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Lat. Am. J. Aquat. Res., 41(3): 490-497, The lactic acid bacteria as growth promoters in fish fed 490 Research Article
The use of lactic acid bacteria isolated from intestinal tract of Nile tilapia
(Oreochromis niloticus), as growth promoters in fish fed low protein diets
Maurilio Lara-Flores1 & Miguel A. Olvera-Novoa2
1Instituto de Ecología, Pesquerías y Oceanografía del Golfo de México, Universidad Autónoma de Campeche, Av. Agustín Melgar y Juan de la Barrera S/N, Col. Buenavista C.P. 24039. San Francisco de Campeche, Campeche, México 2Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional Unidad Mérida Apdo. Postal 73-Cordemex, C.P. 97310, Mérida, Yucatán, México ABSTRACT. In this study, the effect as growth promoter of five lactic acid strains (Enterococcus faecium, E.
durans, Leuconostoc sp., Streptococcus sp. I and Streptococcus sp. II), isolated from intestinal tract of Nile
tilapia (Oreochromis niloticus), was evaluated. Eight isocaloric diets were formulated: one containing 40% of
protein as positive control, and seven with 27% protein. Five diets with 27% protein were supplemented with
one of the isolated lactic acid bacteria in a concentration of 2.5x106 cfu g-1 of diet. A commercial probiotic
based on S. faecium and Lactobacillus acidophilus was added at the same concentration to one 27% protein
diet as a comparative diet, and the last diet was not supplemented with bacteria (negative control). Tilapia fry
(280 mg basal weight) stocked in 15 L aquaria at a density of two per liter were fed for 12 weeks with
experimental diets. Results showed that fry fed with native bacteria supplemented diets presented significantly
higher growth and feeding performance than those fed with control diet. Treatment with Streptococcus sp. I
isolated from the intestine of Tilapia produced the best growth and feeding efficiency, suggesting that this
bacteria is an appropriate native growth promoter.
Keywords: probiotics, Nile tilapia, Oreochromis niloticus, growth promoter, lactic acid bacteria.
Uso de bacterias ácido lácticas aisladas del tracto intestinal de tilapia
nilótica (Oreochromis niloticus) como promotores de crecimiento en peces
alimentados con dietas bajas en proteína
RESUMEN. Se evaluó el efecto como promotores de crecimiento de cinco cepas de bacterias ácido lácticas
(Enterococcus faecium, E. durans, Leuconostoc sp., Streptococcus sp. I y Streptococcus sp. II) aisladas del
tracto intestinal de tilapia nilótica (Oreochromis niloticus). Se formularon ocho dietas isocalóricas: una
conteniendo 40% de proteína como control positivo y siete con 27% de proteína. Cinco dietas con 27% de
proteína fueron suplementadas con cada una de las bacterias aislada a una concentración de 2,5x106 ufc g-1 de
alimento. Un probiótico comercial a base de S. faecium y Lactobacillus acidophilus a la misma concentración
de inclusión bacteriana a una dieta con 27% de proteína como dieta comparativa, y la última dieta no fue
suplementada con bacterias (control negativo). Juveniles de tilapia (280 mg de peso basal) fueron distribuidos
en acuarios de 15 L de capacidad, a una densidad de dos juveniles por litro, alimentados durante 12 semanas
con las dietas experimentales. Los resultados mostraron que los organismos alimentados con las dietas
suplementadas con bacterias nativas presentaron crecimiento y asimilación del alimento significativamente
mayor que las dietas control. El tratamiento con Streptococcus sp. I, aislada del intestino de la tilapia, produjo
el mejor crecimiento y la mejor eficiencia alimenticia, sugiriendo que esta bacteria es apropiada como un
promotor de crecimiento nativo de tilapia.
Palabras clave: probiótico, tilapia nilótica, Oreochromis niloticus, promotor de crecimiento, bacterias ácido
lácticas.
Corresponding author: Maurilio Lara-Flores (maurilio_lara@yahoo.com.mx) 491 Latin American Journal of Aquatic Research INTRODUCTION
morphology and produce certain enzymes and inhibitory compounds causing improved digestion Aquaculture is a fast-growing and rapidly expanding and absorption of nutrients, as well as enhanced multibillion dollar industry. Marine capture fisheries immune response (Verschuere et al., 2000). Several and aquaculture supplied the world with about 104 studies have demonstrated that use of probiotics million ton of fish in 2004 (FAO, 2007). Of this total, improves health of larval and juvenile fish, disease marine aquaculture accounted for about 18%, where resistance, growth performance and body compo- shrimp from aquaculture continues to be the most sition, however, the mode of action in fish species important commodity traded in terms of value (2.4 may vary between farmed fish species cultured in million ton). Worldwide, the aquaculture sector has been expanding at an average compounded rate of The use of probiotic in feeds to improve growth of 9.2% per year since 1970, compared with only 1.4% different fish species including African catfish, (Al- for capture fisheries and 2.8% for terrestrial-farmed Dohail et al., 2009); Senegalese sole (Sáenz de meat production systems. During the last decades, Rodrigáñez et al., 2009), Nile tilapia (Lara-Flores et antibiotics used as traditional strategy for fish al., 2003, 2010; El-Haroun et al., 2006), Japanese diseases management but also for the improvement of flounder (Taoka et al., 2006), gilthead sea bream and growth and efficiency of feed conversion. However, sea bass (Carnevali et al., 2006) has been inves- the development and spread of antimicrobial resistant tigated. The effects of probiotics have been linked to pathogens were well documented (Kim et al., 2004; modulation of gut microbiota and establish-ment of the beneficial microorganisms, higher specific and There is a risk associated with the transmission of total digestive enzyme activities, in the brush border resistant bacteria from aquaculture environments to membrane, which increases the nutrient digestibility humans, and risk associated with the introduction in and feed utilization (Verschuere et al., 2000; Balcazar the human environment of nonpathogenic bacteria, et al., 2006; Kesarocodi-Watson et al., 2008). In containing antimicrobial resistance genes, and the addition, the production of vitamins by these gut subsequent transfer of such genes to human microbiota could also increase vitamin synthesis and pathogens (FAO, 2005). Considering these factors, as improve fish health (Holzapfel et al., 1998). This well as the fatal effect of residual antibiotics of study was carried out to find the effect of isolated aquaculture products on human health, the European acid lactic bacteria from intestinal tract of Nile tilapia Union and USA implemented bans on, or restricted (Oreochromis niloticus), on feed efficiency and the use of antibiotics (Kesarcodi-Watson et al., growth of fry Nile tilapia fed with low protein diets. In connection with the ban of antibiotic growth MATERIALS AND METHODS
promoters new strategies in feeding and health management in fish aquaculture practice have Bacterial strains
received much attention (Balcázar et al., 2006). In Five strains of lactic acid bacteria isolated from Nile addition, the global demand for safe food has tilapia intestine were characterized on the basis of prompted the search for natural alternative growth morphological, physiological and biochemical test by promoters to be used in aquatic feeds. There has been Bergey´s Manual of Systemic Bacteriology (Holt et heightened research in developing new dietary al., 1993). Axenic cultures of the purified bacteria supplementation strategies by promoting various were tentatively identified, using Mini-API System health and growth compounds as probiotics (Denev, Bio-Merieux, as Enterococcus faecium, E. durans, Leuconostoc sp., Streptococcus sp. I and Strepto- The importance of probiotics in human and coccus sp. II. Commercial probiotic containing animal nutrition is widely recognized (Fuller, 1992; mixture of Lactobacillus acidophilus and S. faecium Rinkinen et al., 2003), in recent years, the role of probiotics in nutrition and health of certain All bacteria were grown aseptically in 10 mL of aquaculture species have also been investigated MRS broth for 24 h at 35 ± 2°C. Five mL were (Gatesoupe, 1999; Verschuere et al., 2000; Kesarcodi- transferred under aseptic conditions into 250 mL of Watson et al., 2008; Ringo et al., 2010; Merrifield et MRS broth and held on a shaker at 150 rpm for 24-48 al., 2010). It appears that probiotics provide benefits h at 35 ± 2°C. The cells of each isolate were by establishing favorable microbial communities, harvested by centrifugation at 10,000 rpm for 15 min such as lactic acid bacteria and Bacillus sp. in the and washed twice with phosphate buffer (PB) having gastrointestinal track, which may alter gut The use of lactic acid bacteria as growth promoters in fish fed 492 Experimental diets
after which the different treatments were randomly Eight isocaloric diets were formulated: one assigned to the aquaria, with four replicates per containing 40% protein, and the other seven with 27% of protein level. The lower protein inclusion in Feed was manually administered ad libitum four the latter diets was used as a stress factor since that times a day, for 12 weeks. A daily record was kept of the optimum protein level for fry tilapia is 40% feed offered. Bulk weight was measured weekly to (Tacon, 1984). Each one of the lactic acid bacteria follow growth in weight and calculate survival and isolates was added to lower protein diets in a feeding ration. Briefly, the fish were taken from each concentration of 2.5x106 cfu g-1 of diet. The tank using a net previously disinfected with a 1% commercial probiotic was added to one diet with 27% benzalkonium chloride solution. Initial mean weight protein in a concentration of 2.5x106 cfu g-1 of diet (IMW), final mean weight (FMW), specific growth for comparison. Finally, positive and negative control rate (SGR), Feed conversion ratio (FCR), survival, diets were formulated with 40 and 27% of protein protein efficiency ratio (PER), apparent nitrogen level, respectively, both diets without bacterial utilization (ANU), apparent organic matter diges- supplements. To all diets, 0.5% chromic oxide was tibility (AOMD) and apparent protein digestibility added for determining digestibility. Tables 1 and 2 (APD) were measured using the following equations: shows diet formulation and proximate composition SGR = 100[(log. final body weight-log initial body FCR = individual food intake/individual weight gain Experimental setup
PER = individual protein intake/individual weight Population density was also used as a stress factor, under the assumption that overpopulation is one of the main growth-inhibiting factors in intensive ANU = 100(carcass nitrogen deposition/N intake) aquaculture systems. To this end, 32 glass aquaria of Beginning in the third week of the experiment, 15 L capacity were stocked at a 30 organisms per feces were collected by siphoning the aquaria 30 min aquaria (2 fry per liter). All fry had similar average after the second daily feeding, to minimize leaching. initial weights (280 ± 10 mg). The different diet Scales were removed from the collected feces, the formulations were assigned within the aquaria. The feces were oven dried at 105°C for 24 h, and then animals were allowed to adapt to the experimental stored in hermetic containers under refrigeration to system for a week, and fed with a conventional diet, Table 1. Formulation of experimental diets.
CON 40: Positive control, CON 27: Negative control, ED: Diet supplemented with E. faecium, B2: Diet supplemented with E. durans, B3: Diet supplemented with Leuconostoc sp., A1: Diet supplemented with Streptococcus sp. I, A2: Diet supplemented with Streptococcus sp. II; ALL 27: Diet supplemented with commercial probiotic. 1Jauncey & Ross, (1982). 2Tacon, (1984). 493 Latin American Journal of Aquatic Research Table 2. Proximate composition of experimental diets (% dry matter).
Gross energy (MJ kg-1) 19.95 19.75 20.09 For water quality control, temperature and dissolved oxygen were measured daily, and weekly analyses were done of total ammonium, nitrite, nitrate The growth performance including IMW, FMW, and pH levels, using standard methods (APHA, SGR, FCR, PER, ANU, AOMD, APD and survival 1989). The following values (±SD), appropriate for rate of Nile tilapia are shown in Table 3. No tilapia cultivation, were used: temperature, 28.83 ± significant differences were observed in IMW among 0.45°C; dissolved oxygen, 5.71 ± 1.16 mg L-1; pH treatments. Fish fed with CON 27 diet showed significantly lower survival (66.7%) than those fed 7.98 ± 0.45; ammonia, 0.09 ± 0.04 mg L-1; nitrite, with bacteria-supplemented and positive control diets 0.08 ± 0.02 mg L-1 and nitrate, 5.93 ± 0.61 mg L-1. (P < 0.05). The highest survival was recorded for Every third day, each aquaria was partially cleaned CON 40 and diet supplemented with E. durans and the water partially changed (1:l). Once a week, (100%). The treatment CON 27 presents the lower the same day bulk weight measurement was done, the FMW (5.95 g). Fish fed with diets supplemented with aquaria were completely cleaned and a total change native bacteria exhibited higher FMW compared to The ALL 27 treatment resulted with the signifi- Chemical analysis
cantly higher FCR (2.02) among the bacteria- Proximate chemical analyses of diet ingredients were supplemented diets, thought all the other bacteria- made and a sample of fish, at the beginning and end containing diets showed FCR significantly lower than of the experiment, according to standard methods those for the controls diets (P < 0.05). The best FCR (AOAC, 1995). Gross energy in the feed was determined by combustion in a Parr adiabatic The PER was significantly higher in treatment A1 calorimeter. To evaluate digestibility, the chromic (2.53) than in the others treatments. The lower PER oxide content of each diet and the collected feces was recorded for the CON 40 treatment (1.36). Fish were analyzed using the acid digestion method from A1 treatment presented ANU significantly (Furukawa & Tsukahara, 1966). Protein content was greater (48.4%), in comparison with the other also determined for the feces, to assess protein treatments. The lowest biological value was observed In general, AOMD and APD were variable among Statistic
treatments. The maximum value were obtained in the Growth performance and feed utilization efficiency A2 treatment (AOMD = 95.08%; APD = 94.28%), parameters were statically compared using one-way which was statically different from the rest of the ANOVA (P < 0.05), and differences among means were identified using Duncan Multiple Range Test. Whole body composition data are presented in Analyses were carried out with the StatGraphics Plus Table 4. The moisture content showed no significant Version Centurion XV computer software. Arcsin difference among fish fed with the experimental transformation of raw data were made when diets, and it ranged from 72.9 to 76.4%. The uppermost two values (18.7 and 18.4%) of crude The use of lactic acid bacteria as growth promoters in fish fed 494 Table 3. Growth and feeding performance of fish feed with diets supplemented with bacteria.
1Values with the same superscript in the same row are not statistically different (P > 0.05), 2Standar error, calculated from mean-square error of the ANOVA, 3Specific Growth Rate, 4Food Conversion Ratio, 5Protein Efficiency Ratio, 6Apparent Nitrogen Utilization, 7Apparent Organic Mater Digestibility, 8Apparent Protein Digestibility. Table 4. Body composition of fish fed diets supplemented with bacteria.
1Values with the same superscript in the same row are not statically different (P > 0.05). protein were achieved for fish fed diets A1 and promotion (Gatesoupe, 2002; Lara-Flores et al., ALL27, with no significant difference. Fish from B2 treatment showed lower lipid content (4.7%) in In this study, groups administered diets with lactic comparison with the other treatments. Statistical acid bacteria showed similar and superior survival differences were observed also in the body ash results when compared with positive and negative content among fish fed with the different diets, with control groups. Similar results were observed by significantly lower content in fish from ALL 27 Suyanandana et al. (2002) when administered Lacto- bacillus sp. isolated from the intestine of Nile tilapia. Probiotics are biopreparations containing living DISCUSSION
microbial cells that optimize the colonization and composition of the growth and gut micro flora in Many studies on probiotics in aquaculture have used animals, and stimulate digestive processes and in vitro models of specific bacteria as antagonists of immunity (Bomba et al., 2002). The results of the pathogens (Vine et al., 2004, 2006), measured the present study confirm the results from other studies survival of probiotic in fish gut (Andlid et al., 1998), that the incorporation of probiotic in the diets can or evaluated the beneficial effect of probiotic on improve growth performance in terms of SGR, FCR health management, disease resistance and immune and PER. Gatesoupe (1991) reported increased response of fish (Li & Gatlin III, 2004; Shelby et al., weight gain in Scophital mus larvae fed a diet 2006). Other important effect of the use of probiotic, incorporating lactic acid bacteria and Bacillus toyoi. that it is not extensively study, but demonstrated an In the present study, fish fed lactic acid bacteria grew important effect, is the feed efficiency and the growth faster than those fed a control. It has been reported 495 Latin American Journal of Aquatic Research that the improvement of growth by using probiotics is The present investigation showed that the addition related to an enhancement of nutrition (El-Haroun et of native bacteria in Nile tilapia fry diets improved al., 2006), as some probiotic strains may serve as a animal growth and mitigated the effect of stress supplementary source of food and their activity in the factors, such as the low protein level in diets. All digestive tract may be a source of essential nutrients native bacterial strains used in the present study were (Balcazar et al., 2006). According with Ghosh et al. effective in stimulating fish performance. Strepto- (2007), most of this enhancement is reflected in the coccus sp. I produced the best results, and it could be whole body proximal composition of fish. In the a good candidate for optimizing growth and feed present experiment, and regardless of the treatments utilization in intensive tilapia culture. with lactic acid bacteria, the whole body composition of O. niloticus showed a trend of higher values of REFERENCES
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