Selection of One Probiotic Bacterium and Assessment of its Effects on Digestive Enzyme activity of Young Pufferfish, Takifugu Rubripes

Bacterial Isolates

Healthy juvenile pufferfish (~1 g) were obtained from a local hatchery farm (Dalian Fugu Fishery Co. Ltd., Dalian, China). The intestines were removed from seven fish and homogenized in a sterile glass homogenizer with sterile physiological saline solution (0.9 g sodium chloride dissolved in 100 mL distilled water, pH 7.0). Appropriate dilutions were prepared and 0.01 mL volumes were spread over the surface of plates of ZoBell 2216E agar with incubation at 25℃ for 5–7 days. A total of 30 colonies were picked randomly from the plates. Pure cultures were obtained by repeated subculture on new agar plates and stored at –80℃ as suspensions in 30% (v/v) glycerol.

Enzyme-Producing Isolates

Thirty strains were inoculated into media containing varied substrates to screen isolates producing extracellular protease, amylase and lipase according to the methods of Zhao, et al. [11] and Zhang, et al. [12]. For proteolytic activity, the isolates were inoculated on casein supplemented agar plates and incubated at 28℃ for 72 h. Appearances of clear zones around the colonies were recorded. To assess amylolytic activity, the isolates were inoculated on soluble starch supplemented agar plates and were incubated at 28℃ for 72 h. Appearances of clear zones around the colonies were recorded after Lugol’s iodine solution was flooded on the culture plates. For lipolytic activity, the isolates were inoculated on Tween 80 supplemented agar plates and were incubated at 28℃ for 72 h. Appearances of opaque halos around the colonies were recorded.

Antagonism Study

The antagonistic activity of 30 test strains was examined against the indicator pathogenic bacterium V. harveyi following a method described by zhao, et al. [11]. Briefly, a culture of the indicator bacterium was spread on ZoBell

2216E agar plates, and then the test strains were inoculated on the plates. The plates were incubated at 28℃ and zones of inhibition around the test strains colonies were observed and recorded after 24 h.

Hemolytic Test

The bacterial isolates were spotted onto blood-agar plates and incubated at 28℃ for 24 h. The plates were observed for hemolytic reaction. Strains that produced no change on the agar plates around the colonies were considered to be non-hemolytic and strains displaying clean hemolysis zones around colonies were considered to be hemolytic (β-haemolysis).

Safety of a Probiotic Strain Evaluation

The safety of F15 was evaluated using healthy pufferfish (~22 g) by a challenge study. Pufferfish were acclimated to the rearing conditions for two weeks. Then pufferfish were distributed randomly into 2 plastic tanks (200 L), each tank containing 15 pufferfish. The bacterium was cultured in ZoBell 2216E broth at 25℃ with constant shaking until the early stationary phase before centrifuging at 1000×g for 10 min at 4℃, washing twice and re-suspending in 0.9% (w/v) saline. The pufferfish from one tank were intraperitoneally injected with 0.2 mL of F15 fresh culture suspension containing 109 cells mL-1 as determined by means of a haemocytometer slide. Rest one tank served as a control and injected with 0.2 mL sterile saline. All pufferfish were fed with basal diet containing the mixture of surimi from frozen Ammodytes personatus and fish meal (4:1, w/w), and kept under observation for 30 days, and the disease symptoms and mortality were recorded.

Feeding Trial

The safety test showed that F15 did not induce disease symptoms and mortality of fish. The F15 was cultured in ZoBell 2216E broth at 25℃ with constant shaking until the early stationary phase. The cellular suspension was centrifuged and the recovered pellet re-suspending in 0.9% (w/v) saline was incorporated into the diet. A basal diet containing the mixture of surimi from frozen A. personatus and fish meal (4:1, w/w) was used as a control. The experimental diets were prepared by supplementing graded doses of F15 at 107 and 109 cells g-1 of diet, respectively, which were prepared every day in order to guarantee the vitality of F15.

Young pufferfish were purchased from Dalian Tianzheng Industry Co. Ltd., Dalian, China. The pufferfish were transferred to the laboratory and acclimatized in a concrete pool filled with 25 m3 aerated filtered seawater at ~20℃ for

2 weeks. Then fish of similar sizes (22.13 ± 1.47 g, mean ± SD) were reared in 9, 200 L plastic tanks, at a density of 15 pufferfish per tank. Pufferfish were assigned to three groups (three tanks per group) and received the control diet (basal diet) and diets supplemented with F15 at 107 and 109 cells g-1. During the period of 2 weeks feeding, all pufferfish were fed with diets twice (08:00 and 14:00) daily at a rate of 2%–2.5% body weight. Uneaten feed residue and feces were removed by siphoning 1 h after each feeding. Water in each tank was replaced with fresh sand-filtered seawater twice a day. Aeration was provided to maintain dissolve oxygen levels at >5 mg L-1. Water temperature ranged from 20℃ to 24℃, salinity from 32.4 to 32.8 and acidity from pH 7.9 to 8.1. All experimental protocols were approved by the Experimental Animal Ethics Committee of Dalian Ocean University, China.

Sample Collection and Digestive Enzyme Assay

At the end of the feeding trial, pufferfish were starved for 48 h and anaesthetized administration of an overdose of MS-222. The intestines /hepatopancreas from six fish were removed, rinsed three times with sterile 0.9% (w/v) saline, pooled and immediately stored at −80℃ for further analysis. Frozen intestinal/hepatopancreas homogenates were thawed at 4℃ and homogenized in nine volumes of sterile 0.9% (w/v) saline using a manual glass homogenizer. The homogenates were then centrifuged and the supernatants were used for digestive enzyme activity analysis. The soluble protein content of the supernatants of intestinal/ hepatopancreas homogenates were measured according to the method described by Bradford [13] using bovine serum albumin as standard.

Pepsin, trypsin, amylase and lipase activities were measured by colorimetric analysis using commercial test kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions except that the temperature for the reaction was changed to room temperature (~25℃).

Statistical Analysis

Statistical analysis was performed using the SPSS 22.0 for windows. The differences in digestive enzyme activity between different diets were compared using one-way analysis of variance. If significance was detected, Tukey’s multiple range test was used to compare the means between groups. Differences were considered significant if P < 0.05.

Selection of Probiotic Bacteria

A total of 30 bacterial strains were isolated from the intestine of juvenile pufferfish. Strains F04, F05, F09 and F15 produced protease, amylase and lipase (Table 1). The in vitro antibacterial assay showed that the strain F15 induced an inhibition zone against V. harveyi (Table 1). Hemolysis was tested in four strains and the results showed that F15 did not secrete hemolysin. The intraperitoneal injection of F15 was thought to be harmless to the pufferfish although three pufferfish (one injected with suspension of F15, two injected with sterile saline) died during the 30 days of post- challenge observation. Nevertheless, no disease symptoms were found in the skin, intestine and liver tissues of the dead pufferfish. Thus, the cause of pufferfish death should be that the pufferfish kill each other during rearing. Pufferfish were ferocious in nature and mutual biting occurred from teeth growth of juveniles to adults [14]. F15 was identified as Pseudoalteromonas sp. by 16S rRNA gene sequencing [15].

Dh denotes diameter of hydrolysis zone; Dc denotes colony diameter; Di denotes diameter of inhibition zone Table 1: Qualitative extracellular enzyme activity (Dh/ Dc) and antagonistic activity (Di/Dc) produced by the bacterial strains from the intestines of juvenile pufferfish Takifugu rubripes.

Enzymatic Activity

After 14 days of feeding, pufferfish fed with F15 at 107and 109 cells g-1 of diet showed a significant increase in intestinal pepsin, trypsin, lipase and amylase activities compared with those fed the control diet (Table 2). Moreover, the intestinal pepsin and amylase activities of pufferfish fed the 109 cells g-1 F15 diet were significantly higher than those of pufferfish fed the 107 cells g-1 F15 diet (Table 2).

Means with different superscript lowercase letters in the same column differ significantly (P < 0.05) among different groups. Data represents means ± SDs (n = 3). Table 2: Intestinal digestive enzyme activity of pufferfish fed with control diet and F15-supplemented diets at 107 and 109 cells g-1 for 14 days.

After 14 days of feeding, all pufferfish fed the F15-supplemented diets had a significant enhanced hepatopancreas pepsin, trypsin, lipase and amylase activities as compared with the controls (Table 3). Moreover, there were significant differences in four digestive enzymes activities in pufferfish fed with two doses of F15.

Means with different superscript lowercase letters in the same column differ significantly (P < 0.05) among different groups. Data represents means ± SDs (n = 3). Table 3: Hepatopancreas digestive enzyme activity of pufferfish fed with control diet and F15-supplemented diets at 107 and 109 cells g-1 for 14 days.