom extract. (A) Growth of S. cerevisiae in medium containing 100% Jerusalem artichoke or mushroom extracts as an only carbon source. For these symbionts, visible colonies could be counted (B) Growth of A. niger in medium containing 100% Jerusalem artichoke or mushroom extracts as an only carbon source. For this experiment, colonies were not visible, therefore, dilutions series were made and dry weight was measured as an indicator for the growth of this fungus

Fig. 4

Growth performance and survival of zebrafish (Danio rario) fed with different supplementation diets for 60 days. (A) Initial length (mm), (B) final length (mm), (C) Initial body weight (mg), (D) final body weight (mg), (E) Weight gain percentage (%), (F) Specific growth rate (% day^− 1), (G) Feed conversion ratio, (H) Protein efficiency ratio, (I) Feed intake (g), (J) K Factor (mg mm³), K) Survival (%). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)

Fig. 5

Digestive enzyme activity of zebrafish fed with different diets for 60 days. The group fed no feed supplement was maintained as control; (A) Amylase (U g protein^− 1), (B) Protease (U g protein^− 1). B) Lipase (U g protein^− 1). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)

Fig. 6

Metabolic enzymes in zebrafish fed with different diets for 60 days. The group fed with no feed supplement was maintained as control; (A) ALP (U L^− 1), (B) ALT (U L^− 1), (C) AST (U L^− 1). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)

Fig. 7

Stressed indicators of zebrafish fed with different diets for 60 days. A group without site supplement was maintained as control; (A) Glucose (mg mL^− 1), (B) Cortisol (µg mL^− 1). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)

Fig. 8

Non-specific immunity responses in zebrafish fed with different diets for 60 days. A group without site supplement was maintained as control; (A) Total protein (g dL^− 1), (B) Total immunoglobulin (g dL^− 1), (C) Albumin (g dL^− 1), (D) Lysozyme activity (U mg protein^− 1). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)

Fig. 9

Immunity responses in zebrafish fed with different diets for 60 days. A group without site supplement was maintained as control; (A) Total protein (g dL^− 1), (B) Total immunoglobulin (g dL^− 1), (C) Albumin (g dL^− 1), (D) Lysozyme activity (U mg protein^− 1). Bars with different letters represent significant differences among groups (Duncan’s test, P < 0.05)