Dietary administration of Bacillus (B. licheniformis and B. subtilis) and isomaltooligosaccharide influences the intestinal microflora,immunological parameters and resistance against Vibrio alginolyticus in shrimp,Penaeus japonicus (Decapoda: Penaeidae)

The experiment was conducted to investigate the effects of oral administration of probiotics (Bacillus licheniformis and Bacillus subtilis) in combination with prebiotic isomaltooligosaccharide (IMO) on the intestinal microflora and immunological parameters of Penaeus japonicus and its resistence against Vibrio alginolyticus. A basal diet was supplemented with 0% probiotics and prebiotic (control), 10⁸ colony forming unit (CFU) g^?1B. licheniformis and 0.2% IMO (T1), 10⁸ CFU g^?1B. subtilis and 0.2% IMO (T2), 10⁸ CFU g^?1B. licheniformis in combination with 10⁸ CFU g^?1B. subtilis and 0.2% IMO (T3). The results showed that the total bacterial counts significantly increased (P<0.05) and Vibrio counts significantly decreased (P<0.05) in the intestine of shrimp with supplementation of dietary synbiotics. Shrimp fed the diet with both Bacillus probiotics and IMO (T3) produced significantly higher immune parameters (phenoloxidase activity, lysozyme activity and nitric oxide synthase activity and superoxide dismutase activity) than the control group (P<0.05). Significant lower cumulative mortality after challenge with V. alginolyticus was also observed in shrimp fed diet with both Bacillus probiotics and IMO (T3) than that in the control group (P<0.05). The results indicated that the oral administration of probiotics (B. licheniformis and B. subtilis) and prebiotic IMO has positive effects on bacterial flora in P. japonicus gut, and can activate non‐specific immunity of shrimp.     

芽胞杆菌B13抑真菌作用的研究

对芽胞杆菌Ｂ１３抑真菌的效果进行了研究，研究结果表明：芽胞杆菌Ｂ１３对霉菌、酵母菌均有不同程度的抑制作用。将该菌加在饲料原料中，也具有一定的防霉效果。     

枯草芽孢杆菌B7的分离和净化水质的初步研究

对水产养殖水体及底泥中的芽孢杆菌进行分离,得到四株芽孢杆菌。筛选各项指标较好的B7,研究其对水质的净化作用。结果表明,添加枯草芽孢杆菌后,池水的氨氮和亚硝酸盐浓度比对照池显著降低,即枯草芽孢杆菌B7对水质具有明显的净化作用。     

Antimicrobial activity of partially characterized analytes from Bacillus pumilus(B2)

This study was carried out to characterize the antimicrobial substance produced by the strain of Bacillus pumilus (B2) obtained from Novozymes Biologicals Inc. to compare its antimicrobial activity by agar well diffusion assay and bacteriocin activity assay via critical dilution method against seven different strains of Vibrio spp., specifically V. alginolyticus (A01), V. cholerae (C01), V. fluvialis (F01, F02), V herveyii (H), V. mimicus (M01), V. parahaemolyticus (P01) and V. vulnificus (V01, V02). All Vibrio spp. were isolated from the hemolymph and intestine of the white faeces disease‐infected moribund pacific white‐leg shrimp Litopenaeus vannamei (Boone 1931) and one strain (V. harveyi) from its diseased postlarva. The cell‐free neutralized supernatant (CFNS) of B2 showed moderate thermo‐stability being stable up to 70°C for 60 min with, however, reducing activity above 80°C for 20 min. B2 antimicrobials showed a stable activity within the pH ranging from 6 to 10 at room temperature and at 4°C, while residual antimicrobial activity of crude CFNS showed tolerance to salinity up to 7% of sodium chloride below 4°C. No B2 activity was obtained while exposed to proteolytic enzyme, such as proteinase k and pepsin, while its activity kept stable exposed to lipase. Initial B2 characterization for antimicrobial substance in CFNS revealed proteinaceous in nature owing to activity loss against proteolytic enzymes and no lipid moiety activity against lipase, which could be categorized as bacteriocin‐like inhibitory substance having potential application against several strains of Vibrio spp. in aquaculture.     

Effects of the probiotic Bacillus subtilis (BP11 and BS11) on the growth and survival of Pacific white shrimp,Litopenaeus vannamei

The effect of Bacillus S11 (BS11)‐ and/or Bacillus P11 (BP11)‐supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated. Four feeding treatments of (i) regular feed (control), (ii) BS11‐supplemented feed, (iii) BP11‐supplemented feed and (iv) BS11‐and BP11‐supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL‐30 shrimp at 60 and 90 days, respectively. The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L. vannamei in cultivation, because the average weight and survival of shrimp fed BS11‐supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups. The survival of shrimp fed either BS11‐or both BS11‐and BP11‐supplemented feed was significantly higher (P < 0.05) than that of the control group. In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11‐supplemented feed. After challenge with Vibrio harveyi 639 (~10⁷ CFU mL^?1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11‐supplemented feed.     

枯草芽孢杆菌B2的生长及其对仿刺参的益生特性

从大连湾地区健康的仿刺参养殖池塘沉积物中分离得到一株芽孢杆菌菌株B2,经16SrDNA鉴定以及形态学分析确定其为枯草芽孢杆菌。该菌株生长适宜温度为15~35℃,适宜pH为6~8,适宜盐度为30~50。电镜观察发现,枯草芽孢杆菌B2周围有明显可见的芽孢。枯草芽孢杆菌B2可产淀粉酶和蛋白酶,但不具有产脂肪酶的能力。将枯草芽孢杆菌B2按照仿刺参体质量的0.1%、0.2%和0.3%配成不同密度的菌液,添加至饲料中,投喂7d后发现,该菌株可有效提高仿刺参肠道内的消化酶以及体腔液中多种非特异性免疫酶的活性。结果表明,枯草芽孢杆菌B2可作为益生菌剂的基础菌株添加至仿刺参饲料中。     

Effect of ultraviolet (UV) radiation on the abundance and respiration rates of probiotic bacteria

Effects of ultraviolet radiation (UV) on probiotic bacteria (Bacillus subtilis and B. licheniformis) were tested in two experiments, with the following treatments: (i) UV treatment – using fluorescent and UV‐lamps and (ii) Control – CTRL, using fluorescent lamps. Bacterial abundance and respiration were evaluated every 24 h for 3 days for Experiment 1, and at 0, 6 and 24 h for Experiment 2. In the Experiment 1, total UV dose was 4 336.41 mW cm^?2. UV treatment presented small respiration rates only on day 3, while in the CTRL oxygen consumption was always high. On all days, the abundance of the Bacilli exposed to UV was significantly smaller than that of the CTRL. The second experiment, with total UV dose of 1 445.47 mW cm^?2, presented oxygen consumption in the UV treatment only during the first 6 h. In the CTRL, oxygen consumption increased from the beginning due to the bigger abundance Bacilli cells. Small coccus‐shaped bacteria ocurred in the UV treatment of both experiments. It may be concluded that exposure to UV, normally used for water disinfection, can inactivate probiotic bacteria.     

Effect of probiotic (Bacillus spp.) addition during larvae and postlarvae culture of the white shrimp Litopenaeus vannamei

The effect of the addition of Bacillus probiotic during larvae and postlarvae culture of the white shrimp Litopenaeus vannamei was examined in three separate experiments: (I) Nauplius_4‐5 to Zoea₃, which were exposed to probiotic in the water (P_w), only in the microalgae (P_m), in the water and microalgae (P_wm) and a control with no probiotic (C); (II) Mysis₁ to Mysis₃, which were exposed to probiotic in the water (P_w), only in Artemia (P_a), in the water and Artemia (P_wa) and a control (C); (III) PL₁ to PL₁₀, which were exposed to similar treatments for mysis experiment. The use of probiotic Bacillus spp. resulted in an increase in the survival and growth of zoea and mysis phases, especially when the probiotic was added only in the water. For postlarvae, the use of the probiotic had no influence on the zootechnical parameters, however, there was a reduction in the count of presumptive Vibrio both for water and shrimp.     

草鱼肠道芽孢杆菌的鉴定及产酶能力分析

从100 g左右的健康草鱼(Ctenopharyngodon idellus)肠道中分离出3株疑似芽孢杆菌(Bacillus)菌株(G1、G2、G3),并通过对其进行生理生化特征、16S rDNA全序列和产酶能力的分析,筛选出1株高产酶能力的益生菌株。结果显示:G1、G2和G3菌株与枯草芽孢杆菌在16S rDNA序列相似性≥99%的水平上聚为同一分支,结合形态观察和生理生化特征,最终鉴定G1、G2和G3菌株均为枯草芽孢杆菌(Bacillus subtilis);以水解圈直径/菌落直径比值评定了G1、G2和G3菌株的产纤维素酶、淀粉酶和蛋白酶的能力,其中G1菌株产纤维素酶、淀粉酶和蛋白酶能力(比值分别为2.9、2.2和3.3)均高于G2、G3菌株,具有作为益生菌的潜力。     

Effect of Bacillus subtilis on growth development and survival of larvae Macrobrachium rosenbergii (de Man)

A feeding experiment was conducted to investigate the effect of Bacillus subtilis bacterium, on larval growth and development rate of Macrobrachium rosenbergii (de Man) during February 28 to April 8, 2005 in University Putra Malaysia hatchery. Newly hatched larvae of M. rosenbergii were reared with two dietary treatments consisting of newly hatched Artemia salina nauplii with B. subtilis (10⁸ cells ml⁻¹), and newly hatched A. salina nauplii without B. subtilis carried out in triplicate in 60‐L aquarium (50 L⁻¹). After trial, the larvae that fed B. subtilis‐treated Artemia naupli were found to have higher survival and a faster rate of metamorphosis than larvae that were fed with nontreated Artemia naupli. There were significant differences between B. subtilis‐treated Artemia naupli and nontreated Artemia diet in larval growth and development rate of metamorphosis (P < 0.05). Larval survival after 40 days was significantly greater (P < 0.05) in the B. subtilis‐treated groups (55.3 ± 1.02) compared with the nontreated groups (36.2 ± 5.02%).     

泥鳅养殖水体中一株芽孢杆菌的筛选及其净水效果研究

从泥鳅（Misgurnus anguillicaudatus）养殖池塘水体中分离到4株芽孢杆菌,筛选后获得1株优势目的菌株NQ1;根据形态学特征和生理生化特性结果,将其鉴定为枯草芽孢杆菌（Bacillus subtilis）。安全性试验证实,试验浓度（最高为1×107 cfu/mL）的枯草芽孢杆菌NQ1对泥鳅是安全的。水质净化试验结果显示,在泥鳅养殖水体中加入1×107 cfu/mL浓度的NQ1,14天后氨氮和亚硝酸盐含量较对照组分别降低34.97%和89.46%,表明该菌的净水效果明显,具有作为水质改良微生态制剂开发应用的潜力。     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

山姜对益生芽孢杆菌体外抑菌活性试验研究

为研究中草药山姜粗提液对益生芽孢杆菌体外抑菌效果,特采用试管二倍稀释法和管碟法检测不同浓度的山姜煎煮液和乙醇提取液在体外对蜡样芽孢杆菌PAS38菌株和枯草芽孢杆菌Pab04菌株的最小抑菌浓度(MIC)和抑菌直径,并以肠毒性大肠杆菌(ETEC)、4μg/mL硫酸庆大霉素作阳性对照,生理盐水作阴性对照。结果表明,山姜煎煮液和乙醇提取液对两株益生芽孢杆菌和ETEC无抑菌作用,硫酸庆大霉素对蜡样芽孢杆菌PAS38和枯草芽孢杆菌Pab04菌株MIC均为1.25μg/mL,对ETEC的最低抑菌浓度MIC为0.625μg/mL。山姜煎煮液和乙醇提取液对受试菌株抑菌直径与生理盐水相比差异不显著(P>0.05),硫酸庆大霉素与其相比差异极显著(P<0.01)。山姜粗提液对两株益生芽孢杆菌无明显抑菌作用。     

两种芽孢杆菌体外体内拮抗气单胞菌的试验

将蜡样芽孢杆菌Y1和巨大芽孢杆菌BM1259混合培养,结合平板活菌计数、抑菌活性测定及体内拮抗试验,研究其对水产动物气单胞菌的抑菌效果。体外拮抗试验结果表明,这两种芽孢杆菌均能明显抑制嗜水气单胞菌NQ的增殖,抑制率分别为53.74%和56.12%。不同温度下的抑菌活性试验显示,2种芽孢杆菌的抑菌活性温度分别为32℃和29℃。体内拮抗试验结果表明,巨大芽孢杆菌能显著降低异育银鲫肠道气单胞菌的数量。提示巨大芽孢杆菌可作为潜在的水产专用微生态制剂。     

The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum)

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 x 10(8) cells g(-1) of feed, JB-1 led to a reduction in mortalities to 0-13% after challenge with a range of bacterial pathogens compared to 80-100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0-16% following the challenge compared to 80-100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.     

地衣芽孢杆菌De在优质草鱼养殖中的应用研究

运用综合对比分析法探讨了地衣芽孢杆菌Bacillus licheniformis De在优质草鱼（Ctenopharyngodon idellus）养殖中的应用效果，其评价指标分别为成活率、水体pH、透明度、溶解氧及水中氨氮、硝酸盐浓度等。结果表明，施用地衣芽孢杆菌De可在一定程度上使水体环境和养殖生产性能得到优化，提高养殖草鱼的成活率，显著降低水体透明度及水中氨氮、硝酸盐含量（P〈0．05），使水体pH、溶解氧有利于草鱼的生长。其中施菌组较对照组的成活率、水体pH、溶解氧分别提高了3．2％、3．9％、25．5％，而水体透明度、氨氮及亚硝氮浓度则分别降低了38．5％、74．6％、69．3％。     

Dietary supplement of fructooligosaccharides and Bacillus subtilis enhances the growth rate and disease resistance of the sea cucumber Apostichopus japonicus (Selenka)

The effects of two dietary supplements, entrapped fructooligosaccharide (FOS) and microencapsulated Bacillus subtilis, on the growth and disease resistance of the sea cucumber Apostichopus japonicus were investigated. Sea cucumbers (a total of 810 animals, with initial weight of 3.72 ± 0.16 g each) were fed with basal diet only or basal diet supplemented with three different doses of B. subtilis (0.2, 0.4 and 0.8%), three different doses of FOS (0.4, 0.8 and 1.6%), 0.2% B. subtilis plus 0.4% or 0.8% FOS. Growth and lysozyme activity were measured after 50 days feeding. The results showed that dietary 0.2% and 0.4% B. subtilis alone or 0.2% B. subtilis plus 0.8% FOS significantly increased the specific growth rate (SGR) of sea cucumbers compared with control (P < 0.05). The addition of B. subtilis with or without FOS had no significant effect on lysozyme activity. The animals were also challenged with Vibrio splendidus to assess the level of disease resistance provided by the two dietary supplements. Sea cucumbers fed with 0.8% B. subtilis or 0.2% B. subtilis plus 0.8% FOS exhibited the highest survival (P > 0.05). Under these experimental conditions, dietary B. subtilis and FOS had no synergistic effect on enhancing SGR and disease resistance of sea cucumber.     

多抗间接ELISA方法的建立及其在海洋细菌快速检测中的应用

为了从对虾体内分离出的菌株中快速筛选出蜡样芽孢杆菌和溶藻胶弧菌,将蜡样芽孢杆菌与溶藻胶弧菌作为抗原,免疫SPF新西兰大白兔,获得免疫血清,效价均高于1:2000。将免疫兔血清作为一抗,HRP-羊抗兔血清作为二抗,建立了蜡样芽孢杆菌和溶藻胶弧菌快速检测的间接ELISA方法。此方法中,兔抗血清最佳稀释度为1:10000,菌液最佳包被浓度为106 CFU/ml;HRP-羊抗兔血清最佳稀释浓度为1:1000,可检测细菌最低浓度为104 CFU/ml。抗蜡样芽孢杆菌血清与苏云金芽孢杆菌有交叉反应,抗溶藻胶弧菌血清与其亲缘相近菌株无交叉反应,具有较强的特异性。2012年和2013年,分别从斑节对虾、中国对虾、凡纳滨对虾等9批样本中分离纯化到109株海洋细菌,利用建立的多抗间接ELISA方法对其中部分菌株进行快速检测,共检测出6株溶藻胶弧菌,未检测出蜡样芽孢杆菌。     

一株功能益生菌的简易发酵及其在凡纳滨对虾(Litopenaeus vannamei)生物絮团养殖中的应用

对一株有脱氮作用的巨大芽孢杆菌(Bacillus megaterium)进行简易发酵,以对虾饲料及赤砂糖为培养基,通过正交实验及发酵条件优化,在对虾饲料3 g/L、赤砂糖6g/L、接种量1×108 CFU/ml、发酵温度31℃、装液量40％条件下,发酵24 h可获得活菌数为1.16× 1010 CFU/ml的发酵产物.使用发酵得到的巨大芽孢杆菌进行凡纳滨对虾(Litopenaeus vannamei)生物絮团养殖实验.结果显示,添加芽孢杆菌和赤砂糖的增强絮团组的絮团形成速度较添加赤砂糖的絮团组及传统养殖的对照组明显提升(P＜0.05),整体上增强絮团组的亚硝酸氮水平与絮团组和对照组差异显著(P＜0.05).养殖结束时,添加巨大芽孢杆菌组的对虾体长、体重水平均显著高于另2组.本研究建立了一种简单可行的功能益生菌发酵方式,并验证添加功能益生菌可提高生物絮团技术在对虾养殖中的效果.