Growth and survival promotion of a probiotic bacterium Enterococcus durans enriched Artemia nauplii on the prawn Macrobrachium rosenbergii

This work accentuate the growth performance of Macrobrachium rosenbergii post larvae (PL) fed with a probiotic bacterium, Enterococcus durans enriched live feed Artemia franciscana nauplii at five different serially diluted concentrations (10-1, 10-3, 10-5, 10-7 and 10-9). After 45 days of the feeding trial, the survival rate, growth rate, activities of protease, amylase and lipase, and contents of total protein, amino acid, carbohydrate and lipid were significantly (P<0.05) increased, particularly at 1071×10-7 CFU. The gut microflora of un-enriched Artemia nauplii fed prawns showed the presence of Escherichia coli, Klebsiella sp., Citrobacter sp., Acinetobacter sp., Streptococcus sp., Bacillus sp., Staphylococcus sp., and Pseduomonas sp. The optimized concentration of E. durans enriched Artemia nauplii fed prawns showed the presence of Enterococcus sp., E. coli, Bacillus sp., and Klebsiella sp. The pathogenic bacteria, Citrobacter sp., Acinetobacter sp., Streptococcus sp., Staphylococcus sp., and Pseduomonas sp., were competitively excluded in the gut of prawns due to the colony establishment of Enterococcus sp., Hence, E. durans can be used as a enrichment material of Artemia nauplii for sustainable culture of M. rosenbergii.


Introduction
Aquaculture is one of the fastest growing food producing sectors in the world. It offers proteinaceous food supply to fight against malnutrition, poverty and considerably generate employment opportunity to improve rural economy. In recent years both seafood production and overall worldwide consumption has increased considerably [1]. Among crustacean, prawns provide protein, vitamins, minerals and nutritious delicacy for human consumption [2].The growth of aquaculture has resulted in environmental damages, which can be minimized through sustainable practices. The use of antibiotics and chemicals can be reduced and the outbreaks of diseases can be prevented by applications of probiotics [3,4].
The genus Enterococcus is the most controversial group of lactic acid bacteria. Enterococci are ubiquitous microorganisms, widely distributed in nature (water, plant, soil, foods, and gastrointestinal tract of humans and animals) and considered as bacteria of low pathogenicity. Enterococcus durans, Enterococcus faecium, Enterococcus faecalis and Enterococcus avium produces antimicrobial bacteriocins called enterocins, they used in food biopreservation, play an important role in the ripening of cheeses, probably through proteolysis, lipolysis, and citrate breakdown. Enterococci are nowadays have shown a very appreciable probiotic characteristics in animal nutrition and used as potential probiotic candidates in aquaculture, live cells with different beneficial characteristics such as stimulation of immunity, anti-inflammatory activity, hypocholesterolemic effect, and prevention/treatment of some diseases in hosts. At the same time, Enterococci have been associated with a number of human infections [31][32][33][34][35].
It has been reported that high ammonia and high dissolved oxygen in water decreased the virulence of Enterococcus in Macrobrachium rosenbergii culture medium [36,37], and thus, Enterococcus can be used as probiotics. Enterococcus genus is isolated from the intestine of common carp and freshwater prawn M. rosenbergii [38]. Enterococcus gallinarum and Enterococcus hirae have proved with probiotic functions in M. rosenbergii [39,40] when ingested in sufficient quantities. E. durans possessed quorum quenching activity (ability to grow in low pH) and to produce antimicrobial agents, hence, this bacterium could be used for therapeutics [31,41]. Therefore in this study, E. durans was used as a probiotic bacterium to enrich live Artemia nauplii for feeding M. rosenbergii PL in order to assess the ability of E. durans to increase the survival and growth, activities of digestive enzymes (amylase, protease and lipase) and the contents of basic biochemical constituents (total protein, total amino acid, total carbohydrate and total lipid).

Procurement of Enterococcus durans (3031) and its sub culture
The lyophilized spores of E. durans (MTCC 3031) was purchased from Microbial Type Culture Collection (MTCC), Chandigarh, India. It was subjected to sub-culture with Nutrient broth (Hi-media, India, pH, 6.5 at Temperature, 25 °C), contained peptic digestion of animal tissues (5 g L -1 ), Beef extract (1.5 g L -1 ), Sodium chloride (5.0 g L -1 ), and Yeast extract (1.5 g L -1 ). The medium (13 g) was mixed with 1 L of double distilled water in a screw cap container and autoclaved at 121°C for 15 minutes. A loop full of E. duranswas inoculated into the broth and incubated for 24 hours at 37°C. The appearance of turbid broth indicates the growth of E. durans ( Figure 1). The cultured E. duranswas harvested by centrifugation at 5000 rpm for 10 min, washed twice with phosphate buffered saline (pH, 7.2), weighed and resuspended in the same buffer. For further usage it was stored at 4 °C. The suspension (30μl) was spread over the agar plate and the appearance of white colony indicates the growth of E. durans (Figure 1). 20 µl of serially diluted broth (up to 10 -9 ) was spread on nutrient agar for enumerating the CFU in order to optimize it, and the count was 3874 at 10 -1 , 2671 at 10 -3 , 1864 at 10 -5 , 1071 at 10 -7 , and 684 at 10 -9 .

Enrichment of Artemia nauplii with E. durans
The brine shrimp, Artemia franciscana cyst was purchased from Aqua world, Paris Corner, Chennai, India. The cysts (2 g/ 20 L and 15 g kg -1 body biomass of the prawns) were taken and hydrated in 1 L -1 of purified artificial saltwater (prepared from artificial sea salt powder 35.0 g L -1 , pH of 6.5) for 12-15 h. The cysts burst and the embryo surround by the hatching membrane become visible for few hours. The brownish orange colored nauplii came out. The 48-hr old Artemia nauplii were filtered and transferred to 1 L capacity glass beaker. Five such groups were enriched with 3874×10 -1 , 2671×10 -3 , 1864 ×10 -5 , 1071×10 -7 , and 684×10 -9 concentrations of E. durans for 1 hour (Figure 2). The Artemia nauplii were washed with freshwater and fed to M. rosenbergii PL.

Procurement and acclimatization of experimental animal
The post larvae (PL-8) of M. rosenbergii were procured from a prawn hatchery, Marakkanam, Chennai, India. They were transported to the laboratory in polythene bags filled with oxygenated water and acclimatized with ground water (Temperature, 28±2.2 °C; pH, 7.4±0.10; TDS, 0.94±0.05 g/L; DO 4.25±0.25 mg/L; Salinity, 0.70±0.02 mg/L; EC, 1.01±0.01 Ms/cm; Ammonia, 0.028±0.006 mg/L) for two weeks in cement tanks [1,2]. During acclimatization the prawns were fed with Artemia nauplii, boiled egg albumin threads and artificially feed formulated in our laboratory. Nearly half of the tank water was renewed every day and adequately aerated in order to maintain a healthy environment. The unfed feed, faecal material, exuvia/moults, and dead prawns if any were routinely removed by siphoning without disturbing the prawns while renewing the water medium [39,40].

Feeding trial
The feeding trials were conducted for a period of 45 days. Seven group of M. rosenbergii (1.1±0.05 cm and 0.05±0.003 g) were taken. The group 1 was fed with artificial feed, group 2 was fed with unenriched Artemia nauplii, and groups 3-7 were fed with E. durans (3874×10 -1 , 2671×10 -3 , 1864 ×10 -5 , 1071×10 -7 , and 684×10 -9 ) enriched Artemia nauplii. Each group comprised of 30 individuals accommodated in 25L of ground water. The water medium was renewed daily by siphoning and aerated. While renewing the water medium, the unfed feed, feces and moult were removed. At the end of the feeding trial the morphometric measurements were taken for calculating the nutritional indices and estimating concentrations of basic biochemical constituents, such as total protein, amino acid, carbohydrate and lipid, and activities of digestive enzymes, such as protease, amylase and lipase.

Estimations of basic biochemical constituents
The basic biochemical constituents, such as total protein, amino acid and carbohydrate were estimated in test prawns adopting standard methodologies [43][44][45], respectively, and the total lipid was extracted gravimetrically [46] and spectrophotometrically estimated [47]. The contents of ash and moisture were analysed [48].

Assays of digestive enzymes activities
Activities of digestive enzymes (protease, amylase and lipase) were assayed at 45 th day of feeding trial. The digestive tract of three prawns from each replicate were carefully dissected out and homogenized in ice-cold distilled water and centrifuged at 9000 g under 4 °C for 20 min. The supernatant was used as a source of crude enzyme. Total protease activity was determined by casein-hydrolysis method of Furne et al., [49], where one unit of enzyme activity represented the amount of enzyme required to liberate 1 μg of tyrosine per minute. Amylase activity was determined according to Bernfeld [50], the specific activity of amylase was calculated as milligrams of maltose liberated per gram of starch per hour (mg/g/h). Lipase activity was assayed by the method of Furne et al., [49], one unit of lipase activity was defined as the amount of free fatty acid released from triacylglycerol per unit time.

Gut microbial colonization
The bacterial culture was performed in the gut homogenate of experimental prawns fed with E. durans (1071×10 -7 ). The prawns were deactivated by kept them in freezer at -20 °C for 10 minutes. The surface of the prawn was sterilized with 50 ppm formalin for 30 seconds for removing external flora. Then the digestive tract was dissected out and homogenized with phosphate buffered saline (pH, 7.2) under aseptic condition. The homogenate was serially diluted up to 10 -7 . The aliquot (0.5 mL) was mixed with agar nutrient broth and incubated at 35 °C for 24 h. The broth (0.1 ml) was seeded on the surface of freshly prepared nutrient agar plate and incubated at 37 °C for 24 h. Different bacterial colonies seen were identified and confirmed through routine bacteriological tests, such as Gram's staining, motility test, Indole test, methyl red test, Voges Proskauer test, citrate utilization test, starch hydrolases, gelatin hydrolases, nitrate reduction test, oxidase test, catalase test and carbohydrate fermentation test [51]. The bacterial colony was enumerated by using the formula, Bacterial count (CFU/ g) = Number of colonies × Dilution factor/ Volume of sample (g).

Statistical analysis
All the data were subjected to statistical analysis through one-way ANOVA and subsequent post-hoc multiple comparisons (DMRT using SPSS v20). The P value less than 0.05 was considered as statistically (95%) significant.

Activities of digestive enzymes
Activities of protease, amylase and lipase were found to be significantly (P<0.05) elevated in E. durans enriched Artemia nauplii fed prawns when compared with un-enriched Artemia nauplii and artificial pelletized feed. Among the different concentrations of E. durans, 1071×10 -7 has produced the best performance (Table 2).

Contents of basic biochemical constituents
Concentrations of total protein, amino acids, carbohydrate, and lipids were found to be significantly (P<0.05) elevated in E. durans enriched Artemia nauplii fed prawns when compared with un-enriched Artemia nauplii and artificial pelletized feed. Among the different concentrations of E. durans, 1071×10 -7 has produced the best performance (Table  3).  Each value is mean ± standard deviation of three individual observations. *, unit×10 3 . Mean values within the same row sharing different alphabetical letter superscripts are statistically significant at P<0.05 (one-way ANOVA and subsequent post-hoc multiple comparison with DMRT. Each value is mean ± standard deviation of three individual observations. *, unit×10 3 . Mean values within the same row sharing different alphabetical letter superscripts are statistically significant at P<0.05 (one-way ANOVA and subsequent post-hoc multiple comparison with DMRT. Similar increases in total protein, amino acid, carbohydrate and lipid levels have been reported by Seenivasan et al., [52] and Jain et al., [39,40] in M. rosenbergii PL fed with L. sporogenes, E. gallinarum and E. hirae enriched Artemia nauplii. Increased contents of these basic biochemical constituents have also been recorded in M. rosenbergii PL fed with many probiotics supplemented diets [21, 22, 25-30, 59, 60, 70].

Analysis of gut microbial consortium
We have previously identified the presence of Escherichia coli, Klebsiella sp, citrobacter sp., Acetinobacter sp., Streptococcus sp., Bacillus sp., Staphylococcus sp., and Pseudomonas sp., in the un-enriched Artemia nauplii fed control prawn gut through colony morphology and biochemical tests [39,40], when a parallel experimental set-up was maintained to study the probiotic effects of E. gallinarum, E .hirae and E. durans. In the present study, in the gut of experimental of PL fed with 1071×10 -7 concentration of E. durans enriched Artemia, the presence of Enterococcus sp., E. coli, Bacillus sp., and Klebsiella sp., were identified through colony morphology and biochemical tests (Tables 4 and 5; Figure 4). Therefore, the pathogenic bacteria, Citrobacter sp., Acinetobacter sp., Streptococcus sp., Staphylococcus sp., and Pseduomonas sp., were competitively excluded due to the colony establishment of Enterococcus sp.
Aquatic animals, particularly at larval stage are monogastric have difficulty in digesting complex or fibrous feedstuffs de facto, thus bacteria colonized in the gut enhanced secretion of digestive enzymes to expedite the digestion [71]. Actually, probiotics have the ability to modulated gut microbiota and subsequently improve digestive enzymes secretion which in turn improves digestion and feed utilization [20,62,72,73]. Probiotics are excellent source of growth promoter and provide vast nutritional benefits, among them the genera of Bacillus, could biosynthesize a wide range of extracellular enzymes, such as protease, lipase, amylase and cellulase, and other growth factors, such as biotin, vitamin B12, short-chain fatty acids, and essential amino acids [74,75].   It is well known that the microbiota in the gut of aquatic animals can be modified by ingestion of other microorganisms, and therefore, microbial manipulation constitutes a viable tool to eliminate the incidence of opportunist pathogens by maintaining bacterial antagonism (Balcazar, 2002). Competitive exclusion of potential pathogenic bacteria effectively reduces or eliminates the need for antibiotic prophylaxis. For example, Vibrio alginolyticus has been used to increase survival and growth of L. vannamei PL [76]. Bacillus sp., and E. faecalis improved growth performance in L. vannamei [77]. The lactic acid bacteria enhance the crude protein and ash content in juveniles of P. indicus [78].
Enterococcus lactis isolated from shrimp samples of P. vannamei possesses antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The bacteriocin produced by E. lactis was thermostable, active in the pH range from 4.0 to 9.0, has a bactericidal mode of action, showed lack of significant antibiotic resistance genes, virulence determinants and absence of haemolytic activity, and thus recommended to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic [79]. It has been reported that E. durans and L. lactis have in vitro suppression of growth of Escherichia coli, Staphylococcus aureus and Shigella flexneri and therefore, they emerged as new probiotics [80].

Conclusion
The present study concluded that the probiotic bacterium, E. durans has got colonized in the gut of M. rosenbergii, eliminated some pathogenic bacteria Citrobacter sp., Acinetobacter sp., Streptococcus sp., Staphylococcus sp., and Pseduomonas sp., and also enhanced the survival and growth. Furthermore, E. durans increased activities of digestive enzymes which in turn improve digestibility and absorption of nutrients in M. rosenbergii, which ultimately improved the contents of basic biochemical constituents. Therefore, it can be taken as a probiotics to maintain sustainability in prawn culture.