Enhanced Spore Production of <i>Bacillus subtilis</i>Grown in a Chemically Defined Medium

Spores of Bacillus subtilis are being used as probiotics and competitive exclusion agents for animal consumption. Commercial production media often include relatively expensive components of animal origin that are a potential source for the presence of adventious agents, therefore undesirable for use in production scale. In this study a new animal-free component, chemically defined medium, was tested for B. subtilis spore production. Medium composition was optimized with respect to vitamin composition, carbon, nitrogen and calcium concentrations. A fed-batch bioprocess was developed, being the effect on sporulation of the carbon to nitrogen ratio at the end of the exponential growth phase studied. The developed strategy consisted of an initial and a final batch phase and an intermediate fed-batch phase with the addition of a feeding solution containing glucose and calcium and the addition of a feeding solution of ammonium sulphate, using an exponential and a constant feeding profile, respectively. Using the fed-batch strategy, it was possible to achieve a maximum spore production of 3.6 × 1010 spores/mL, corresponding to a 5 folds increase when compared to the preliminary batch experiments.

Characteristics of Bacillus cereus Spore Counts in Soils w ith Underlying Gold Mineralization in Northwestern Sichuan, China: Results from a Pilot Study

A pilot survey on a microbial mineral exploration method based on so il Bacillus cereus spore counts was carried out across three different gold mi ning regions, which vary in soil type, climate condition and geological setting in northwestern Sichuan, China. B horizon soils from these sites were analyzed for B. cereus spores, Au, Ag, Cu, Pb and Zn. The results show that the numbe rs of B. cereus spores generally increased in soils overlying gold mineraliz ation. Specifically, elevated spore counts were found in samples slightly offset from the outcrops of orebodies, whereas soils directly above the outcrops of or ebodies usually contained low spore counts. However, the background counts of B. cereus spores varied from place to place and were complicated by environmen tal and pedological factors, but the relative ratios of spore counts still were indicative of the underlying gold mineralization.

Modified Green Tea Polyphenols, EGCG-S and LTP, Inhibit Endospore in Three <i>Bacillus</i>spp.

Endospores have the ability to withstand extreme temperature, desiccation, ultraviolet radiation and chemicals which make them a threat to the food and healthcare industry. Green tea polyphenols (GTP), contain anti-microbial and anti-spore properties but not stable. In this study, two modified lipophilic green tea polyphenols, epigallocatechin-3-gallate-sterate (EGCG-S) and crude lipophilic green tea polyphenols (LTP), were used to compare their anti-spore effect with EGCG and crude GTP. Purified endospores from Bacillus cereus (B. cereus), B. megaterium and B. subtilis were treated with 1% or 5% of four tea polyphenols. Log reduction showed colony forming units (CFU) reduced significantly in all treated samples, ranging from 1.27 to 4.31 with no survivals (CFU = 0) in four samples (P < 0.05). Average percentage of inhibition for these poly-phenols treatment ranged from 91.68% to 100%. The EGCG-S and LTP have equal or better anti-spore activities compared with EGCG and GTP. EGCG-S and LTP were further used to carry out time course study on B. cereus. The results indicated that 15 min of treatment of 1% and 5% LTP and EGCG-S are able to inhibit 98.7% to 100% of germination. Transmission and scanning electron microscopy studies showed that EGCG-S caused surface disruption and damaged spores structural integrity. EGCG-S and LTP are stable anti-spore agents may aid in preventing food and beverage spoilage caused by spore-forming bacteria as well as preventing contamination in the medical industry.

Insecticidal Activity of Crystals and Spores of Purified Bacillus thuringiensis 00-50-5 Strain against Trichoplusia ni Hubner

[ Objective] The paper was to study pathogenic mechanism and action mode of Bacillus thuringiensis （Bt） 00-50-5 strain against Trichoplusia ni Hub- ner. [Method] The crystals and spores of B. thuringiensis 00-50-5 strain were first isolated and purified, and their insecticidal activities against T. ni were com- pared. [ Result] Either the mixture of pure crystals and pure spores of 00-50-5 strain, or only pure crystals could kill T. n/larvae under the concentration of 100 p.g/mL after 48 h, and the mortality rate reached 100% ; pure spores could not kill the larvae. Determination results of median lethal concentration showed that in- secticidal activity of pure crystal of Bt 00-50-5 strain （LGs0 =0.32 p~/mL） was higher than the mixture of crystals and spores （LCs0 =0.48）, but the insecticidal acti^ty of pure spores was very low （LC50 〉 500.00）. Therefore, the crystals were primarily responsible for causing death of larvae. [ Conclusion] The paper provides theoretical basis for application of Bt 00-50-5 strain.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i>subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt);and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h;while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores;malachite green-saffranin stained both δ-crystals and endospores;and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

The structure of Bacillus subtilis and their roles in spore resistance

目的 通过检测e IF4E在正常宫颈上皮、CIN及宫颈癌组织中的表达,探讨e IF4E与宫颈癌发生发展的关系。方法 选取2手术治疗的HPV阳性的宫颈病变组（CINI、CINII、CINIII各10例）共30例及宫颈癌组30例作为研究组,选取同期我院因子宫良性病变行子宫全切术的HPV阴性的正常宫颈上皮组织30例作为对照,应用免疫组织化学方法检测各组中e IF4E的表达。结果 e IF4E在正常宫颈上皮组织中无表达,在CINI级组织中无表达,在CINII级组织中偶有表达,阳性率30%;在CINIII级中低表达,阳性率70%,强阳性率10%;在宫颈癌组织中,无论是鳞癌还是腺癌均有强表达,阳性率100%;且其表达与宫颈癌期别呈正相关;II期组〉I期组,III～IV期组〉II期组;P〈0.05。e IF4E在低分化癌中染色灰度明显高于中分化及高分化组,差异有统计学意义。结论 e IF4E可能作为宫颈癌的预测肿瘤标志物之一。

兰花炭疽病拮抗细菌Bacillus megaterium1-12菌株的产芽孢条件优化

为了提高兰花炭疽病拮抗菌巨大芽孢杆菌(Bacillus megaterium)1-12菌株的芽孢形成率及芽孢数量,在摇瓶发酵基础上对芽孢形成的主要影响因素进行了考察.通过单因素实验和正交实验对1-12菌株产芽孢条件进行了分析,确定了摇瓶发酵最佳条件:培养基组成(质量分数)为玉米粉1%,大豆蛋白胨1%,CaCl2.2H2O 0.1%,MnSO4.H2O 0.05%;培养液初始pH 6.0;培养条件为种龄20 h,装瓶量30 mL/250 mL,接种量8%,温度30℃,摇瓶转速200 r/min.在此条件下,B.megaterium1-12菌株发酵液中的芽孢数量为1.92×109个mL-1,芽孢形成率达到了95%.

一株凝结芽孢杆菌(Bacillus coagulans)发酵培养基的优化

以凝结芽孢杆菌(Bacillus coagulans)为研究对象,芽孢数为响应值,对其发酵培养基进行响应面优化分析。在单因素试验的基础上,采用Plackett-Burman试验筛选出对芽孢数有显著影响的因素为麸皮和牛肉膏。由中心组合试验设计及响应面分析优化确定最优发酵培养基配方为麸皮32.12g/L,牛肉膏15.24g/L,硫酸镁0.49g/L,硫酸锰0.34g/L,磷酸二氢钠0.31g/L。在此最佳条件下,发酵液中凝结芽孢杆菌芽孢数达到3.38×108CFU/mL,是优化前的5.28倍。

枯草芽孢杆菌Bacillus subtilis BS1发酵培养基的优化

为提高枯草芽孢杆菌BS1发酵液中芽孢含量,通过单因素试验、正交试验及回归正交组合试验优化其发酵培养基,最终确定培养基配方为:麦芽浸粉1 g/l、黄豆粉15 g/l、CaCO36.68 g/l、MnSO40.4 g/l、MgCl22 g/l,初始pH值7.0。使用该配方,在37℃、180 r/min条件下于恒温摇床中振荡培养48 h,枯草芽孢杆菌BS1菌株发酵液中芽孢含量可达8.55×1010cfu/ml。

Bacillus细菌的分离及其芽孢漆酶对染料脱色的影响

利用含铜离子的富集培养基,从东北林业大学实验林场蒙古栎林下的土壤中筛选出具有较高漆酶活性的1株细菌,采用形态学、生理生化反应以及16S r DNA序列同源性分析等方法,鉴定其为芽孢杆菌属细菌,命名为Bacillus sp.5`MS。以丁香醛连氮为底物,以芽孢干重计算,其芽孢漆酶的活性高达56.73 U/g。菌株5`MS芽孢漆酶的最适pH值为6.6,最适反应温度为70℃。在100℃条件下,该芽孢漆酶反应活性仍可达最适条件下的34.61%。当染料脱色体系中添加介体乙酰丁香酮时,菌株5`MS芽孢漆酶1 h内对活性黑、靛红及结晶紫的脱色率均达92%以上,4 h内对活性蓝的脱色率达82.8%。

小麦全蚀病菌拮抗菌Bacillus methylotrophicusZ-9菌株产芽孢条件优化

甲基营养型芽孢杆菌(Bacillus methylotrophicus)Z-9菌株对小麦全蚀病病原菌有显著拮抗活性,以芽孢产率和活菌数为主要指标,采用单因素试验和正交试验对Z-9菌株摇瓶产芽孢条件进行优化,为其工业生产提供试验依据。通过单因素试验确定最佳碳源、氮源和无机盐分别为玉米粉、黄豆饼粉和MnSO_4·H_2O、Mg SO_4·7H_2O。通过正交试验确定最适培养基组分为玉米粉1.0%、黄豆饼粉2.0%、MnSO_4·H_2O 0.04%、MgSO_4·7H_2O 0.04%、Na_-2HPO_4·2H_2O 0.4%、Na H_2PO_4·2H2O 0.2%;最适发酵条件为:初始p H值7.5,250 m L三角瓶装液量50 m L,37℃培养36 h。优化后发酵液的芽孢数量为1.60×109cfu/m L,较优化前提高了13.7倍,芽孢产率达到96.5%。

混凝土修复功能菌Bacillus cohnii DSM6307芽孢萌发条件探究

目的:研究具有矿化能力的科氏芽孢杆菌(Bacillus cohnii,DSM6307)芽孢的萌发条件。方法:运用光吸收法对芽孢的萌发结果进行检测。结果:最适萌发缓冲液为Na2CO3/Na HCO3,芽孢萌发率为66.16%;萌发剂为肌苷,芽孢萌发率为83.89%,肌苷最适浓度为15 mmol/L;热激温度60℃,热激时间5 min,芽孢萌发率达到90.85%;而营养底物的加入,也促使芽孢迅速萌发,变成营养体。结论:萌发缓冲液、萌发剂的种类及浓度、热激温度及时间、营养底物均能影响芽孢的萌发率。

Safety Properties and Probiotic Potential of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895

This study reports on the safety and putative probiotic properties of Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933. According to the bacterial reverse mutation (Ames) test, cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 were not mutagenic. The two strains co-aggregated with Escherichia coli and Pseudomonas aeruginosa, and cell-free supernatants inhibited the growth of Streptococcus intermedius and Porphyromonas gingivalis. Endospores of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 were tolerant to 0.3% (w/v) bile salts and survived incubation for 4 h in MRS broth at pH 2.0 to 3.0. The ability of the two strains to produce antimicrobial compounds potentiates their application in health care formulations, personal care products, food and animal feed.

生防菌Bacillus safensis的鉴定及其对核桃病原菌的抑菌特性

为发掘对核桃真菌病害防效较好的生防细菌资源,对前期分离筛选到的细菌菌株7-3进行形态特征观察、生理生化特性测定和16S rRNA系统发育分析,并开展平板对峙和对病原菌孢子的影响试验。结果表明,菌株7-3为沙福芽孢杆菌(Bacillus safensis),对多种核桃病原菌均有一定的抑制作用,抑菌谱广,抑菌能力强。其中,对间座壳菌(Diaporthe nobilis)的抑制率最高,达到82.9%。生防菌发酵液能导致病原菌孢子明显畸形,且热稳定性强,菌群具有较强的黏附性。该菌株具有被开发成生物农药的潜质。

Study on Optimizing Nutrition and Fermentation Conditions for Compound <i>Bacillus spp.</i>

In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L;fermentation temperature is 37&#8451;, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.

Aerobic Spore-Forming Bacteria in the Ultra High Temperature Milk Produced in the North West of Algeria

The occurrence of the spore-forming bacteria in different segments of production lines of Ultra High Temperature （UHT） milk of a commercial plant and its transfer to the final product was studied. The samples were collected from different segments of production lines from reception to packaging sections, over a period of 75 days at a rate of one sampling a week, from plants in North West of Algeria. The total colony count of aerobic spore-forming bacteria was done using Plate Count Agar （PCA） method. The results showed that there was more than 1 ×10^3 CFU/mL at the level of 50%, 25%, 37.5%, 62.5% and 37.5% of the analyzed samples of milk powder, pasteurized milk, UHT milk （before storage）, UHT milk stored at 55℃ for 7 days and UHT milk stored at 30℃ for 14 days, respectively. The mean total colony count of aerobic spore-forming bacteria was considerably lower in the UHT milk than in the other samples, while the higher proportion of aerobic spore-forming bacteria was noticed to be 32.58% and 30.90% in the UHT milk stored at 55℃ and 30℃, respectively. The results obtained by the classic biochemical identification （Bacillus API 20E）, interpreted by software of calculation for microbial identification reflected the presence of especially Bacillus sphaericus. The other species found were： Bacillus cereus, Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis and Bacillus stearothermophilus. The study reveals that the proportion of aerobic spore-forming bacteria in the final product depends on temperature and storage periods.

THINDOWN IN RADIOBIOLOGY：E．COLI B／r，Bs－1，B．SUBTILUS SPORES，AND V－79 CHINESE HAMSTER CELLS

Track theory rested on the foundation of the radial distribution of dose from δ rays as the central contribution of atomic physics to heavy ion radiobiology.Here,a new calculation of the radial distribution of dose is applied, in which the classical angular distribution of dose of delta rays and a logarithmic polynomial representation of the electron range-energy relation are used,to form the basis of the present thindown calculation.Calculations of inactivation cross sections for heavy ions in the track width regime displaying thindown for E.Colt B/r and Bs-1,and for Bacillus Subtilus are straightforward for these are 1-hit detectors,Calculations for V-79 hamster cells are more complex.They follow the original development of this model for eucaryotic cells,and make use of the cross sections calculated for hypothetical internal targets which are then asserted to be proportional to the measured cellular inactivation cross sections.The results are in reasonable agreement with experimental data.

Role of the Putative Polysaccharide Deacetylase BA1836 from <i>B. anthracis</i>in Spore Development and Germination

Putative and known polysaccharide deacetylases (PDAs) from B. anthracis have key roles in resistance to host lysozyme, stabilization of the cell wall, biogenesis of peptidoglycan (PG) and for neutral polysaccharide modification and attachment to PG. Here we elucidated the physiological role of the putative PDA BA1836 from B. anthracis. The ba1836 gene was expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. The Δba1836 knockout strain had normal growth rate, did not exhibit any significant alterations in PG pattern of stationary phase cells and was not sensitive to lysozyme, but showed a defect in cell separation. Strikingly, the Δba1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and had normal morphology. Additionally, digestion of Δba1836 mutant spore PG with mutanolysin produced an almost identical muropeptide pattern compared to peptidoglycan from wild type spores, although the amount of all muropeptides was significantly reduced. Finally, knockout spores exhibited a lower germination rate. To our knowledge, BA1836 has a unique role, among the presently characterized PDAs from B. anthracis, in spore development and germination.

Single-cell analysis reveals microbial spore responses to microwave radiation

To determine the effects of microwave radiation at the molecular level as well as on the germination,growth and morphology of dry spores at the single-cell level.Dry Bacillus aryabhattai MCCC 1K02966 spores were microwave-treated at different powers and characterized using single-cell optical technology.As determined by laser tweezers Raman spectroscopy,the Ca^(2+)-dipicolinic acid content increased and nucleic acid denaturation occurred in response to microwave treatment.Livecell microscopy revealed that the germination and growth rates decreased as the microwave power increased.With respect to morphology,atomic force microscopy(AFM)demonstrated that spores became wrinkled and rough after microwave treatment.Furthermore,spores became smaller as the microwave power increased.Microwave treatment can damage DNA,and high-power microwaves can inhibit the germination of spores and reduce spore volumes.These results provide a new perspective on the responses of living single cells to microwave radiation and demonstrate the application of various new techniques for analyses of microorganisms at the single-cell level.

生防枯草芽孢杆菌Czk1固体发酵工艺条件优化

枯草芽孢杆菌(Bacillus subtilis)Czk1是一株具广谱拮抗、促生和诱导植株产生抗病性的根际益生菌。为了全面资源化利用农业废弃物中的有机养分和促进对植物土传病害的生物防治,本研究以Czk1为研究对象,以鸡粪为底肥,分别利用玉米秸秆、菌糠、椰糠堆肥为原料,采用液固两相发酵工艺,以稀释平板菌落计数法测定活菌总数和芽孢数,经过单因素和正交试验方法,明确Czk1固体发酵物料的配方、工艺参数和培养条件。得出Czk1固体发酵的最优条件:最佳原料为菌糠鸡粪堆肥,Czk1接种重量为10%,黄豆粉添加量为10%,含水量为40%,发酵温度为37℃,翻抛1次/72h,在此条件下,Czk1菌株中的芽孢活菌数量总量可以达到8.63×10^(8)CFU/g,芽孢数为8.37×10^(8)CFU/g。其次,发酵效果较佳的原料为秸秆鸡粪堆肥,Czk1接种量为10%,添加5%黄豆粉,40%含水量,发酵温度为37℃,翻抛1次/72 h,此条件下,Czk1接种菌株中的活菌数量总量可达到6.53×10^(8)CFU/g,芽孢数为5.97×10^(8)CFU/g;另外,在此配方条件下再添加30%菌糠,Czk1菌株的活菌数量总量及芽孢数分别可达到8.70×10^(8)CFU/g和6.90×10^(8)CFU/g。4种发酵生物肥原料的理化性质测定表明,其有机质含量、碳氮比均有较大差异,其中以鸡粪为原料其pH、全磷和全钾含量均最高,碳氮比最低,氮、磷、钾总含量均高于秸秆、椰糠和菌糠;秸秆为原料其电导率、有机质含量最高;椰糠为原料其全氮含量最高,菌糠为原料碳氮比最高。不同鸡粪与菌糠、秸秆及椰糠组合的生物有机肥的种子发芽指数均大于80%。因此,枯草芽孢杆菌Czk1具有良好的开发利用价值,选择合适的发酵生产条件可将农业废弃物制备出高质量的生物有机肥。