Growth Promoting Potential and Colonization Ability of Probiotics ( Bacillus coagulans and Bacillus subtilis ) on the Freshwater Prawn Macrobrachium rosenbergii Post-Larvae

The probiotic effects of Bacillus coagulans and Bacillus subtilis were studied on survival, growth, concentrations of basic biochemical constituents, activities of digestive enzymes, and their colony establishments in the gut of Macrobrachium rosenbergii post-larvae (PL). Eleven groups of PL (2.03±0.05 in length and 0.18±0.01g in weight), each consists of 35 individuals maintained in 25 L of ground water and fed ad libitum with ﬁ ve serially diluted concentrations, 10 -1 , 10 -3 , 10 -5 , 10 -7 and 10 -9 of B. coagulans , and B. subtilis incorporated diets containing 40% protein, for 45 days. Diet without incorporation of any of these probiotics was served as control. These probiotics were found to be alive in the respective feed even on day-15 after their formulations. Signi ﬁ cant improvement in survival, nutritional indices (weight gain, speci ﬁ c growth rate, food conversion ratio and protein ef ﬁ ciency ratio), contents of basic biochemical constituents (total protein, amino acid, carbohydrate and lipid) and activities of digestive enzymes (protease, amylase and lipase) were observed (P<0.05), particularly in 10 -7 concentration of B. coagulans , and B. subtilis incorporated diets fed PL when compared with control. The biochemical con ﬁ rmation tests revealed that presence of Escherichia coli , Acetonobacter sp., Salmonella sp., and Pseudomonas sp., in the gut of control PL. In the gut of PL fed with B. coagulans incorporated diet, Acetonobacter sp., Salmonella sp., and Pseudomonas sp., were found to be competitively excluded, whereas, in the gut of PL fed with B. subtilis incorporated diet, Acetonobacter sp., and Salmonella sp., only were found to be excluded competitively. Actually, colonies of Bacillus sp., and Lactobacillus sp., were found to be establishment in the gut of PL fed with B. coagulans , and B. subtilis incorporated diets. Overall, these probiotics incorporated diets produced better growth and survival due to better FCR and activities of digestive enzymes, which in turn led to better nutritional pro ﬁ le. Therefore they are recommended as feed additives for sustainable culture of M. rosenbergii .

In the present study, B. coagulans and B. subtilis was individually incorporated at different concentrations with formulated diets and fed to M. rosenbergii PL for assessing their ability on promotion of growth and survival by enhancing the contents of basic biochemical constituents (total protein, amino acid, carbohydrate and lipid), and activities of digestive enzymes (protease, amylase and lipase). This was further to recommend the aquaculture industry with their optimum concentrations. Furthermore, to evaluate their competitive exclusion abilities of pathogenic bacteria, their colony establishments in the gut of PL were con irmed biochemically.

Procurement of Macrobrachium rosenbergii PL and acclimation
The post larvae (PL-20) of M. rosenbergii were procured from the Nursery pond at Singanallur, Coimbatore, Tamil Nadu, India. They were transported to the laboratory in polythene bags half illed with oxygenated pond water. They were then acclimated to the ambient laboratory condition in cement tanks (6×3×3 feet) illed with groundwater for 2 weeks. The ground water satis ied the required physico-chemical parameters (Table 1).
During acclimation the prawns were fed with boiled egg albumin, live Artemia nauplii, and commercially available scampi feed. About 50% of tank water was routinely renewed every day to maintain a healthy environment. Aeration was also provided. These ensured suf icient oxygen supply to the prawns and an environment devoid of accumulated metabolic wastes. The unfed feeds, feces, molt and dead prawns were removed by siphoning.

Procurement of probiotics and their sub-culture
The lyophilized powder of Bacillus coagulans (MTCC 2302) and Bacillus subtilis (MTCC 121) were procured from Microbial Type Culture Collection (MTCC), Chandigarh, India. It was subjected to sub-culture with nutrient broth (Hi-media, India) containing the following ingredients as per manufacturer's protocol (Table 2) and incubated at 37 °C for 12 hours to observe their growth. Agar (12.0 g L -1 ) was added with the nutrient broth, and the agar plates were incubated for 24 h at 37°C for checking the sterility. Then 20 μL broth culture of B. coagulans, and B. subtilis were separately spread over the agar media (nutrient+agar) and kept for 24 h at 37 °C to observe their growth ( Figure 1).

Feed preparation
The branded feed ingredients, included ishmeal, soybean meal, groundnut oilcake, wheat bran, tapioca lour, cod liver oil and egg were purchased from a local market. A Vitamin-B complex with vitamin-C purchased from a local pharmacy was also added. The experimental diets were prepared with selected feed ingredients as per "Pearson's square-method" using pre determined value of 45% protein content. Fishmeal, soybean meal and groundnut oilcake were used as protein sources; wheat bran and tapioca lour were used as carbohydrate sources; sun lower oil was used as lipid source; tapioca lour and egg albumin were served as binding agents; vitamin B complex with vitamin C was also added as essential micronutrients ( Table 3).
The proportion of each ingredient required was calculated precisely for the premix along with tapioca powder, stream cooked and cooled at room temperature (28ºC). Egg albumin, sun lower oil, and vitamin B-complex with vitamin-C were then added one by one. B. coagulans, and B. subtilis were separately incorporated into the basal diet at 10 -1 , 10 -3 , 10 -5 , 10 -7 and 10 -9 concentrations, each (diet-1 to diet-10). Diet '0' (without incorporation of any probiotic served as control. The dough was prepared for each formulation and pelletized separately to 2 mm pellets. These were dried in a thermostatic oven (M/s. Modern Industrial, Mumbai, India) at 40ºC until they reached constant weight, and stored in airtight jars at room temperature (28ºC). The proximate composition of organic matters was determined according to AOAC [22]. The feeds were then placed in a hot air oven at slightly >100ºC and the loss of weight determined as the moisture content. The concentrations of minerals were estimated by using atomic absorption spectrophotometer (AAS) method ( Table 4).

Viability of probiotics in the respective diets
The diet was freshly prepared once every 15 days to ensure high probiotic viability throughout the feeding trail. One gram of each diet prepared with10 -7 diluted probiotic  concentration was taken and dissolved in autoclaved double distilled water (10 mL). A volume of 20μL was spread over MRS agar medium, incubated at 37ºC for 24 h and the colony morphology observed was compared with the original B. coagulans, and B. subtilis sub-cultures morphology. The growth was recorded in all the culture plates except control. Therefore, the incorporated B. coagulans, and B. subtilis were alive in their respective diets even on day-15 after their formulation ( Figure 2). Table 3: Ingredients used to formulate the basal diet.

Fish meal 25
Groundnut oil cake 25

Feeding trial
M. rosenbergii PL-35 (2.03±0.05 in length and 0.18±0.01g in weight) were acclimated in plastic aquaria for three days during which they starved for 24 hr before the commencement of the experiment. The feeding trail lasted 45 days. Four groups of 35 prawns were placed in 25 L aquaria in triplicate groups. The experimental groups were fed with the respective concentrations of B. coagulans, and B. subtilis incorporated feeds twice a day (8:00 AM and 8:00 PM) at 10% of body weight. All the water was renewed daily and aerated constantly. The uneaten food particles, faeces, moults and dead prawns were removed by siphoning.

Analysis of nutritional indices
On the inal day of feeding trial the morphometric data, such as the inal length and weight were measured for calculating the growth parameters, such as survival rate (SR), Length gain (LG), weight gain (WG), speci ic growth rate (SGR), food conversion ratio (FCR), food conversion ef iciency (FCE) and protein ef iciency ratio (PER) [23]. SR (%)=Total No. of PL alive at the end of experiment/ Total No. of PL introduced at the beginning of the experiment×100.

Estimation of concentrations of biochemical constituents
On the inal day, the concentrations of basic biochemical constituents were determined. Concentration of total protein was estimated by the method of [24]. The concentration of total amino acid was analyzed by according to Moore and Stein [25]. The concentration of total carbohydrate was estimated by the method of Roe [26]. The concentration of total lipid was determined according to [27] and estimated by the method of [28]. For these parameters, tissues from ive prawns were pooled together from each group to constitute a single observation and three such observations were made to ful ill the triplicate analysis.

Assays of digestive enzymes activities
Activities of digestive enzymes were assayed before and after the feeding trial.
The digestive tract of three prawns from each replicate were carefully dissected and homogenized in ice-cold distilled water and centrifuged at 9300 g under 4ºC for 20 min. The supernatant was used as a crude enzyme source. Total protease activity was determined by casein-hydrolysis method of Furne et al. [29], where one unit of enzyme activity represented the amount of enzyme required to liberate 1μg of tyrosine per minute. Amylase activity was determined according to Bernfeld et al. [30], and the speci ic activity of amylase was calculated as milligrams of maltose liberated per gram of protein per hour (mg/g/h). Lipase activity was determined by the method of Furne et al. [29]. One unit of lipase activity was de ined as the amount of free fatty acid released from triacylglycerol per unit time estimated by the amount of NaOH required to maintain pH constant and represented as milli equivalents of alkali consumed.

Analysis of gut microbial colonization
The gut of control PLs, and the gut of experimental PLs fed with the best concentration of B. coagulans, and B. subtilis supplemented diet (10 -7 dilution of each probiotic) were subjected to bacterial analysis. The PLs were deactivated by kept them in freezer at -20ºC for 10 minutes. The PL surface was sterilized with 50 ppm formalin for 30 seconds to remove the external lora. The digestive tract was dissected and homogenized with phosphate buffered saline (pH-7.2) under aseptic condition. The homogenate was serially diluted up to 10 -4 dilution. A volume of 0.5 mL of aliquot was mixed with agar nutrient broth and cultured for 24 h at 35ºC. Then, 0.1 mL broth culture was seeded over the surface of freshly prepared nutrient agar plates and incubated at 37 ºC for 24 h. The appearance of different bacterial colony was identi ied and con irmed through routine bacteriological tests [31]. The bacterial colonies were enumerated [Bacteria count (CFU/g)=Number of colonies×Dilution factor/ Volume of sample (g)].

Statistical Analysis
One way analysis of variance (ANOVA) using SPSS package (version 20.0) was used to determine the variations between control and treatment values, and between treatments, followed by Duncan multiple range test (DMRT) and the signi icances at P<0.05 are mentioned. The data are represented as means±standard deviations.

Growth and nutritional indices
The survival, growth (LG and WG), speci ic growth rate, protein ef iciency ratio were signi icantly increased in the prawns fed with B. coagulans, and B. subtilis supplemented diets when compared with control (P<0.05). Among the different concentrations, 10 -7 dilution of each probiotic incorporation was produced the best performance on these parameters. The lowest FCR recorded in this concentration of B. coagulans, and B. subtilis incorporations re lected the superior quality of the diets formulated (Tables 5 and 6).

Concentrations of basic biochemical constituents and activities of digestive enzymes
The concentrations of total protein, amino acid, carbohydrate and lipid were signi icantly increased (P<0.05) in test prawns fed with B. coagulans, and B. subtilis supplemented diets when compared with control (Tables 7 and 8). These were because of the signi icant increases (P<0.05) in activities of protease, amylase and lipase (Tables  7 and 8), which ultimately produced better digestion and absorption of nutrients. Among the different concentrations, 10 -7 dilution of each probiotic incorporation was produced the best performance on these parameters, which in turn responsible for better survival and growth of M. rosenbergii.

Conclusion
Overall, these probiotics incorporated diets produced better growth and survival due to better FCR and activities of digestive enzymes, which in turn led to better nutritional pro ile. B. coagulans, and B. subtilis incorporated diets possessed the ability of competitively exclude Acetonobacter sp., Salmonella sp., and Pseudomonas sp., and Acetonobacter sp., and Salmonella sp., respectively. Therefore they are recommended as feed additives for sustainable culture of M. rosenbergii.