大闸蟹肠道中产酶菌株及其胞外酶的研究

从健康成年大闸蟹肠道中分离出细菌36株。革兰氏染色结果表明，11株为革兰氏阳性菌，25株为革兰氏阴性菌。52．8％的菌株能分泌蛋白酶，44．4％的菌株能分泌脂肪酶，58．3％的菌株能分泌淀粉酶，41．7％的菌株能分泌纤维素酶。36株菌均能产酶，其中产4种酶的有1株，产3种酶的有9株，产2种酶的有14株。由于这些产酶菌株的作用，使得大闸蟹的肠道菌群能够维持平衡，提高饲料的消化利用率，促进大闸蟹生长。     

草鱼和银鲫肠道产消化酶细菌的研究

检测了分别从草鱼(Ctenopharyngodon idellus)和银鲫(Carassius auratus gibelio)肠道中分离的180株细菌的蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果显示,两种鱼肠道内可分泌胞外消化酶的细菌包括Aero-monas(气单胞菌属,Aer.)、Vibrio(弧菌属,Vib.)、Bacillus(芽孢杆菌属,Bac.)、Pseudomonas(假单胞菌属,Pse.)四个种属的细菌,Aer.在其中占主要优势,45.71%的Aer.可分泌胞外消化酶。草鱼可分泌上述四种胞外消化酶的菌株共有33株,占肠道菌总数的36.67%;银鲫43株,占47.78%。产酶菌的分布上,草鱼中肠内产消化酶细菌数量显著多于前肠和后肠(P<0.05),前、中、后肠分别是6株、20株和7株;银鲫中肠和后肠数量差异不显著,前肠分布最少。草鱼分泌蛋白酶、脂肪酶、淀粉酶和纤维素酶的菌株分别有21株(23.33%)、10株(11.11%)、30株(33.33%)和16株(17.78%)。银鲫肠道内未检测到可分泌纤维素酶的细菌,蛋白酶、脂肪酶和淀粉酶菌株的数量分别是21株、37株和17株。可见鱼类肠道细菌对食饵消化有重要作用。     

尼罗罗非鱼肠道中产酶菌株的研究

从尼罗罗非鱼肠道中分离出41株好氧菌和6株厌氧菌。好氧菌中31株为革兰氏阴性菌，10株革兰氏阳性菌；厌氧菌中，革兰氏阳性菌5株，革兰氏阴性菌1株。41株好氧菌中有31．7％的菌株能分泌蛋白酶(13株菌)；有39．0％的菌株能分泌脂肪酶(16株菌)；有17．1％的菌株能产淀粉酶(7株菌)，有36．6％的菌株能产纤维素酶(15株菌)。其中，产3种酶的有7株菌，产2种酶的有9株菌，产1种酶的有12株菌，不产酶的有13株菌。好氧菌在尼罗罗非鱼消化食饵过程所起的作用大；6株厌氧菌中仅有1株菌能产脂肪酶和纤维素酶，对尼罗罗非鱼消化食饵所起作用相对不大。优化罗非鱼肠道中微生物菌群的结构很有必要。     

福建大连1号杂交鲍肠道及养殖水体中细菌产酶能力的分析

从大连1号杂交鲍养殖水体及肠道中分离出18株弧菌和32株异养型细菌,对其进行革兰氏染色及产胞外酶能力的分析。结果表明,养殖水体中分离出的14株弧菌和22株异养细菌,共有22株(61.1%)具有分泌脂肪酶、卵磷脂酶、明胶酶、蛋白酶、淀粉酶或溶血毒素的能力;鲍肠道分离出的4株弧菌和10株异养细菌,共有9株(64.3%)具有此产酶能力。但总体上,从水体中分离出的菌株的产酶能力要强于从鲍肠道中分离出的菌株,且数量更多,尤其是水体中异养细菌。在所有的50株菌株中,有2株(4%)具有产3种酶(脂肪酶、卵磷脂酶和溶血素)的能力;有9(18%)株具有产2种酶的能力。产酶菌株大部分为革兰氏阴性。本次试验对鲍肠道及养殖水体中菌株产酶能力进行了分析,为大连1号杂交鲍在南方的健康养殖提供科学依据。     

近江牡蛎肠道厌氧菌及其产酶能力的研究

为了解近江牡蛎肠道厌氧菌在食饵消化过程中的作用，为微生态饲用添加剂的开发研究打下基础，从近江牡蛎肠道中分离出8株厌氧菌，其中有5株为革兰氏阳性菌。并研究了它们蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力，结果表明仅有3株菌具有产酶能力：2株菌能分泌脂肪酶，1株菌能分泌蛋白酶。由此可见．厌氧菌在分泌消化酶、消化食饵中所起的作用不大。     

虾池有机污染物降解细菌的筛选

在实验室条件下对612株海洋细菌和10株海水驯化淡水菌进行筛选,检测其快速降解水体中富营养有机物的能力。利用对虾饵料培养基、BOD仪、胞外酶检测等方法进行筛选,最后筛选到10株对富营养有机物具有 较高降解性能的细菌。所有的细菌均能产生明胶酶和脂酶（Tween-80),其中9株细菌能产生淀粉酶,8株产生卵磷脂酶,2株产生酪蛋白酶,1株产生褐藻胶酶。通过测量BOD来衡量10株细菌利用对虾饵料的效果,2天时能消化46.6%-59.5%的对虾饵料,5天时能消化50.8%-70.2%的对虾饵料。用常规生理生化方法将细菌鉴定到属,其中3株为弧菌属细菌（Vibrio spp.),3株为假单胞菌属细菌（Pseudomonas spp.),2株为发光杆菌属细菌（Photobacterium spp.),1株为气单胞菌属细菌（Aeromonas spp.),1株为枯草杆菌（Bacillus subtilis)。     

近江牡蛎养殖水体中细菌产酶能力的研究

为了解近江牡蛎养殖水体中细菌的产酶特性 ,筛选出有应用潜力的有益菌 ,本实验从近江牡蛎养殖水体中分离到 32株菌。革兰氏染色表明 ,10株为革兰氏阳性菌 ,2 2株革兰氏阴性菌。在此基础上研究了它们蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果表明 ,6 1.9% ( 17株 )的菌株能分泌蛋白酶 ,71.9% ( 2 3株 )菌株能分泌脂肪酶或淀粉酶 ,34.4 % ( 11株 )的菌株产纤维素酶。这些产酶菌株均以革兰氏阴性菌为主。在这 32株菌中 ,产四种酶的有 5株菌 ,产三种酶的有 9株菌 ,产两种酶的有 9株菌 ,产一种酶的有 9株菌 ,也就是说 ,所有的菌株都能产酶。由此表明近江牡蛎养殖水体中细菌在该环境物质循环中的重要地位。     

凡纳滨对虾肠道内产消化酶益生菌的分离与筛选

为获得具有消化酶活性且安全的益生菌,从凡纳滨对虾肠道中初步分离得到576株细菌,对菌株进行产蛋白酶、淀粉酶和脂肪酶能力的定性及定量测试,筛选出产酶种类多且产酶能力强的菌株11株。对筛选出的11株菌进行了幼虾浸浴实验、药敏性实验和溶血性实验,以评价其生物安全性。将11株菌的菌悬液添加到凡纳滨对虾幼虾的养殖水体中进行浸浴实验,浸浴结束后用鳗弧菌进行刺激,测定不同浸浴组幼虾相关免疫基因的相对表达量,以确定其对幼虾的保护效果。综合消化酶活性、菌株对幼虾的保护效果及生物安全性,筛选得到4株效果较好的菌株。菌株的16S r DNA分子鉴定结果表明,细菌1号、2号和4号分别与芽孢杆菌(Bacillus sp.PCSAS2-38,GQ284495.1)、蜡样芽孢杆菌(B.cereus strain N419,JN400121.1)及苏云金芽孢杆菌(B.thuringiensis strain EA26.1,KC758847.1)的相似性均为100%,9号菌株与荚膜红细菌(Rhodobacter capsulatus strain PSB-03,FJ866782.1)相似性达到99%,为后续益生菌制剂的开发奠定了前期基础。     

黄鳝肠道细菌及产酶菌株的研究

从健康的黄鳝肠道中分离出细菌32株.革兰氏染色结果表明,8株为革兰氏阳性菌,24株为革兰氏阴性菌.研究了其产蛋白酶、脂肪酶、淀粉酶、纤维素酶的能力.结果表明,有53.13%的菌株能分泌蛋白酶,43.75%的菌株能分泌脂肪酶,34.38%的菌株能分泌淀粉酶,21.88%的菌株能分泌纤维素酶.其中产4种酶的有1株,产3种酶的有5株,产2种酶的有9株,产1种酶的有12株,不产酶的仅有5株.产酶菌株的比例高达84.38%.     

饲用微生物添加剂的研究开发现状与展望（下）

产生多种酶:地衣芽孢杆菌在肠道生长繁殖过程中能产生多种酶。柏建玲报道,该菌能产生淀粉酶、蛋白酶和植酸酶,笔者通过淀粉酶平板试验,也证实该菌能富产淀粉酶。如用于动物,在动物体内可形成一个小型的"酶工厂"。不但能防病治病,而且有促进消化和提     

九孔鲍肠道及其养殖水体中弧菌产酶能力的分析

从汕尾健生鲍鱼场养殖水体和九孔鲍肠道中共分离筛选到25株弧菌,其中14株来自成鲍肠道,11株来自养殖水体,并对它们产多种胞外酶的能力进行了比较分析。试验结果表明:78.6%的肠道弧菌能分泌蛋白酶,21.4%能分泌脂肪酶,64.3%能分泌卵磷脂酶或淀粉酶,92.9%能分泌明胶酶;水体中的弧菌63.6%能分泌蛋白酶,54.5%能分泌脂肪酶或明胶酶,72.7%能分泌卵磷脂酶,100%能分泌淀粉酶。同时筛选出5株胞外酶分泌能力超强的菌株,除分泌蛋白酶能力最强的弧菌(Bh14)来自鲍鱼肠道外,其余分泌其它4种胞外酶能力最强者(Sh05、Sh03、Sh11、Sh08)均来自养殖水体。研究还发现来自水体的弧菌分泌多种胞外酶能力强于来自肠道的弧菌。由此提示,应用有益微生物以改善鲍鱼肠道中的菌群结构,可能会提高鲍鱼饲料利用率。     

Enzyme producing bacterial flora isolated from fish digestive tracts

Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried out.Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media. The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability. Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities. Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities. Maximumactivity of lipase was exhibited by a strain isolated from silver carp. Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut. The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics.     

海南西岛九孔鲍养殖水体中细菌胞外产物的分析

2004年11月,自海南西岛的鲍养殖水体中分离出90株菌株。TCBS和2116E培养基分别分离40株(弧菌)和50株(异养菌)。比较了不同分离方法所得到的2批菌株分泌胞外产物的能力。试验结果表明,鲍苗掉板死亡期间,养殖水体中TCBS培养基分离的菌株能分泌脂肪酶、淀粉酶、磷脂酶和明胶酶的比例总体上高于2116E培养基分离的菌株,2116E培养基分离的菌株中能分泌溶血素的比例要略高于TCBS培养基分离的菌株。总体上,后者具有较强分泌胞外产物的能力。因此,这部分具有较强分泌胞外产物能力的菌株应视为鲍苗的潜在致病菌。此外,在对鲍的细菌性病害研究中,除了具有较强分泌胞外产物的菌株外,养殖环境中的菌群结构也是不可忽视的因素。     

Diversity and activity of cellulolytic bacteria,isolated from the gut contents of grass carp (Ctenopharyngodon idellus) (Valenciennes) fed on Sudan grass (Sorghum sudanense) or artificial feedstuffs

Herbivorous grass carp (Ctenopharyngodon idellus) has a powerful capability to digest cellulose from aquatic plants, depending on the cellulase complex produced by the cellulolytic bacterial community in the gastrointestinal (GI) tract. However, it remains uncertain which bacteria taxa may actively participate in the digestion of food fibre. In this study, a total of 499 cellulolytic bacteria from the gut content of grass carp fed on Sudan grass (242 strains) and artificial feedstuffs (257 strains) were randomly isolated and characterized using carboxymethyl‐cellulose, microcrystalline cellulose and cellobiose agar media. The results showed that more than half of the isolates were capable of degrading carboxymethyl‐cellulose and cellobiose, while the remaining isolates were restricted to microcrystalline cellulose decomposition, exclusively. The cellulolytic bacterial community was dominated by Aeromonas, followed by Enterobacter, Enterococcus, Citrobacter, Bacillus, Raoultella, Klebsiella, Hydrotalea, Pseudomonas, Brevibacillus and some unclassified bacteria, as revealed by 16S rDNA sequence analysis. Notably, grass carp fed on grass with high‐fibre content harboured a higher diversity of cellulolytic bacteria than the ones fed on low‐fibre feedstuffs. Our results provided evidence for a positive correlation between the content of food fibre and the diversity of cellulolytic bacteria in grass carp intestines. Thus, improving growth conditions and cellulase activities for GI cellulolytic microorganisms in grass carp intestines are critical for effective utilization of feedstuffs containing high fibre levels.     

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability,isolated from freshwater aquaculture pond

Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca²⁺ 0.8, Mg²⁺ 0.2, Mn²⁺ 0.4, Fe²⁺ 0.6, Al³⁺ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics.     

不同水平的蚯蚓和蚯蚓粪对草鱼消化能力的影响

本研究首次在基础饲料中添加不同浓度的蚯蚓(5%、7.5%、10%)和蚯蚓粪(2%、4%、6%),饲养平均体重(54.03±0.15)g的草鱼100d,研究蚯蚓和蚯蚓粪对草鱼消化能力的影响。结果表明:添加7.5%～10%蚯蚓和6%蚯蚓粪显著提高了草鱼肝胰脏和前肠中胃蛋白酶、胰蛋白酶、淀粉酶和脂肪酶的活性,以添加7.5%蚯蚓效果最好;采用石蜡切片技术观察各试验组草鱼前肠肠道内部结构,结果表明,添加7.5%～10%蚯蚓可显著提高草鱼肠道褶皱高度、宽度和基层厚度。综上所述,在提高草鱼消化能力方面,以饲料中添加7.5%蚯蚓的效果最好。     

穿心莲对草鱼肠内细菌的影响

给不同组草鱼分别投喂含0％、0.5％、1％和2％穿心莲的人工配合饲料，在不同时期取样检测各组草鱼前、中、后肠需氧和兼性厌氧菌的种类和数量。结果显示，草鱼肠内细菌的种类，特别是优势菌群的种类没有受到穿心莲的影响。各组草鱼肠内各部位的优势菌群均为气单胞菌Aeromonas和肠杆菌Enterobacteriaceae。草鱼肠内细菌数量为105～108CFU·g-1，分析后发现，各组草鱼肠内细菌数量之间没有显著差异(P＞0.05)。但1％及2％的穿心莲能降低气单胞菌的组成。上述结果表明，一定浓度的穿心莲可以通过降低优势菌群气单胞菌的组成而对草鱼肠内微生态系产生影响。     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

鲤、鲢、鳙、草鱼消化道消化酶种类和活性的比较研究

采用聚丙烯酰胺凝胶电泳方法研究了鲤、鲢、鳙、草鱼亲鱼消化道消化酶的种类；用比色法及滴定法分别测其消化酶相对活性。结果表明：鲤、鲢、鳙、草鱼4种鱼消化道的蛋白酶、淀粉酶活性与食性有明显的相关性。其中，蛋白酶活性依次为鲤〉鲢〉鳙〉草鱼；淀粉酶活性依次为草鱼〉鲤〉鳙〉鲢。脂肪酶活性与食性的关系不明显。四种鱼消化酶的表达与消化道的组织结构特征相一致，消化道前2／5部位（G1、G2）的消化酶活性最高，是无胃鱼消化道的主要消化场所。消化道中央部位（G3）的消化酶活性次之，能将未消化完全的食糜进一步消化，而消化道后段（G4、G5）消化酶活性最低。可见，消化酶活性及消化功能呈递减形式，食物在消化道中随着消化、吸收，逐渐排出体外。本研究为鲤、鲢、草鱼、鳙4种鱼的营养生理学、高效饲料配比的研制及种质资源的科学管理提供理论依据。