Comparison of Antibacterial Activities of Six Bacillus amyloliquefaciens Strains

In this study, six bacterial strains were isolated from the sediment, probiotic fermentation products, and lake sediments, they were identified as Bacillus amyloliquefaciens using genetic evolution analysis, which were named B3, B4, B5, XD3, YF6, and YF8. The comparison of the antibacterial activity, hemolytic activity, and antibiotic sensitivity of six Bacillus amyloliquefaciens strains laid a foundation for the development and application of antimicrobial peptide products. A surface activity assay was used to determine the production of biosurfactants in six Bacillus amyloliquefaciens strains. With Staphylococcus aureus and Escherichia coli as indicator bacteria, their antibacterial activity was determined using the agar diffusion method;the same diffusion method was used to determine the antibiotic susceptibility of Bacillus amyloliquefaciens. The results showed that the six Bacillus amyloliquefaciens strains had obvious biosurface activity, and the bacteria inhibited Staphylococcus aureus and Escherichia coli, from strong to weak: YF8, XD3, B3, B4, YF6, and B5. Strain YF8 had the best broad-spectrum bacteriostatic effect, followed by strain XD3. All Bacillus amyloliquefaciens strains were susceptible to 16 common drugs, except for Bacillus amyloliquefaciens strain YF8, which was intermediate to neomycin. The study shows that Bacillus amyloliquefaciens and secondary metabolites have the ability to produce a variety of active peptides, exert a certain inhibitory effect on common pathogens, and have the potential to develop as animal probiotics.

利用Bacillus amyloliquefaciens SQR9研制复合微生物肥料

针对化肥、传统有机肥和生物肥料的优缺点,开发集化肥速效、有机肥长效和生物肥增效为一体的复合微生物肥料具有重要意义。本文鉴于Bacillus amyloliquefaciens SQR9易在逆境中形成芽孢,具有耐盐性好,抗病,广谱促生等特点,利用其通过固态发酵研制的生物有机肥添加一定配比的无机化肥研制"全元"复合微生物肥料。同时通过粉状、颗粒复合微生物肥料存放试验和田间试验对"全元"复合微生物肥料的配方、工艺和肥效进行了初步的研究。结果表明:粉状和颗粒复合微生物肥料储存6个月时,功能菌活菌数量均超过国家标准(2×107 cfu/g,干重);田间试验结果表明,复合微生物肥料能明显促进番茄的生长,与等养分有机无机复合肥、化肥处理和不施肥处理相比,在总养分100 g/kg施肥水平下,番茄增产幅度分别达8.23%、10.26%和37.73%;在总养分140 g/kg施肥水平下,番茄增产幅度分别达7.36%、13.92%和44.77%。因此,利用Bacillus amyloliquefaciens SQR9能够生产粉状和颗粒状复合微生物肥料。

生防解淀粉芽胞杆菌(Bacillus amyloliquefaciens)最新研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)是与枯草芽胞杆菌相似,可产芽胞、革兰阳性(G+)、具有生防活性的益生细菌。其代谢过程中产生的抗菌物质种类多、抗菌谱广,其抗菌制剂或抗菌提取物具有无毒、无害、无残留、抑菌时效长等优点,可广泛应用于果蔬保鲜、果蔬生长期及采后微生物病害的生物防治,是极具开发价值的生防菌。本文从抗菌机理、抗性基因组成、基因工程菌构建、抗性产品开发及对微生物病害防治等几个方面,对解淀粉芽胞杆菌的研究及应用现状进行了归纳和总结,为其在农业生物工程中的规模化安全应用提供参考。

环境因素对Bacillus amyloliquefaciens TF28在土壤中定殖的影响

为了明确生防菌株 Bacillus amyloliquefaciens TF28在土壤中的定殖动态,采用活菌计数及菌株特异PCR验证相结合的方法,研究温度、含水量、pH、接种量、土壤类型和病原菌对其在离体土壤中定殖的影响。结果表明,适于菌株TF28定殖的温度、pH、含水量范围较广,在20~35 ℃,pH 4.5~8.6,含水量 5%~35%范围内均能有效定殖;25 ℃、弱酸性和中性、15%含水量更利于其定殖;接种量和病原菌显著影响其定殖,接种量越大,定殖能力越强;病原菌在接种后14 d内显著促进其定殖;另外,土壤有机质质量分数高更利于其定殖。该研究为菌株TF28的推广应用提供了理论依据。

南海深海细菌Bacillus amyloliquefaciens GAS 00152抗菌代谢产物研究

采用多种色谱分离技术,从南海深海细菌Bacillus amyloliquefaciens GAS 00152发酵液中分离得到12个化合物,经波谱数据分析分别鉴定为4-苯基-3-丁烯酰胺（1）、苯乙酰胺（2）、苯乙酸-2-（4-羟苯基）乙酯（3）、对羟基苯乙醇（4）、环（甘氨酸-2-氨基丁酸）（5）、环（甘氨酸-亮氨酸）（6）、环（甘氨酸-L-脯氨酸）（7）、环（D-脯氨酸-L-缬氨酸）（8）、环（4-羟基脯氨酸-亮氨酸）（9）、环（N-甲基甘氨酸-苯丙氨酸）（10）、环（L-2-羟基脯氨酸-苯丙氨酸）（11）、环（2-哌啶酸-苯丙氨酸）（12）。其中,化合物1为新天然产物。测试化合物1-12对番木瓜炭疽菌和香蕉黑星菌的抑制活性,发现化合物9对两种热带水果致病菌显示出中等抗菌活性。

木薯渣、桉树渣固体发酵Bacillus amyloliquefaciens WJ22研制生物有机肥的配方研究

Bacillus amyloliquefaciens WJ22为实验室保存的香蕉根际促生功能菌。利用牛粪有机肥和菜籽饼为基本原料,海南当地农业废弃物木薯渣和桉树皮为添加原料,研究该菌株的固体发酵配方工艺,最终获得利用该菌株研制的促生生物有机肥。结果表明,拮抗菌WJ22在菜籽饼含量为15%、木薯渣含量为20%、桉树渣含量为20%的原料混合物中固体发酵效果最佳,菌株数量超过2×109cfu/(g·干重),远远超过国家固体生物有机肥的标准[2×107cfu/(g·干重)]。盆栽试验结果表明,该配方研制的生物有机肥能够很好地促进香蕉苗期的生长。

Bacillus amyloliquefaciens MBRC1的鉴定与抗菌活性研究

从台州市剑门港海区的海底淤泥中采集样品分离得到4株具抗菌活性的微生物,其中一株具有较强的抗菌活性。对该菌作了形态学、生理生化、分子生物学、(G+C)mol%含量等研究,确定该菌株属于芽孢杆菌属,命名为Bacillus amyloliquefaciens MBRC1。采用BLAST Clustal W(V1.83)及Mega(V 5.1)等软件对其进行了系统发育树的分析,确立了该菌株在系统发育上的地位。采用不同的培养基对该菌株进行发酵培养,利用牛津杯法对该菌胞外的抑菌活性物质的抑菌效果进行了研究,结果显示:Bacillus amyloliquefaciens MBRC1胞外具有较强的抑菌活性物质,且对金黄色葡萄球菌(S. aureus)、枯草杆菌(B. subtilis)和大肠杆菌(E. coli)均有较好的抑菌效果。

Effects of Bacillus amyloliquefaciens Biofertilizer on Tea Yield and Quality

[Objective] This study was aimed to explore the effects of Bacil us amy-loliquefaciens biofertilizer on tea yield and quality. [Method] The field plot experiment was conducted with the biofertilizer treatments and control to investigate 100-bud weight and main biochemical components. [Result] The treatments by Bacil us amy-loliquefaciens biofertilizer, which was fermented using sweet potato starch wastewa-ter （SPSW） as culture medium, improved 100-bud weight and tea quality significant-ly under the concentration of 0.8×108, 1.6×108 and 3.2×108 cfu/ml with the dose of 1 L/m2 for 4 times. At the optimum concentration of 1.6 ×108 cfu/ml, the biofertilizer treatment increased the 100-bud weight by 22.3%, water extracting materials by 21.9%, amino acids content by 8.83%, tea polyphenol content by 9.76%, and de-creased theine content by 8.32%, respectively. Compared with the control, there was no significant difference between the SPSW treatment and the control. [Con-clusion] The production of the B. amyloliquefaciens biofertilizer could consume SP-SW, and the application of the biofertilizer could improved the tea yield and quality, which provided references for the development of ecological agriculture.

海洋产蛋白酶Bacillus amyloliquefaciens MSP05发酵南美白对虾虾壳脱蛋白条件优化

对利用产蛋白酶海洋B.amyloliquefaciens MSP05发酵南美白对虾虾壳脱蛋白的发酵条件进行优化研究。根据B.amyloliquefaciens MSP05发酵南美白对虾虾壳脱蛋白条件单因素优化试验结果,选取对脱蛋白率有显著影响的转速、温度和装液量3个因素,采用Box-Benhnken中心组合法设计,利用响应面法对3个因素进行优化。结果表明,菌株B.amyloliquefaciens MSP05发酵南美白对虾虾壳脱蛋白的最佳条件为转速200 r/min、温度39℃和装液量20%。在此条件下脱蛋白率为96.47%,较优化前提高了16.7%,该研究结果为利用B.amyloliquefaciens MSP05发酵南美白对虾虾壳进行脱蛋白的工业化应用奠定了基础。

Bacillus amyloliquefaciens KHQH-1的鉴定及其抗菌活性初步研究

为了探寻新的具有防腐保鲜功能的微生物资源,前期从柑橘防腐保鲜研究过程中,分离纯化得到1株具有抗菌活性的细菌。该研究对该菌进行了形态、生化和分子生物学等鉴定,确定该菌株属于芽孢杆菌属,命名为Bacillus amyloliquefaciens KHQH-1。通过NCBI平台BLAST进行序列比对,结合Clustalw和Mega 7.0等软件进行序列分析和构建系统发育树,确立了该菌株在系统发育上的地位。采用点植法和滤纸片法研究了KHQH-1对几种食品中常见霉菌和细菌的拮抗效果,结果表明,KHQH-1对根霉(Rhizopus spp.)、米曲霉(Aspergillus oryzae)有一定抑制效果,对食品中常见霉菌黄曲霉(Aspergillus flavus)、青霉(Penicillium spp.)、绿色木霉(Trichoderma viride)具有较强抑制效果,其抑菌圈大小分别为13.70、9.30和10.87 mm;KHQH-1对食品中常见细菌金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)均有一定抑制作用,其抑菌圈分别为3.20和8.20 mm。这为更进一步探索天然产物防腐保鲜技术和微生物制剂的开发与应用提供了一定的理论与技术参考。

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)XZ-1发酵条件的优化

采用单因素试验和正交试验,对解淀粉芽孢杆菌菌株XZ-1的发酵培养基成分和发酵条件进行了优化。结果表明:XZ-1的发酵培养基的最佳碳源、氮源和无机盐分别为糊精、蛋白胨和氯化钾;其培养基最佳组分为糊精10 g/L,蔗糖5 g/L,蛋白胨10 g/L,氯化钠7 g/L;最佳发酵培养条件为培养基初始p H值6,发酵温度27℃,发酵时间36 h,装液量100 m L/250 m L,接种量3%,转速150 r/min。

Effects of dietary Bacillus amyloliquefaciens supplementation on growth performance,intestinal morphology, inflammatory response, and microbiota of intra-uterine growth retarded weanling piglets

Background: The focus of recent research has been directed toward the probiotic potential of Bacillus amyloliquefaciens(BA) on the gut health of animals. However, little is known about BA's effects on piglets with intra-uterine growth retardation(IUGR). Therefore, this study investigated the effects of BA supplementation on the growth performance,intestinal morphology, inflammatory response, and microbiota of IUGR piglets.Methods: Eighteen litters of newborn piglets were selected at birth, with one normal birth weight(NBW) and two IUGR piglets in each litter(i.e., 18 NBW and 36 IUGR piglets in total). At weaning, the NBW piglet and one of the IUGR piglets were assigned to groups fed a control diet(i.e., the NBW-CON and IUGR-CON groups). The other IUGR piglet was assigned to a group fed the control diet supplemented with 2.0 g BA per kg of diet(i.e., IUGR-BA group). The piglets were thus distributed across three groups for a four-week period.Results: IUGR reduced the growth performance of the IUGR-CON piglets compared with the NBW-CON piglets. It was also associated with decreased vil us sizes, increased apoptosis rates, reduced goblet cel numbers, and an imbalance between pro-and anti-inflammatory cytokines in the smal intestine. Supplementation with BA improved the average daily weight gain and the feed efficiency of the IUGR-BA group compared with the IUGR-CON group(P < 0.05). The IUGR-BA group exhibited increases in the ratio of jejunal vil us height to crypt depth, in ileal vil us height, and in ileal goblet cel density. They also exhibited decreases in the numbers of jejunal and ileal apoptotic cel s and ileal proliferative cel s(P < 0.05). Supplementation with BA increased interleukin 10 content, but it decreased tumor necrosis factor alpha level in the smal intestines of the IUGR-BA piglets(P < 0.05). Furthermore, compared with the IUGR-CON piglets, the IUGR-BA piglets had less Escherichia coli in their jejunal digesta, but more Lactobacil us and Bifidobacterium in their ileal digesta(P < 0.05).Conclusions: Dietary supplementation with BA improves morphology, decreases inflammatory response, and regulates microbiota in the smal intestines of IUGR piglets, which may contribute to improved growth performance during early life.

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)DC-4产豆豉溶栓酶发酵条件的优化

解淀粉芽孢杆菌DC 4是从豆豉中筛选得到 ,产生的豆豉溶栓酶具有较好的体外溶栓效果。利用单因素实验和正交分析获得该菌株产生豆豉溶栓酶的最佳发酵条件为 :接种量为 7% ;发酵培养基组分为纤维蛋白 2 0 % ,蛋白胨 0 5 % ,糊精 2 0 % ,酵母膏 0 1 5 % ,K2 HPO40 4% ,NaH2 PO40 0 4 % ,CaCO30 3% ,pH7 0 ;培养温度为 32℃ ;2 5 0mL三角瓶装量为 5 0mL ;2 1 0r/min振荡培养 72h ,其发酵上清液纤溶活性可达到 82 0U/mL。

Bacillus amyloliquefaciens Y-3保鲜剂的制备及对番茄货架期的影响

为获得对番茄货架期腐烂率控制具有良好效果的解淀粉芽孢杆菌Y-3可湿性粉生物保鲜剂,采用单因素和平板活性检测相结合的方法,筛选出对解淀粉芽孢杆菌Y-3活性影响最小的最佳载体和助剂及其配方,并在此基础上采用活体试验和货架试验对制剂的最佳稀释倍数和货架期腐烂控制效果进行测定。结果表明:解淀粉芽孢杆菌Y-3生物可湿性粉保鲜剂最佳配方为:发酵液70%、膨润土10%、PEG8000 2.4%、净洗剂LS 3.6%、羧甲基纤维素钠(CMC-Na)2%、膨润土补足100%;药剂稀释倍数为≤300倍;红熟番茄经解淀粉芽孢杆菌Y-3可湿性粉生物保鲜剂浸泡15 min后,可20℃货架11 d,且使病果率和腐烂率分别控制在7.35%和10.75%,明显低于对照。说明该生物保鲜剂不仅对番茄采后早疫病和灰霉病病原具有良好防治效果,而且可有效减少红熟番茄货架期腐烂率和病果率,在延长番茄货架供应时间方面具有重要意义。

Bacillus amyloliquefaciens C-1胞外抑菌脂肽的分离与鉴定

Bacillus amyloliquefaciens C-1为本实验室从即食食品中分离出的菌株,其发酵液上清具有一定的抑菌活性。通过酸沉淀法从发酵液上清中分离粗脂肽,并确定脂肽是发酵液上清中具有抑菌活性的物质。本实验以蜡样芽孢杆菌MS10362R(产黑色素)、蜡样芽孢杆菌CMCC63301(不产黑色素)和大肠杆菌O157∶H7 CDC157为指示生物进行粗脂肽的抑菌活性测定,结果显示0.1 mg/m L的脂肽对这三个菌的6 h生长抑制率分别96%、75%、93%;同时在不同温度、蛋白酶和有机溶剂处理下分别测定了该脂肽的抑菌活性稳定性,结果显示C-1抑菌脂肽对这些处理均不敏感,其抑菌活性非常稳定,具有很好的开发应用潜力。

Modulations of genes related to gut integrity,apoptosis,and immunity underlie the beneficial effects of Bacillus amyloliquefaciens CECT 5940 in broilers fed diets with different protein levels in a necrotic enteritis challenge model

Background:The ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis(NE)to emerge in the flocks.Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease.Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940(BA)in broiler chickens under NE challenge and/or fed diets with different protein levels.Methods:In both experiments,480 day-old mix-sexed Ross-308 broilers were arranged in a 2×2 factorial arrangement of treatments.In experiment 1,the factors were NE challenge(yes or no)and probiotic(yes or no).In experiment 2,the factors were dietary crude protein levels(standard or reduced)and probiotic(yes or no)and were used under NE challenge condition.Oral administration of Eimeria oocysts(day 9)followed by inoculation with Clostridium perfringens(day 14 and 15)was used to induce NE challenge.On day 16,two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran(FITC-d)and blood samples were collected for gut integrity evaluation,and jejunal samples were collected for gene expression assay.Results:In experiment 1,BA supplementation decreased caspase-3(CASP3)(P<0.001)and caspase-8(CASP8)(P<0.05)and increased occludin(OCLD)(P<0.05)expression regardless of the challenge.Additionally,BA supplementation downregulated interfron-γ(IFN-γ)expression(P<0.01)and upregulated immunoglobulin-G(IgG)(P<0.01)and immunoglobulin-M(IgM)(P<0.05)only in challenged birds.In experiment 2,the expression of genes encoding mucin-2(MUC2)(P<0.001),tight junction protein-1(TJP1)(P<0.05)and OCLD(P<0.05)were upregulated by the addition of BA in the diet,regardless of the crude protein level.Further,BA supplementation downregulated INF-γ(P<0.01)and upregulated immunoglobulin-A(IgA)(P<0.05),IgM(P<0.05)and IgG(P<0.01)regardless of the crude protein level.Conclusion:These findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier,tight junction,and immunity in broilers challenged by unfavourable conditions such as NE challenge.

玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库的构建

构建玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库,以期获得丢失玉米赤霉烯酮降解功能的突变子,克隆玉米赤霉烯酮降解酶基因,阐明MQ01降解玉米赤霉烯酮的分子机制。将携带转座子TnYLB-1的穿梭载体pMarA电转化至玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01中,50℃高温条件下,把穿梭载体pMarA上转座子TnYLB-1随机插入到菌株MQ01基因组中,获得转座子插入突变的阳性克隆,构建MQ01菌株的突变体文库,随机挑选突变子采用PCR和Southern杂交方法进行验证。本研究成功获得了3 000多个TnYLB-1转座子插入突变的阳性克隆,构建了B.amyloliquefaciens MQ01的突变子文库,结果显示TnYLB-1转座子以单拷贝的形式随机插入到B.amyloliquefaciens MQ01的基因组DNA中,从而可以从转座子突变文库中筛选丢失玉米赤霉烯酮降解功能的转座突变子,从菌株MQ01中克隆玉米赤霉烯酮降解酶基因。

Isolation of Secondary Metabolites with Antimicrobial Activities from Bacillus amyloliquefaciens LWYZ003

The strain LWYZ003, which can restrain multiple pathogens, was screened from the sediment of the ocean and identified as Bacillus amyloliquefaciens. Large-scale fermentation and modern chromatographic separation technologies(macroporous resin column chromatography, silica gel column chromatography, thin-layer chromatography and high-performance liquid chromatography) were used to separate antimicrobial products from the fermentation broth of marine-derived Bacillus amyloliquefaciens LWYZ003. Bioactive-guided separation was used in the term of seeking antimicrobial products from the secondary metabolites of Bacillus amyloliquefaciens LWYZ003. As a result, two natural products cycloheximide( 1) and trehalose( 2) were obtained. Their structures were elucidated by Fourier transform infrared spectroscopy, high-resolution mass spectrometry, ~1 H and ^(13) C nuclear magnetic resonance analysis. In the cylinder plate method, compound 1 exhibited stronger antimicrobial activities than compound 2 against Micrococcus luteus, and also exhibited wider antimicrobial spectrum than compound 2. In conclusion, isolation of bioactive secondary metabolites from marine Bacillus sp. has enormous potentials in finding suitable antibiotics to inhibit multiple pathogens.

Physicochemical Properties and Bioactivities of Rice Beans Fermented by Bacillus amyloliquefaciens

The purpose of this study was to investigate the physicochemical properties and bioactivities of rice beans(Vigna umbellata)fermented by Bacillus amyloliquefaciens.The fermentation conditions were optimized on the basis of the fibrinolytic activity.Under the optimal fermentation conditions,the fibrinolytic activity reached a maximum of 78.0 FU·g^(-1)(4890 IU·g^(-1),fibrin plate method,FU:fibrin degradation unit).The contents of peptides(which increased from 2.1 to 10.9 g per 100 g),total phenolics(from 116.7 to 388.5 mg gallic acid per 100 g),total flavonoids(from 235.5 to 354.3 mg rutin per 100 g),and anthocyanin(from 20.1 to 47.1 mg per 100 g),as well as the superoxide dismutase activity(from 55.3 to 263.6 U·g^(-1))in rice beans were significantly increased after fermentation.The 2,2-diphenyl-1-picrylhydrazyl(DPPH)and 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt(ABTS)free radical scavenging activities and ferric reducing antioxidant power(FRAP)of fermented rice beans were 1.9–4.8 times higher than those of unfermented rice beans.Moreover,fermentation induced an increase in the dipeptidyl peptidase IV(DPP-IV)inhibition,a-glucosidase inhibition,and anticoagulant activities of rice beans.Rice beans fermented by Bacillus amyloliquefaciens may serve as a functional food with potential benefits for the prevention of thrombotic diseases.

实时荧光定量聚合酶链反应检测核桃根际土壤生防菌Bacillus amyloliquefaciens和Trametes versicolor的定殖

以铁核桃根腐病为研究对象,运用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)扩增技术,分别对铁核桃根际土壤中解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和白腐菌(Trametes versicolor)2种生防菌及腐皮镰刀菌(Fusarium solani)病原菌的数量动态变化进行研究。结果表明:2种生防菌在接种后短期内都能够迅速生长繁殖,B.amyloliquefaciens相对含量在第20天达到峰值,20d后呈下降趋势,40d后趋于稳定,而T.versicolor在各时期的相对含量变化均较前者延迟10d;60d后,在施入B.amyloliquefaciens和T.versicolor的根际土壤中F.solani的相对含量极显著降低;在生防菌的相对含量达到峰值时,B.amyloliquefaciens与其对照组相比差异有统计学意义(P<0.05),T.versicolor则差异有高度统计学意义(P<0.01);定殖稳定后,B.amyloliquefaciens与其对照组相比差异无统计学意义(P>0.05),T.versicolor对照组与其相比差异有统计学意义(P<0.05),2种生防菌在铁核桃根际土壤中均有很强的生防效果和定殖能力,生防效果为B.amyloliquefaciens>T.versicolor,定殖能力为B.amyloliquefaciens>T.versicolor。该研究为由腐皮镰刀菌引起的病害实时监测提供了重要参考,同时为2种生防菌在土壤中的定殖检测提供了有力的技术支持。