Effect of Feeding Bacillus sp. As Probiotic Bacteria on Growth of Giant Freshwater Prawn (Macrobrachium rosenbergii de Man)

MATERIALS AND METHODS

Experimental conditions: The experiment was run for 120 days at the Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand. The experiment was divided in four experimental groups, each with triplicates, in 12 concrete tanks (1.50x2.00x1.00 m). The total water in each tank was maintained at 1,500 L and aeration was continuously provided to all the containers. All the tanks were cleaned and about 30% of water was replaced with fresh water every week.

The details of the experimental groups were as follows: T₁ Commercial diet + Bacillus KKU03; T₂ Commercial diet +Bacillus KKU02; T₃ Commercial diet +Bacillus KKU02 +Bacillus KKU03 and T₄ Commercial diet (control). The commercial feed used was from Charoenpokapan Company, Samut Sakhon, Thailand (CP No. 9041) The feed was autoclaved at 110°C 15 min before mixing with the cultures.

Bacterial strains and feeding regime: Bacillus KKU 03 and Bacillus KKU 02 used as probiotics in this research were obtained from the culture collection, Department of Biotechnology, Khon Kaen University. The isolated Bacillus sp. were grown in nutrient broth (NB) for 24 h at 37°C on a shaker at 150 rpm to a final concentration of about 10⁷ CFU mL^-1 and mixed at the rate of 200 mL to 1 kg feed. Prawns were fed twice daily, at 8.00 and 16.00 h. The daily feeding rate was ~10% of total body weight.

Preparation of experimental animals: Post larvae of giant freshwater prawn (PL-15) were obtained from a hatchery located in Supanburi, Thailand. The prawns were acclimatized in the tanks for 2 weeks feeding with only commercial feed. Two hundred PL-15 of uniform size (0.11-0.12 g) were kept in each tank.

Analysis of samples: Ten randomly collected live prawns from each tank were measured for lengths and weights once every 30 days.

Water quality was checked weekly for pH, dissolved oxygen and temperature. The amount of ammonium, nitrate, nitrite and total alkalinity were also determined by test kits obtained from Prima Tech Company, Bangkok, Thailand.

Statistical analysis: One-way analysis of variance (ANOVA) to determine any significant differences among the treatment groups and comparison between four treatments were done using Duncan’s Multiple Range Test (DMRT) using SPSS for window XP.

Longevity of the probiotics in prawn feed: Aerobic plate counts were performed on nutrient agar plates and incubated at 37°C for 24 h for comparison of the test feeds and the commercial probiotic mixed feed. The commercial probiotic (Biopro®) was obtained from M.D. Synergy Co. Ltd., Bangkok, Thailand. The probiotic (10⁷ CFU mL^-1) was mixed in the feed at the rate of 200 mL to 1 kg feed.

Enzyme production by the probiotic cultures: Amylase, lipase and protease production by the selected cultures, Bacillus KKU02 and Bacillus KKU03, were tested using starch agar, tributyrin agar and skim milk agar, respectively.

The formulations of agars were as follows:

Starch agar: Soluble starch (Diffco) 10.0 g L^-1, beef extract 3.0 g L^-1 (BDH), agar 12.0 g L^-1 adjust volume by deionised water to 1,000 mL, pH 7.5.

Tributyrin agar: peptone from meat (Oxoid) 2.5 g L^-1, proteose peptone (Oxoid) 2.5 g L^-1, yeast extract (Diffco) 3.0 g L^-1, agar 12.0 g L^-1, tributyrin (Oxoid) 100 mL. Adjust volume to 1 L by deionised water, pH 7.5.

Skim milk agar: skim milk 100 g L^-1, agar 20 g L^-1 adjust volume to 1,000 mL by deionised water, pH 6.3.

The strains were grown in nutrient broth at 37°C on a shaker at 150 rpm for 12 h before testing by the well diffusion method and by streaking on the specific agar plate. One hundered milliliter of cultured medium were loaded into each well (diameter 7 mm). The plates were held at room temperature for 24-48 h (72 h for tributyrin agar). After growth, the starch agar plates were flooded with iodine solution.

RESULTS AND DISCUSSION

Prawn production: There were significant differences (p = 0.05) of body weight and length gain between T₃ and the control group during probiotic feeding (Fig. 1A and B). The prawns fed mixed culture exhibited the highest Figures at 3.325 g and 7.487 cm, respectively. However, the probiotic treatment groups T₁ and T₂ were not significantly different from each other (p≥0.05) or the control group. There have been previous reports that using a mixed culture feed resulted in better shrimp growth. Thimmalapura et al. (2002) investigated shrimp (Penaeus monodon) production in ponds treated with two commercial mixed microbial products. There were significant differences in feed conversion ratio in the pond treated with a product containing Bacillus sp. and Saccharomyces sp. compared with ponds treated with Nitrosomonas sp. and Nitrobacter sp. and untreated controls.

Fig. 1:

The body weight (A) and length (B) of Giant freshwater prawn after rearing for 120 days with isolated bacteria in four feed treatments : ♦ = T₁, (Bacillus KKU03); ♦ = T₂, (Bacillus KKU02); € =T₃ (Bacillus KKU02 + Bacillus KKU03) and ▲ = T₄, Control

Table 1:	Ranges of water quality parameters during experiments

Table 2:	Enzyme production of Bacillus KKU02 and Bacillus KKU03

Likewise, Gatesoupe (1991) reported a significant difference in turbot larvae weight when feeding with bioencapsulated Lactic acid bacteria (LAB) and Bacillus toyoi.

Water quality: The water quality in each shrimp culture ponds is shown in Table 1. Saxena (2003) reported that the optimum parameters for giant freshwater prawn culture were pH = 7.5-8.5, Temperature = 25-32°C, Total alkalinity concentration = 50-150 mg L^-1, DO (Dissolved oxygen) = 4 mg L^-1, Ammonia concentration = 0.1 mg L^-1, Nitrite concentration = 0.1 mg L^-1. Our results revealed that all of the measured parameters were in the acceptable ranges suggested by Amstrong et al. (1976) Hsieh et al. (1990) and New (1990). However, the season changed to winter 60 days into the experiment and the water temperature was slightly low at 24°C for two weeks and then increased.

Enzyme production by the isolates: Both the probiotic strains gave positive results in starch agar (amylase) and skim milk agar (protease) but lipase activity was not detected in tributyrin agar (Table 2). These extracellular enzymes are also the main enzymes in the digestive tract involved in breaking down organic material in the feed matter. Increased growth yield of shrimps might be partly the result of better digestion and assimilation of nutrients in the feed resulting from the exoenzymes produced by the modified gut microflora. Bacteria, by virtue of their exoenzymes, have been reported to play an important role in the process of digestion in turbot larvae (Munilla-Moran et al., 1990). However, an alternative explanation for the increased yields observed in the present trials might be the suppression of pathogenic micro-organisms in the gut by the probiotic strains.

Shelf life of the probiotic in prawn feed: The stability of the pure Bacillus and mixed Bacillus spp. and commercial probiotic were examined in the feeds, which were stored at 4°C and room temperature (Fig. 2). The viability of all the probiotics was detected consistently for 70 days. It was found that the % viability of Bacillus KKU03, Bacillus KKU02 and mixed culture stored at room temperature declined dramatically to 21.88, 2.54 and 10.92% within 2 weeks respectively. Under 4 °C storage, however, the % viabilities of the tested probiotics were reduced more slowly and the Bacillus KKU03 behaved similarly to the commercial probiotics. The percentage survival of the commercial probiotics was the same at both temperatures. The average percentage viability of the commercial probiotic was about 50% after 70 days’ storage. It appears that the commercial preparation is more stable at room temperature than Bacillus KKU02. This was possibly because it has been formulated for having a longer shelf life. Before commercialization can be undertaken, it is important to study and optimize the extended survival of the Bacillus KKU02 probiotics in shrimp feed.

Fig. 2:	Viability of probiotics in prawn feed stored at 4°C and room temperature for 70 days. (A) Bacillus KKU02; (B) Bacillus KKU03; (C) Bacillus KKU02+Bacillus KKU03; (D) commercial probiotic

CONCLUSIONS

The results from these experiments indicate that a significant improvement of growth of M. rosenbergii occurred when the feed included a mixed culture of Bacillus KKU02 and Bacillus KKU03 previously isolated from the intestinal contents of shrimps. There was no difference between the groups fed single strain probiotics and the controls. However, the optimum concentration ratio of these probiotics in the prawn feed is still needed to be determined. These results should also be confirmed in outdoor, earthen pond trials before they are applied commercially. Further investigation of the effect of probiotics on the prawn immune system and a risk assessment to human consumers is continuing and will be reported in a future publication.

ACKNOWLEDGMENT

The authors would like to thank the Thailand Research Fun (TRF), Thailand Toray Science Foundation (TTSF) and the Agro-industry Consortium of Thailand for the financial support of this research. Thank you to the Department of Biotechnology, Khon Kaen University; Department of Biotechnology, Maha Sarakham University; Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand and the Fermentation Research Center for value added agricultural products, Khon Kaen University, Thailand for providing facilities for this research.