Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

Effects of the commercial probiotic Lactobacillus casei on the growth,protein content of skin mucus and stress resistance of juveniles of the Porthole livebearer Poecilopsis gracilis (Poecilidae)

A 11‐week feeding trial was carried out to determine the effects of the probiotic bacteria Lactobacillus casei from the commercial product Yakult^® on the growth performance, proximal composition, protein content of skin mucus and stress resistance of juvenile Porthole livebearer Poeciliopsis gracilis. Triplicate groups of 15 juveniles per tank with an initial weight of 47 ± 9 mg (mean ± standard deviation) were fed with Artemia nauplii enriched with the probiotic, by using the bacteria cells plus the fermented milk (group ProN) and the other (group ProC) by using only the bacterial cells, eliminating the fermented milk by centrifugation. A control of fish was set up, by feeding non‐enriched Artemia nauplii. Growth performance and survival rates did not show significantly differences among the treatments and control group, but a slightly tendency of higher values for body weight, weight gain and specific growth rate was observed in the juveniles of ProC treatment. Whole body proximate composition did not show significant differences among the groups, but higher values of protein and lipid contents were observed in the groups fed with the probiotic. Content of protein in the skin mucus were significantly higher in the ProC treatment than control group. Recovery rates after an air‐dive test were significantly higher on the fish fed with the probiotic cells than the control group. These results show that L. casei might be used as a probiotic for fish and would help during the culture of juvenile of the Porthole livebearer P. gracilis.     

Colonization and probiotic effects of lactic acid bacteria in the gut of the abalone Haliotis gigantea

Strains of lactic acid bacteria (LAB) were isolated from several different sources and evaluated in vitro for potential probiotic effects in abalones. Two isolates (Lactobacillus sp. strain a3 and Enterococcus sp. strain s6) were highly resistant to bile salt and/or gastric juice and inhibited the growth of three abalone pathogens (Listonella anguillarum, Vibrio harveyi, and V. carchariae). Each of the LAB isolates was used to supplement diet of the abalone Haliotis gigantea for a period of 3 weeks. One group of animals received Lactobacillus sp. strain a3 added to commercial dry feed, one group received Enterococcus sp. strain s6 added to the feed, and a control group received only standard commercial feed. Culturable LAB counts of gut homogenates indicate the a3 colonized in the gut of abalones. Digestive enzyme activities and the concentrations of a number of volatile short-chain fatty acids (VSCFA) were elevated in the gut of abalones receiving feed supplemented with the two LAB strains. These results indicate that dietary supplementation can enable LAB colonization or persistence in the gut of abalone species and can potentially enhance probiotic effects.     

Enhancing growth and resistance to Vibrio alginolyticus disease in catarina scallop (Argopecten ventricosus) with Bacillus and Lactobacillus probiotic strains during early development

In bivalve aquaculture, selecting suitable probiotic treatments can be crucial for improving hatchery‐rearing of larvae and juveniles. We assessed the potential of five bacterial strains, previously selected in vitro, to improve survival, growth and resistance of catarina scallop Argopecten ventricosus during early and late larval and juvenile developmental stages, as well as during exposure to the pathogen Vibrio alginolyticus. Hatchery‐reared larvae and juveniles were treated with eight treatments of single or combined strains of Bacillus and Lactobacillus at 1 × 10⁶ CFU mL^?1 every 48 h for 9 days (larvae) and 21 days (juveniles). Compared with the control, significantly higher survival and growth in size and weight of early veliger larvae occurred with the antibiotic and the RL5 (Lactobacillus graminis) treatments. Significantly enhanced settlement of pediveliger larvae occurred with a different probiotic strain, the mix of Lactobacillus and Bacillus (MIX‐LB), while higher survival and growth of early juveniles occurred with C3 (Lactobacillus plantarum). The mix of Bacillus (MIX‐B) significantly increased survival of juveniles from V. alginolyticus after 120‐h infection, consistent with maximum activity of superoxide dismutase enzyme. In contrast, all untreated and infected scallops died by 96 h. The three Bacillus strains performed poorly when used as single treatments and when given to early developing larvae. Our results indicate that the action mechanism of probiotic strains is stage specific and strain specific, generating different responses by the host, including improved survival and growth (likely from better nutrient assimilation) and higher resistance against pathogens (possibly from strengthening the immune system).     

悉生条件下3株嗜盐古菌对卤虫生长及其抗弧菌感染的影响

嗜盐古菌存在于高盐水体中,是高盐环境微生物种群的重要组成部分。本研究利用从日晒盐场结晶池卤水中分离得到的3株红色嗜盐古菌,分别命名为Haloarcula sp.HG-1、Haloferax sp.KN-4和Halorubrum sp.IT-5。采用悉生实验系统,探讨不同盐度(30、100和150)条件下3株古菌能否作为卤虫(Artemia)的唯一饵料,并比较其作为饵料对卤虫存活、生长和抗鳗弧菌(Vibrio anguillarum)感染能力的影响。结果表明,在盐度30的悉生系统中,投喂3株古菌组卤虫存活率和体长均保持较高水平;鳗弧菌攻毒条件下,卤虫存活率有所下降,其中投喂Haloferax sp.KN-4菌株组卤虫抗鳗弧菌感染能力最强。正常和攻毒条件下,在盐度100和盐度150悉生系统中分别投喂3株古菌,卤虫均保持较高存活率和体长,其中投喂Haloarcula sp.HG-1菌株组卤虫具有最高存活率和最长体长,但攻毒组卤虫存活率和生长普遍优于未攻毒组,这可能与高盐条件下V.anguillarum毒力下降有关。本研究利用"卤虫-古菌"悉生实验系统,证实了3株嗜盐古菌均可作为卤虫的唯一饵料,为卤虫提供营养;利用"卤虫-古菌-致病菌"攻毒实验系统,证实3株嗜盐古菌均可以增强卤虫抗鳗弧菌感染能力。本研究结果为研究古菌在高盐环境食物链中的作用提供了基础数据。     

Characterization of a Probiotic Lactobacillus fermentum Isolated from Snakehead,Channa striatus,Stomach

This study sought to isolate and characterize lactic acid bacteria (LAB) from stomach of adult snakehead fish, Channa striatus, to be used as probiotics for freshwater fish. A total of 13 strains were isolated from the stomach of 10 fish, and 4 of these belonged to LAB. Strain LAB‐3 showing highest in vitro growth inhibition of Aeromonas hydrophila in a disk diffusion test was identified as Lactobacillus fermentum by conventional and molecular techniques and evaluated in vitro through various tests. The bacterium could grow at pH 3–8; but the optimum growth was observed at pH 6. Moreover, LAB‐3 grew at 0.15 and 0.3% bile salt concentrations, from 15 to 45 C, and at 4% NaCl. L. fermentum showed in vitro inhibitory activity against three fish pathogens, A. hydrophila, Pseudomonas aeruginosa, and Shewanella putrefaciens, tested by disk diffusion and well diffusion methods. Antibiotic sensitivity tests indicated that L. fermentum was resistant to streptomycin, gentamycin, and kanamycin, intermediate to tetracycline, but sensitive to chloramphenicol, amoxicillin, and ampicillin. Challenge test by using A. hydrophila showed that survival of snakehead was significantly (P < 0.05) improved when 2 × 10⁶ LAB‐3/g was supplemented to the diet. Therefore, this study suggests that L. fermentum might be a promising probiotic in snakehead aquaculture .     

Isolation,Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao Product

The aim of the study was to isolate, select, and identify proteolytic lactic acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by poured plate method. Proteolytic LAB were selected using agar skim milk media. Protease activity of LAB was determined based on the amount of tyrosine released in unit/mL. Proteolytic LAB were identified using API 50 CHL kit and 16S rRNA gene sequence analysis. The result showed that a total of 60 isolates were obtained from chao, and 57% of them were cocci-shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively. Identification by API 50 CHL kit showed that four rod-shape isolates were Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-shape isolates could not be identified as cocci bacterium. Rep-PCR results showed that there were two kinds of bands, namely thick and thin. Two isolates were selected from two types of bands that had the highest R for the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The results showed that Ags1-3 isolate was identified as Lactobacillus plantarum and Ags7-3 as Pediococcus acidilactici.     

Inhibitory effects of Bacillus licheniformis (DAB1) and Pseudomonas aeruginosa (DAP1) against Vibrio parahaemolyticus isolated from Fenneropenaeus indicus

In aquaculture industries, there is an urgent need to develop microbial control strategies, to control disease outbreaks. In recent years, probiotics are considered as a valid alternative for the use of antibiotics in aquaculture to prevent high mortality and promote growth. In the present study, seven strains of bacteria such as Bacillus licheniformis (DAB1), Bacillus pumilus (DAB2), Bacillus sp. (DAB3), Pseudomonas aeruginosa (DAP1), Pseudomonas sp. (DAP2), Pseudomonas aeruginosa (DAP3), Pseudomonas aeruginosa (DAP4), and three pathogenic Vibrio parahaemolyticus (DAV1, DAV2, DAV3) were isolated from healthy and diseased Fenneropenaeus indicus collected from the east coast of Tamilnadu, India. The strains were identified by biochemical analysis and 16S rRNA sequence methods. Among the seven probiotic strains tested, the cell-free extract from DAB1 and DAP1 exhibited higher inhibitory activity of V. parahaemolyticus than other isolates under in vitro conditions. The LC₅₀ of DAV1, DAV2, and DAV3 was found to be ～10³ CFU mL^?1. Pathogenicity of three V. parahaemolyticus DAV1, DAV2, and DAV3 showed significant mortalities (40 %) in Artemia nauplii at inoculation densities of 10³ CFU mL^?1 when compared to the controls (unchallenged nauplii). A significant reduction in mortality (P < 0.001) was found by addition of 10⁶ CFU mL^?1 of DAB1 and DAP1 strains in nauplii against the pathogens. In conclusion, the present study result reveals that DAB1 and DAP1 have potential applications for controlling pathogenic V. parahaemolyticus in Artemia culture systems and aquaculture practices.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

Antibacterial effect of Lactococcus lactis subsp. lactis on Artemia franciscana nauplii and Dicentrarchus labrax larvae against the fish pathogen Vibrio anguillarum

The antibacterial effect of Lactococcus lactis subsp. lactis against the fish pathogen Vibrio anguillarum was determined in Artemia franciscana nauplii and in European sea bass (Dicentrarchus labrax) larvae fed with ten pulses of nauplii enriched with L. lactis. The evaluation of the bactericidal activity of the extracellular products of L. lactis in vitro showed inhibition of growth of Vibrio (Listonella) anguillarum and Photobacterium damselae subsp. piscicida. The incorporation of L. lactis in Artemia nauplii did not affect their survival and offered protection in a challenge with V. anguillarum, significantly increasing LD₅₀. The administration of Artemia nauplii enriched with L. lactis for 48 h to sea bass larvae for five consecutive days had no adverse effect on survival of fish. In an in vivo challenge test with V. anguillarum using sea bass larvae, fish treated with nauplii enriched with the probiotic L. lactis showed significantly (P < 0.001) increased survival rates of 81 % compared with the untreated group of challenged fish (24 %). Our results indicate that L. lactis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep‐PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout‐associated microbiota may provide a defensive barrier against L. garvieae.     

Vibrio spp. Control at Brine Shrimp,Artemia, Hatching and Enrichment

Experiments were conducted to evaluate different prophylactic methods to control the bacterial load in brine shrimp, Artemia, hatching. The first experiment evaluated three treatments to control Vibrio spp. during the Artemia hatching: microalgae (Chaetoceros calcitrans), probiotic (Bacillus spp.), and antibiotic (Florfenicol). In the second experiment, Artemia metanauplius were enriched in distinct treatments with C. calcitrans, probiotic, and emulsion rich in docosahexaenoic and eicosapentaenoic fatty acids. Enriched Artemia metanauplius and nauplii (control) were offered to white shrimp, Litopenaeus vannamei, postlarvae (PL₇–PL₁₉). Presumptive Vibrio were quantified in Artemia, PL, and rearing water. Microalgae and probiotic were effective to control Vibrio spp. in Artemia nauplii. The enrichment process increased the Artemia bacterial load but did not affect Vibrio load in L. vannamei.     

Marine gammarids (Crustacea: Amphipoda): a new live prey to culture Octopus maya hatchlings

The effects of feeding two alternative live prey Hyalella azteca (freshwater gammarids) and Hyale media (marine gammarids) to Octopus maya hatchlings were compared with feeding adult Artemia sp., traditionally used during the first weeks of the life cycle. Hatchlings were fed ad libitum these three live preys during the first 15 days, and a paste elaborated with fresh squid and shrimp during the next 15 days when hatchling can be fed prepared diets. Weight (g) and specific growth rates (% day^?1) were determined every 15 days. Octopus maya hatchlings fed with marine gammarids grew larger (6.9 ± 0.2% day^?1) compared with hatchlings fed Artemia sp. or freshwater gammarids (4.8 ± 0.2% and 5.0 ± 0.3% day^?1 respectively). Survival was also higher (92.2 ± 6.8%) for hatchlings fed marine gammarids, than for those fed Artemia sp. (74.5 ± 23.8%) or freshwater gammarids (41.2 ± 21.2%). The content of acylglycerides, cholesterol and proteins in O. maya fed marine gammarids suggested a better nutrient assimilation by the hatchlings. Also, polyunsaturated fatty acids levels (EPA and DHA) were more abundant in marine gammarids, possibly contributing to the higher growth rates observed. This is the first study revealing a successful use of marine gammarids as alternative prey for octopus hatchlings culture.     

Isolation and characterization of Bacillus spp. M001 for potential application in turbot (Scophthalmus maximus L.) against Vibrio anguillarum

The aim of this study was to screen Bacillus strains from the guts of Scophthalmus maximus, Paralichthys olivacues, Epinephelus coioides and Clupanodon punctatus, for use as probiotics in aquaculture. Eight Bacillus strains were screened, and strain M001 was selected for probiotic study based on its antagonistic activity against multiple aquatic bacterial pathogens including Vibrio anguillarum, V. campbellii, V. vulnificus, V. parahamolyticus, Streptococcus sp. and Edwardsiella tarda. M001 was identified as B. amyloliquefaciens based on the biochemical tests and 16S rRNA gene analysis. In vitro experiments revealed that M001 was able to grow at a wider range of temperature, pH and salinity and was capable to use turbot mucus as nutrient for growth. Additionally, M001 isolate greatly inhibited the growth of V. anguillarum by producing antibacterial substances and was acid tolerance, non‐antibiotic resistance and non‐harmful. Thereafter, the potential probiotic effect of M001 was tested in turbot by dietary administration of M001 at a dose of 10⁸ CFU g^?1 diet for 42 days. No significant differences of weight gain, specific growth rate and feed ratio were found in the M001‐diet group of fish compared with control fish, but which increased, respectively, by 5.5%, 4.7% and 7.0% after 42 days of feeding. Several digestive enzyme activities were found to increase significantly in the M001‐diet group, including protease and amylase activities in hepatopancreas, protease activity in intestine and lipase activity in stomach (P < 0.05). Sera superoxide dismutase activity and total protein content (P < 0.05) were also increased significantly (P < 0.05) in the M001‐diet group. The challenge experiment showed that the M001‐diet group of fish showed a relative per cent of survival of 62.7% against V. anguillarim infection. The Bacillus M001 identified from this study has good potential to provide vibriosis control as probiotic feed additive for turbot aquaculture.     

Effect of feeding Lactobacillus-based probiotics on the gut microflora, growth and survival of postlarvae of Macrobrachium rosenbergii (de Man)

The present study was conducted to study probiotics treatment in the post‐larval diet of Macrobrachium rosenbergii. Three hundred postlarvae (average weight, 114–118 mg±0.11) were divided in five experimental groups each with four replicates. The experiment was conducted for 60 days. Experimental diets were identical in all the aspects except for variation in the probiotics strain. T1 and T2 groups were fed Lactobacillus acidophilus (140×10¹¹ CFU 100 g^?1) and L. sporogenes (24×10⁷ CFU 100 g^?1) respectively. The T3 group was fed L. sporogenes bioencapsulated in Artemia nauplii. T4 was the control group (without probiotic) whereas T5 was fed Artemia along with control diet. The bacteriological study indicated that the gut microflora of postlarvae are devoid of lactic acid bacteria. The probiotic strains were found to have inhibitory effects against the gram‐negative bacterial flora present in the gut. Growth of the probiotic fed groups was significantly higher (P<0.05) than the control group. Significantly higher growth (P<0.05), per cent weight gain (132.5%), specific growth rate (1.41%), feed efficiency ratio (FER) (0.45), protein efficiency ratio (1.29) and protein gain (161.6%) were recorded in T3 group fed Artemia bioencapsulated L. sporogenes over the control group (P<0.05). Although insignificant (P>0.05), growth‐promoting effects of L. sporogenes were found to be higher than L. acidophillus. Survival of the postlarvae was not affected by probiotics in the diet.     

Immune responses and gut morphology of Senegalese sole (Solea senegalensis,Kaup 1858) fed monospecies and multispecies probiotics

The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non‐specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1‐month trial. Fish were fed for 1‐month two diets with 1.0 or 4.6 × 10⁶ CFU kg^?1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 10⁵ CFU kg^?1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A₁ the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A₂ versus B₂). The current study indicate that the use of the multispecies probiotic at 1.0 × 10⁶ CFU kg^?1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.     

Live microbial feed supplement in aquaculture for improvement of stress tolerance

Two bacterial strains, Lactobacillus fructivorans, isolated from sea bream (Sparus aurata) gut, and Lactobacillus plantarum, isolated from human faeces, were administered simultaneously, during sea bream development, using Brachionus plicatilis and/or Artemia salina as vectors. The probiotic treatment significantly affected gut colonization. To test the probiotic influence on stress responsiveness, sea bream fry, 47 days post-hatching (p.h.), were subjected to pH stress (from 8.6 to 6.3) and cumulative mortality, cortisol levels and HSP70 gene expression were analysed. Cortisol was selected, since under stress conditions its level increases. HSP70 was selected with consideration of its wide involvement in response to a great number of injuries, and because it protects cells probably by binding and refolding damaged proteins. The results obtained indicated that the administration of probiotic to sea bream fry induced higher HSP70 levels, indicating a greater potentiality to respond to the harmful conditions possibly present in fish farms. This hypothesis is supported by the fact that the levels of cortisol found were significantly lower (P < 0.05) in both groups under probiotic treatment. When pH was used as a stressor, it induced a higher cumulative mortality in the control; the mortality was found to be significantly lower in both treated groups. Interestingly, a significant increase (P < 0.05) in HSP70 gene expression was observed in all stressed groups. These results suggest an improvement in tolerance to acute stress of fry fed with probiotics.     

Beneficial properties of lactic acid bacteria isolated from a Rana catesbeiana hatchery

This work addresses the selection of potentially probiotic lactic acid bacteria (LAB) to be used in raniculture. Thus, strains belonging to the genera Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactococcus lactis and Enterococcus faecium isolated from a Rana catesbeiana hatchery were evaluated for their inhibitory properties against RLS-associated pathogens ( Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis ) and food-borne bacteria. Cell-free supernatants of LAB strains inhibited the growth of at least one of the pathogens by organic acids, but L. lactis CRL 1584 also produced a bacteriocin-like metabolite. The ability of LAB strains to produce H₂O₂ in MRS+TMB medium was also studied. Seventy-eight to ninety six per cent of the strains showed some level of H₂O₂ production. Moreover, different organic solvents were used to determine the hydrophobicity and Lewis acid/base characteristic of LAB strain surfaces. Most of the strains presented hydrophilic properties, but no acidic or basic surface characters. However, some strains isolated from the skin showed a high degree of hydrophobicity and basic components in the cell surface due to their adhesion to chloroform. These properties were not observed in LAB from balanced feed and freshwater. Taking into account general guidelines and the beneficial properties studied, five strains were selected as potential candidates to be included in a probiotic for raniculture.