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.     

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。     

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.     

Preliminary assessment of in vivo safety of potentially probiotic lactic acid bacteria for American bullfrog culture

The effect of the administration of beneficial lactic acid bacteria (LAB): Lactococcus lactis CRL 1584, L. lactis CRL 1827, Enterococcus gallinarum CRL 1826 and combined CRL 1584+ CRL 1826 on the development of bullfrog embryos from the hatching stage until 31 days (tadpole) was evaluated. In vitro assays indicated that 10³, 10⁶ and 10⁹ CFU mL^?1 single LAB strains remained viable until 24 h in 10% Ringer. Around 10⁹ CFU mL^?1 LAB (individually and combined) were used in an experimental design built to evaluate their effect when administrated at different intervals (three 7‐day cycles with 5‐day rest periods in between) to embryos until day 31. The highest potentially beneficial population (LAB) numbers were detected in the LAB‐treated groups. All the LAB‐treatments increased it and were significantly higher than the controls. Although the highest, potentially pathogenic, population (Red‐Leg Syndrome‐RLS‐related pathogens) numbers were detected in the control and the lowest in the CRL 1584+ CRL 1826‐treated group, they did not differ significantly. Stereoscopic studies showed no malformations in any LAB‐treated group and all the specimens reached the same stage of their biological cycle with a survival >94%. The histological structure of target organs for RLS‐associated pathogens (intestine and skin) and stomach was not affected and the spleen was developed. Only the LAB‐treated groups showed microorganisms associated with the intestinal mucus, without inflammatory reaction in the lamina propria. This is the first report on the evaluation of the absence of adverse effects after LAB administration to bullfrog embryos using indigenous microorganisms.     

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。     

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...     

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.     

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。     

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.     

Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria

ABSTRACT:   The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.     

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.     

海带孢子体、配子体时期附生菌群落结构的比较

为探究海带(Saccharina japonica)生活史孢子体、配子体不同阶段的附生菌群落结构的差异,采用Illumina测序技术,分析了种海带(S1)、配子体(S2)、幼孢子体(S3)和大孢子体(S4)这4个不同阶段的藻体上附生菌16S rRNA序列,研究其附生菌群落结构。结果显示,4个阶段共识别出23门156属共244个操作分类单元(OTU)。从门分类上来说,变形菌门(Proteobacteria)在海带各阶段中均表现出较高的丰度,在S1、S2、S3和S4藻体样本中相对丰度分别达53.0%、94.3%、77.2%和36.7%,蓝细菌门(Cyanobacteria)只在S4藻体样本中表现出较高的丰度,为43.8%;从属分类上来说,假交替单胞菌属(Pseudoalteromonas)在各样本间差异较大,S2、S3和S4样本中相对丰度分别为65.00%、44.88%和25.57%,但在S1样本中仅为0.18%。各样品间附生菌群落结构差异较大,S1与S4样本相邻聚为一支,S2与S3样本为另一支。S2样本的菌群种类单一且分布不均匀,S1、S3与S4样本细菌种类丰富,但S4样本中菌群间的丰度差异较大。海带的苗种繁育阶段(S2和S3)存在褐藻酸降解菌丰度增加的趋势,孢子体阶段(S4)则具有高丰度的共生型蓝细菌。     

The Effects of Fermentation Process with Acid and Lactic Acid Bacteria Strains on the Biogenic Amine Formation of Wet and Spray-Dried Fish Silages of Discards

The impacts of fermentation process with acid and lactic acid bacteria strains (Lactobacillus plantarum, Lactobacillus brevis, Pediococcus acidilactici, Enterococcus gallinarum, and Streptococcus spp.) on the biogenic amine formation of wet and spray-dried fish silage obtained from whole gibel carp (Carassius gibelio, freshwater discard), whole ponyfish (Equulites klunzingeri, seawater discard), and processing by-products of seabass (Dicentrarchus labrax) were investigated. The results showed that among biogenic amines, cadaverine, putrescine, spermidine, spermine, serotonin, dopamine, and agmatine were predominant in all groups. Significant differences (p < 0.05) in biogenic amine concentrations of wet and spray-dried fish silage were observed. Raw fish and wet silages contained histamine level lower than the allowable limit of 50mg/kg, indicating the use of raw fish material with low microbial counts. In addition, no histamine was detected in spray-dried fish silage, except for seabass by-products with a trace quantity of histamine (<0.04mg/100g). The starter culture used for silage preparation did not effectively retard formation of biogenic amines compared to acid silage. It can be concluded that there is potential use of fermented fish silage as a protein source and possibly as a probiotic ingredient for animal feed in both wet and dry form.     

Dietary supplementation of host‐associated lactic acid bacteria modulates growth,metabolic activities,and immune‐related gene expression in giant freshwater prawn,Macrobrachium rosenbergii

The present study was carried out to evaluate the dietary effects of host‐associated lactic acid bacteria on growth performance, metabolic enzyme activities, and immune response of Macrobrachium rosenbergii juveniles. To formulate the test diets, a control (Con) diet was supplemented with a commercial probiotic and three host‐derived bacteria Enterococcus faecalis (EC), Lactococcus lactis I (LC‐I), and L. lactis II (LC‐II), which were previously isolated from the gastrointestinal tract of adult individuals of M. rosenbergii. Juvenile M. rosenbergii (0.65 ± 0.008 g) were randomly stocked at 20 individuals/100 L of fiberglass tanks with three replications for each test diet. After 50 days, juveniles fed the diets LC‐I and LC‐II showed significantly higher (p < .05) weight gain, specific growth rate, and the lowest feed conversion ratio. The analyses of glutamic oxaloacetate transaminase and glutamic pyruvate transaminase in muscle and hepatopancreas revealed significantly (p < .05) reduced values in LC‐I fed juveniles. The total hemocyte count and phenoloxidase activity were significantly increased (p < .05) in LC‐I and LC‐II fed juveniles. The relative mRNA expression patterns of immune‐related α2‐M, LGBP, ProPO, Cu, Zn‐SOD, TG, PE, AKP, and ACP genes were significantly (p < .05) upregulated in juveniles fed with LC‐I followed by the diet LC‐II. Finally, the study suggests that the growth performance and immune response of M. rosenbergii can be improved through supplementation of host‐associated L. lactis bacteria for its higher production.