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.     

Phylogenetic analysis of intestinal bacteria of freshwater salmon Salmo salar and sea trout Salmo trutta trutta and diet

The microbial diversity of culturable intestinal microflora of wild freshwater salmonid fishes salmon Salmo salar and sea trout Salmo trutta trutta juveniles (0+ years old) from the same environmental conditions were investigated by means of molecular identification techniques and analysis of diet. Significant differences in the intestinal microbial diversity were observed in different fish species. The predominant group in the intestinal tract of the salmon comprised representatives of the Enterobacteriaceae family (23%), Plesiomonas (19.2%) and Carnobacterium (15.3%). Predominant microbiota in sea trout intestinal tract were Enterobacteriaceae (52%), Aeromonas (22%) and Pseudomonas (14%). The results show that Enterobacteriaceae were predominant in the intestinal tract of the salmon and the sea trout juveniles raised on diets of different compositions. However, molecular identification of the intestinal microbiota at the species or genus level revealed differences in these fish species. Bacteria in the hindgut of salmon included Pragia and Serratia. However, bacteria in the gut contents of sea trout from the Enterobacteriaceae family were Buttiauxella, Enterobacter, Moellerella, Pantoea, Rahnella and Tiedjeia arctica. A novel phylotype of T. arctica is harbored in the intestinal tract of wild salmon, and may correspond to a previously undescribed species.     

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。     

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.     

Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS‐PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS‐PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N‐α‐p‐tosyl‐l ‐lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N‐tosyl‐l ‐phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin‐ and chymotrypsin‐like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.     

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.     

Assessment of microbial diversity in the river trout Salmo trutta fario L. intestinal tract identified by partial 16S rRNA gene sequence analysis

ABSTRACT:   The microbial diversity of the intestinal tract content of the river trout from two Lithuanian rivers has been investigated by molecular methods: polymerase chain reaction amplification and sequencing of partial 16S rRNA genes. Predominant bacterial populations detected in the river trout intestinal tract from the Skroblus River were Rahnella (21%), from the Žeimena River, Aeromonas (41.7%) and Plesiomonas (22.9%). Buttiauxella agrestis, Budvicia aquatica, Erwinia persicinus , Yersinia mollaretii , Y. kristensenii , Y. rohdei , Moellerella wisconsensis , Obesumbacterium proteus , Pantoea cedenensis , Rahnella aquatilis , and Rahnella sp. from the Enterobacteriaceae family have been detected in the intestinal tract of freshwater salmonid fish for the first time.     

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.     

养殖澳洲宝石鱼迟缓爱德华氏菌的分离鉴定及致病基因的检测

从患病澳洲宝石鱼体内分离到一株致病菌(编号Et4),对该菌的生化特性及致病基因进行检测,并进行了药敏和人工感染实验。结果显示:菌株Et4的生化鉴定结果与迟缓爱德华氏菌(Edwardsiella tarda)标准菌株(AY77513)一致;其16S rRNA序列,经同源性比对与迟缓爱德华氏菌核苷酸相似度最高,达99%;根据迟缓爱德华氏菌的致病基因Ⅲ型分泌系统装置蛋白esaV基因序列设计引物,进行PCR扩增,得到708 bp序列,该序列与迟缓爱德华氏菌的esaV基因序列相似性达99.3%,说明菌株Et4具有esaV基因。菌株Et4对环丙沙星等较敏感,对其它药物中度或不敏感,具有较强的致病力(LD50=3.74×104CFU/mL),从病鱼中可以重新分离出此菌。综合形态学、生化特性、16S rDNA序列及致病基因序列鉴定其为迟缓爱德华氏菌。     

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.     

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。     

Salmonid gill bacteria and their relationship to amoebic gill disease

16S ribosomal RNA gene analysis was used to assess the bacterial community associated with Atlantic salmon, Salmo salar L., gills which were either affected by amoebic gill disease (AGD) or were AGD-negative, in order to determine the role that bacteria may play in the development of AGD. AGD-positive specimens were either infected in the laboratory with Neoparamoeba pemaquidensis, the causative agent of AGD, or were obtained from commercial salmon cages. Samples from laboratory fish maintained in sea water possessed a marine-type community while field samples which had been treated by a series of freshwater baths possessed a more diverse community which included variable proportions of different bacterial ecotypes, including groups typically associated with soil, skin surfaces and faeces. Samples from fish infected with AGD in the laboratory and a sample from one of two salmon cage fish specimens were dominated by a phylotype belonging to the strictly marine bacterial genus Psychroserpens (family Flavobacteriaceae, phylum Bacteroidetes). The phylotype was not detected in any of the AGD-negative samples or in one of two AGD-positive samples obtained from fish subjected to temporary freshwater immersion. The possibility of certain Psychroserpens species as potential opportunistic pathogens associated with salmonid AGD is proposed.     

Secondary stress responses in Indian major carps Labeo rohita (Hamilton), Catla catla (Hamilton) and Cirrhinus mrigala (Hamilton) fry to increasing packing densities

Glycogen content and metabolic enzyme activities viz. lactate dehydrogenase (LDH), malate dehydrogenase (MDH), aspartate amino transferase (AST) and alanine amino transferase (ALT) in Indian major carps, Labeo rohita, Catla catla and Cirrhinus mrigala, were investigated after a 6 h transportation trial to compare the species‐specific variation and the effect of increased packing density on the metabolism. Fish (45±5 mm, 0.5±0.1 g) were packed in three densities (100, 150 and 200 L^?1) for the experiment, and 12 specimens of each species were randomly sampled from all the treatments at the end of transportation. The glycogen content of L. rohita ingerlings decreased significantly (P<0.05) with increasing packing density. The activities of enzymes LDH, MDH, AST and ALT showed a rising trend with increasing packing density in all the three species. Species‐specific differences were observed in various tested parameters at the lowest packing density (100 fry L^?1). Alanine amino transferase and LDH activities were significantly (P<0.05) lower in C. mrigala as compared with the other two species. However, glycogen reserves and MDH activity were not significantly different (P>0.05) among the species. The present study reveals that the optimum packing density for Indian major carp fry (100 fry L^?1) for transportation up to 6 h and metabolic regimes are species specific during transportation.     

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.     

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.     

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。     

A study on bacteria associated with the intestinal tract of farmed yellow seahorse,Hippocampus kuda (Bleeker, 1852): characterization and extracellular enzymes

The microenvironment of bacteria associated with the gastrointestinal tract of an animal influences the host in many ways, including the metabolism of several nutrients. Isolation, molecular characterization and enzymatic activities of culturable bacteria associated with the gastrointestinal tract of hatchery‐reared oceanic yellow seahorse, Hippocampus kuda, were investigated in the present study. 16S rRNA sequencing of bacterial isolates yielded 10 different bacterial genera belonging to two major groups: proteobacteria (62.9%) and firmicutes (37.03%). The predominant microbial flora belonged to the genera Vibrio spp., Enterovibrio sp. and Bacillus spp. Burkholderia cenocepacia, hitherto unreported from the gastrointestinal tract of marine fish, was retrieved from the seahorse gut, albeit in small numbers. Enzymatic studies indicated that the bacterial isolates possess the ability to degrade lipids, cellulose, xylan, starch and proteins. Lipase activity was exhibited by a majority of the isolates (70.4%), indicating that the bacterial flora associated with the intestinal tract of seahorses is capable of digesting foods rich in lipids. The possible implications for the role of these bacteria in the digestion and physiological processes are discussed. To our knowledge, this is the first time that the characterization and enzymatic activities of bacteria associated with the intestinal tract of farmed seahorses have been reported.     

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。     

Cellulase‐producing bacteria of Aeromonas are dominant and indigenous in the gut of Ctenopharyngodon idellus (Valenciennes)

Herbivorous grass carp (Ctenopharyngodon idella) has a powerful capability to digest aquatic weed. Cellulase activity or cellulase‐producing bacteria were found in the gut of grass carp. However, it remains uncertain whether the cellulase‐producing bacteria were a part of indigenous intestinal microbiota that the fish harboured or were introduced with food. In the present study, the bacterial diversities and population abundance in the gut of starved grass carps have been investigated by sequencing 16S rRNA gene libraries. The 16S rRNA gene libraries revealed that 28 parasitic bacteria from gut were affiliated to seven genera of Vibrio, Acinetobacter, Providencia, Yersinia, Pseudominas, Morganella or Aeromonas, respectively, and Aeromonas was identified as the most dominant genus in the gut of C. idella. All of cellulase‐producing bacteria isolated from the gut of C. idella in this research belonged to Aeromonas. On the whole, the results in this research showed that cellulase activity within C. idella should be at least partially resulting from bacteria of Aeromonas with cellulase‐producing capabilities, which were indigenous and dominant intestinal species.