Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC₅₀) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log₁₀ CFU mL^?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log₁₀ CFU g^?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log₁₀CFU mL^?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.     

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.     

乳酸菌在青贮中的应用及研究进展

青贮饲料是一个复杂的微生物共生体系,主要包括乳酸菌、酵母菌、芽孢杆菌、乙酸菌、梭状芽饱杆菌等,其中以乳酸菌的数量和种类最多,也是在青贮饲料发酵中起主要作用的微生物。着重介绍了乳酸菌微生物青贮剂的种类、选作微生物青贮剂的乳酸菌需符合的条件,阐述了乳酸菌微生物青贮剂的作用机制、添加效果以及影响因素,并对乳酸菌微生物青贮剂近年的研究进展和应用现状进行了总结,指出了乳酸菌微生物青贮剂的未来发展趋势将由单一型向复合型方向发展,同时需将现代基因工程、遗传工程技术引入乳酸菌微生物青贮剂,以开发新型制剂。     

Real‐time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus

Whether it is better to use viable or non‐viable probionts in aquaculture is still a matter of debate. In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated. Three forms of this probiont: (1) heat‐killed (HK), (2) live spray (LI) and (3) freeze‐dried (FD) were incorporated into a basal (control) diet for rainbow trout O. mykiss. The LI and FD diets are referred to as viable diets. A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments. The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF‐β), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real‐time polymerase chain reaction. The tested immune genes were up‐regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene. The TNF gene was found to be highly (P<0.05) up‐regulated (5000‐fold) in groups fed both viable forms (LI, FD). With regard to the TGF‐β gene, the spleen of the HK and FD groups showed significant up‐regulation of 20‐ and 30‐folds respectively. The IFN gene was up‐regulated (P<0.05) in all treatments, but more in the viable diet treatments. Kidney and spleen tissues showed similar expression patterns, i.e. all of these genes were up‐regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet.     

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.     

Water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with lactic acid bacteria

To understand the nutrient utilization efficiency and distribution in indoor fish culture system under treatment of probiotics, this study examined water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with four strains of lactic acid bacteria (LAB) isolated from fishery products. The results showed that the concentration of ammonia nitrogen (NH?‐N) and nitrite nitrogen (NO?‐N) in LAB treatments were all significantly lower (p < 0.05) than in the control group. The nitrate nitrogen (NO?‐N) in LAB treatments showed no significant differences (p > 0.05) compared with control group. The fish feed and water exchange accounted for 79.07% and 17.02% of total N input respectively. For N output, the drainage and residual diets in LAB treatment accounted for 24.50%–25.80% and 1.33%–1.60% respectively, and they were significantly lower than in the control group (27.60% and 2.20% respectively). Fish growth and lost N in LAB treatments accounted for 27.10%–30.50% and 11.00%–18.50% respectively, and they were both significantly higher (p < 0.05) than in the control group (22.30% and 5.30% respectively). The results indicated that the indigenous LAB strains were capable of improving fish growth, and reducing NH?‐N and NO?‐N level (at concentration of 10⁵ cfu/ml) by directly adding in S. maximus culture water. Moreover, specific strains of LAB may increase nitrogen loss by promoting denitrification process in culture system.     

Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature

This study examined the effect of potential probiotic bacteria on growth and survival of the tilapia Oreochromis niloticus , under high density and suboptimum temperature. Presumptive Bacillus and lactic acid bacteria (LAB) were isolated from tilapia and from their culture system and were characterized for haemolytic and enzymatic activity, and antagonism against Vibrio . Selected strains were included in the diet of juvenile tilapia and evaluated during a 134-day assay. The experiment was conducted with four treatments: (1) fish fed with commercial feed plus Dry Oil^®; (2) fish fed with commercial feed plus LAB; (3) fish with bacilli in water; (4) fish with a mixture of treatments 2 and 3. Tilapias in all treatments, including bacteria, grew significantly better than fish fed with commercial feed plus Dry Oil^® (control group). Survival was similar in all treatments. The physicochemical parameters of the culture system were maintained within the optimal ranges for the species, with the exception of temperature (19.9–24.82 °C). Animals fed diet supplemented with bacilli and LAB had good survival and the best growth performance, suggesting that bacteria are appropriate growth-stimulating additives in tilapia cultivation.     

褐藻酸降解菌的筛选及其生长条件

采用安藤芳明选择性培养基从自然发病明显的海带幼苗上分离出8株褐藻酸降解菌。研究表明，8株菌能够不同程度地降解褐藻酸钠，菌株A1和A2表现出较强的降解能力，接种1d后液体培养基即变清。温度是该菌大量繁殖的决定性因子，其生长的最适温度为20℃，最适pH7.5。褐藻酸钠质量分数为0.5％-0.6％时，不同氮源和碳源对菌体生长的影响不同，其中有机氮和铵态氮有利于菌体生长，尿素和硝酸钠抑制菌体生长；只有在培养基中含有褐藻酸钠时，菌体才能正常生长。     

Autochthonous probiotic Lactobacillus sp. in the diet of bullfrog tadpoles Lithobates catesbeianus improves weight gain,feed conversion and gut microbiota

Dietary supplementation with probiotics in animal production is an alternative to antibiotics. In frog culture, studies involving native strains of probiotic bacteria and their effects on the performance and intestinal histology of farmed animals are scarce. Therefore, this study aimed to evaluate a diet supplemented with Lactobacillus sp. in tadpoles of Lithobates catesbeianus. This randomized test was performed with two dietary treatments: non‐supplemented control diet and diet supplemented with Lactobacillus sp., with nine replications. The growth performance of Lactobacillus sp., including its bacterial enzymatic activity and stability in feed, as well as colonization and histology of the intestinal tract, was evaluated after 42 days of experimentation. Animals fed with a supplemented diet showed higher weight gain and concentration of lactic acid bacteria in the gut and lower feed conversion. No significant difference was observed in survival, total heterotrophic bacterial count or histological change in the gut between the two treatments. The Lactobacillus sp. strain was able to colonize the intestinal tract and feed and remain at a high concentration of 10⁷ and 10⁶ CFU g^?1, respectively. It produced several enzymes, which might have contributed to the greater weight gain and lower feed conversion in the supplemented animals, thus demonstrating its probiotic potential for use as a dietary supplement in bullfrog tadpoles.     

混合乳酸菌发酵改善罗非鱼蛋白酶解液风味的研究

通过嗜热乳链球菌（Streptococus thermophilus）和保加利亚乳杆菌（Lactococcus bulgarcus）混合发酵,改善罗非鱼（Oreochromis niloticus）下脚料蛋白酶解液风味,采用响应面设计试验考察乳酸菌发酵时间（X1）、温度（五）及接种量（恐）对酶解液风味感官评分值（Y）的影响,并利用同时蒸馏萃取与气相色谱-质谱（GC—MS）联用技术测定挥发性成分。结果表明,响应面分析法（RSM）优化的乳酸菌发酵条件为时间9h,温度45℃,接种量16％,预测风味感官评分值为4．1;酶解液发酵后的挥发性成分由原来的27种增加至33种,增加的主要是酯类物质,另外产生了辛酸及十六醛等风味物质。乳酸菌发酵能减弱罗非鱼酶解液的腥味,但脱苦效果不明显。     

饲喂乳酸菌对凡纳滨对虾幼虾肉质的影响

在凡纳滨对虾(Litopenaeus vannamei)幼虾饲料中添加3株植物乳杆菌[Lactobacillus plantarum YRL45、Lactobacillus plantarum QL、Lactobacillus plantarumKTP(C-2)]和3株副干酪乳杆菌(Lactobacillus paracasei M5、Lactobacillus paracasei X12、Lactobacillus paracasei SB27),采用质构仪TPA模式和气相色谱法分析喂养后肌肉质构和脂肪酸含量变化,研究乳酸菌对凡纳滨对虾幼虾肌肉品质的影响。结果表明,添加乳酸菌能改善凡纳滨对虾幼虾肌肉的弹性和咀嚼性,其中植物乳杆菌[YRL45、QL、KTP(C-2)]的改善效果最好,将幼虾肌肉的弹性和咀嚼性分别提高了35.14%和85.71%(P0.05)。副干酪乳杆菌(M5、X12、SB27)能提高对虾肌肉持水性并且能显著降低对虾肌肉中饱和脂肪酸含量,提高多不饱和脂肪酸含量,其中棕榈酸、十七烷酸和硬脂酸含量显著降低,EPA与DHA的含量分别增加了23.22%和34.40%。综上所述,在饲料中添加植物乳杆菌[YRL45、QL、KTP(C-2)]对凡纳滨对虾幼虾肌肉弹性和咀嚼性有改善作用,副干酪乳杆菌(M5、X12、SB27)能显著改善凡纳滨对虾幼虾肌肉的脂肪酸组成。     

Effects of probiotic Pediococcus acidilactici on growth performance,survival rate,and stress resistance of Persian sturgeon (Acipenser persicus)

This study was conducted to examine the effect of Artemia urmiana nauplii enriched with Pediococcus acidilactici on growth performances, survival rate, and stress resistance of Persian sturgeon (Acipenser persicus). Artemia nauplii were enriched with P. acidilactici at concentration of 10¹⁰ CFU/ml for 3 h (T3), 6 h (T6), 9 h (T9), and one nonenriched nauplii treatment (control). Since nauplii enriched for 9 h had the most significant CFU/g, fish were fed with T9 for 11 days and compared with the control. To evaluate the effect of probiotic on fish resistance, Persian sturgeon larvae were subjected to osmotic shocks of 15, 25, or 35 ppt; four pH treatments, pH 5, pH 6, pH 8 or pH = 9; and air for 5, 10, 15, or 20 s on the 11th day. There were no significant differences in final weight, weight gain, CF, SGR, and FCR between T9 and control (P > 0.05). However, the survival rate of larvae fed nauplii enriched for 9 h increased significantly in comparison with the control group (P < 0.05). In addition, significant higher resistance to all stress challenges was observed in the T9 group.     

Effect of drying on the viability of the probiotic bacterium Lactobacillus plantarum Lab9 (CPQBA 144‐09 DRM 03) impregnated in the feed for tilapia (Oreochromis sp.)

Drying experiments were conducted at different temperatures and air flux velocities to determine the proper drying conditions for reducing moisture in commercial fish feed impregnated with probiotic lactic acid bacteria and to assess the effect on bacteria viability over time. At temperatures of 45°C, the drying time was shorter, without the air flux velocity under study having a relevant influence. The drying conditions influenced the viability of the bacteria in the feed; the least loss of viability was obtained with a velocity of 0.8 m s^?1 and a temperature of 45°C during 15 min. Using these drying conditions, 5 kg of feed was dried and stored for a month at temperature of 26°C and relative humidity of 75%. The viability of the bacteria and the moisture of the feed were measured every 3 days during the storage period. Loss of viability followed first order kinetics, with a constant k of 0.112 days^?1. Thus, the viability of the bacteria in the feed is less than 10⁶ CFU g^?1 after 43 days.     

斜带石斑鱼消化道乳酸菌在模拟胃肠道环境中的存活

从斜带石斑鱼(Epinephelus coioides)幼鱼消化道分离到3株乳酸菌,通过抑菌试验筛选出抑菌效果较好的2株菌(MM1和MM4),经生理生化鉴定结合16SrRNA基因测序分析,MM1菌株鉴定为乳酸乳球菌(Lactococcus lactis),MM4菌株鉴定为屎肠球菌(Enterococcus faecium)。进一步研究了MM1和MM4的体外生长特性,及其对不同pH值的人工胃液、含有不同浓度胆盐的人工肠液及高温的耐受能力。结果表明,MM1和MM4均在培养6h左右进入对数生长期。对人工胃液的耐受能力相似,pH4.0和3.0的人工胃液对2株菌存活的影响较小,而pH2.0的人工胃液对2株菌的影响较大,经2h处理后两者的存活数即下降到105CFU/mL,经4h处理后的存活数低于5.0×103CFU/mL。MM1和MM4对含不同浓度胆盐的人工肠液均具有较好的耐受能力,含中低浓度胆盐(0.1%～0.3%)的人工肠液对2株菌的存活影响很小,处理3h后2株菌的存活数均维持在107CFU/mL以上;高浓度胆盐(0.5%～0.7%)处理3h后2株菌的存活数明显下降,但活菌数仍维持在105CFU/mL以上。MM1和MM4具有一定的耐高温能力,60℃处理30min二者存活数在107CFU/mL以上;80℃处理30min二者存活数在106CFU/mL以上。结论认为,2株乳酸菌具有生长速度快、抑菌能力强、对胃肠道环境和高温耐受能力较强的特点,具有作为益生菌的应用潜力。     

不同乳酸菌对发酵草鱼品质的影响

为探明不同乳酸菌对发酵草鱼微生物数量和理化性质的影响,进一步提高发酵鱼制品的品质,实验对草鱼背部肌肉经不同乳酸菌(植物乳杆菌、戊糖片球菌和副干酪乳杆菌)发酵后制品的微生物数量、pH、总酸、水分含量、白度、氨基酸态氮、硫代巴比妥酸和挥发性盐基氮等进行分析,探究不同乳酸菌在草鱼发酵过程中(0～15d)对其品质的影响.结果显...     

Screening of gastrointestinal bacteria of Indian major carps for a candidate probiotic species for aquaculture practices

Probotic selection could be detrimental for the success of aquaculture practices. While, in most of the cases emphasis is given to the production of antibacterial compounds, and in vitro inhibitory activities of a bacterium for choosing a probiotic strain, however, other criteria like non‐antibiotic resistant, non‐haemolytic nature and non‐pathogenic potentiality of probiotic strain are often overlooked. We have screened gastrointestinal bacteria isolated from all the three species of Indian major carps viz., Labeo rohita, Catla catla and Cirrhinus mrigala for a suitable probiotic. Bacteria like Aeromonas (Aeromonas species, A. hydrophila), Micrococcus, Corynebacterium, Plesiomonas, Bacillus (Bacillus species, B. subtilis) and Pseudomonas (Pseudomonas species, P. aeruginosa) exhibited antibacterial activities against different pathogens. Among these, B. subtilis was found to grow at a wider range of temperatures, pH and salt concentrations and was non‐haemolytic, non‐antibiotic resistant, non‐pathogenic and inhibited all the tested primary and secondary pathogens. Dietary supplementation of the bacteria at 10⁸ CFU g^?1 feed also enhanced the growth of Indian major carp, L. rohita. Based upon these characteristics, we aimed to develop a suitable probiotic for aquaculture practices.     

Short‐term effects of dietary soybean meal and lactic acid bacteria on the intestinal morphology and microbiota of Atlantic salmon (Salmo salar)

The aim of this study was to evaluate the role of the intestinal microbiota in soybean meal enteritis. Three groups of Atlantic salmon (Salmo salar) were fed for 35 days with different diets: a control diet (CD) containing 510 g kg^?1 fishmeal, diet 1 (D1) containing 378 g kg^?1 of soybean meal and diet 2 (D2) containing D1 supplemented with two viable lactic acid bacteria (LAB). As expected, the fish fed with D1 showed clear signs of distal intestinal inflammation during the study compared with the fish fed CD. For the fish fed with D2, the addition of LAB diminished the inflammation at day 28, but did not abolish it. Microbiological analysis demonstrated that specific bacterial groups were not correlated with the development of enteritis, but were correlated with the three diets. Microbacterium, Pseudomonas, Lactococcus lactis sp. cremoris and Aeromonas VIa were correlated with the CD, Aeromonas VIb and Sporosarcina aquimarina were correlated with D1, and Alcaligenes, Acinetobacter, L. lactis sp. lactis and Carnobacterium maltaromaticum were correlated with D2. Shewanella was not affected by the diet and was present in all fish intestines. Our study suggests that LAB may modulate intestinal inflammation; however, the role of the microbiota in the aetiology of enteritis in fish still requires further study.     

3种乳酸菌对乌鳢生长、抗氧化及免疫功能的影响

为研究3种乳酸菌对乌鳢生长、抗氧化及免疫功能的影响,实验选用初始体质量为(3.43±0.05) g的乌鳢360尾,随机分成4组,每组设3个重复,每个重复30尾鱼。分别投喂基础饲料和添加了10~8 CFU/g的乳酸乳球菌L21(L21组)、植物乳杆菌W21(W21组)、粪肠球菌L2(L2组)的实验饲料,8周后,采集乌鳢血清、肝脏、脾脏、肾脏和肠道,以检测相关指标以及基因表达水平的变化。结果显示,与对照组相比,饲料中添加的3种乳酸菌均可显著提高乌鳢的平均增重率(AWGR)、特定生长率(SGR)及饲料效率(FER),其中L21组显著高于其他各组。与对照组相比,L2组超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)及碱性磷酸酶(AKP)活性显著高于对照组,W21和L2组溶菌酶(LZM)活性显著高于对照组,各组免疫球蛋白M(IgM)活性均不显著。各组织中IL-1β、IL-8、IL-10和TNF-α基因表达水平均有不同程度升高,乳酸菌对肠道中免疫相关基因表达均有显著的促进作用。研究表明,本实验条件下,乳酸乳球菌L21对乌鳢的应用效果最好,能够提高乌鳢的生长性能、饲料利用及免疫基因表达,但对血清抗氧化能力并无显著影响。     

Calcium alginate capsules for oral administration of fish probiotic bacteria: assessment of optimal conditions for encapsulation

The application of probiotics in aquaculture represents a promising alternative for disease prevention. These microorganisms must survive adverse conditions during handling and storage to be administered in high concentrations to the host. This is especially important when stressful conditions, such as low water contents, are present in animal feed. Bacterial encapsulation has been used to maintain probiotic viability in human food; however, the usefulness of the encapsulation techniques in animal feed needs to be evaluated. In the present study, optimal conditions for encapsulation in alginate of a fish probiotic Shewanella putrefaciens strain (Pdp11) have been determined. Different alginate (1%, 2%, 3% and 4% w/v) and calcium chloride (0.5%, 1% 2% and 3% w/v)concentrations were used to elaborate alginate beads. The results obtained indicate that S. putrefaciens Pdp11 can be encapsulated successfully in calcium alginate beads. The percentages of encapsulated cells were above 80%, the lowest efficiency rates corresponding to high calcium availability (3%). In addition, capsules containing viable S. putrefaciens Pdp11 can be stored at 4 °C for at least 1 month, survival rates being above 90%. On the contrary, storage of the capsules at 22 °C resulted in 40% viability loss within 30 days. Finally, the survival of encapsulated probiotics through fish gastrointestinal tract has been demonstrated.