辐照对脱水蔬菜细菌ATP生物发光检测的影响

以4种脱水蔬菜为对象,研究ATP生物发光法检测辐照处理脱水蔬菜样品的含菌量时,ATP生物发光强度与细菌含量不一致的原因.结果表明:辐照对样品的本底发光无明显影响,也未改变荧光素酶发光系统的光谱;电离辐射在杀灭样品中细菌的同时降低了细菌体内ATP酶的活性,使死亡细菌中的ATP不能随细茵的死亡而降解,并从死亡细茵体内扩散出...     

辐照对调味品和脱水蔬菜中大肠菌群杀灭效果的研究

6kGy辐照可以使调味品和脱水蔬菜中的大肠菌群最可能数 (MPN)符合国家卫生标准的要求。通过对 1 88个辐照过的调味品的检验 ,未发现在大肠菌群中出现耐辐射的新菌株。目前出现误判“大肠菌群超标”的原因主要是由于菌检技术不过关所致 ,其中复发酵和革兰氏染色挑菌不是来自同一菌株是造成误判的最主要原因。     

辐照对农产品中细菌ATP生物发光检测的干扰

ATP生物发光技术已广泛应用于样品含菌量的快速检测,但在应用该方法检测农产品的辐照灭菌效果时却出现了发光强度均高于对照的现象。本文分别从γ射线对ATP的影响、发光动力学以及发光强度增高的途径等方面对上述现象进行了研究。结果表明,发光强度的增加并非由ATP所造成引起的,而是由农产品中含有的细菌经辐照后引起的;干扰荧光素—荧光素酶制品中存在的能引起光辐射的物质是形成干扰的另一方面的原因。     

辐照调味品和脱水蔬菜在中国商业应用现状

本文介绍了辐照调味品和脱水蔬菜在中国的研究发展、卫生法规、市场试验、商业应用现状及存在的问题和对策。     

南阳牛ATP5B基因启动子区域多态性与生长性状的关联性研究

ATP合成酶亚基β(ATP synthase subunit beta,ATP5B)作为ATP合成的重要关键蛋白之一,参与多种生物代谢活动。南阳牛(Bos taurus)是中国本土五大黄牛良种之一,以良好的抗逆性闻名于世,是重要的黄牛种质资源。本研究以南阳牛为研究对象,探究ATP5B基因启动子区域的多态性与南阳牛生长性状相关关系。利用生物信息学技术分析了不同物种ATP5B蛋白功能保守性,检测了南阳牛不同组织ATP5B基因m RNA表达量,并利用DNA测序方法检测南阳牛ATP5B基因启动子区域的多态性,分析了单个多态位点不同基因型与南阳牛生长性状的关联性,随后对具有多态性位点的不同基因型启动子活性做了双荧光素酶活性检测。结果发现牛的ATP5B和猪(Sus scrofa)等物种存在14个相似的motif结构和5个保守性功能域;南阳牛ATP5B基因在内脏组织和肌肉、脂肪组织中广泛表达,其中,在肌肉组织中表达量最高且显著高于其他组织(P0.05),脂肪中次之;在南阳牛ATP5B基因启动子区域检测到了3个SNP位点,分别为g.-428TA,g.-390TC和g.-322CG。将南阳牛生长性状与SNPs进行关联性分析,结果表明g.-428TA位点上TT基因型个体的体重、体高和胸围显著高于AA基因型个体(P0.01或P0.05);g.-390TC位点上TT基因型个体的体高和胸围与CC基因型个体存在极显著性差异(P0.01);g.-322CG位点CC基因型个体的体重和体长显著高于GG基因型个体(P0.01或P0.05)。双荧光素酶活性报告显示,各SNP位点的不同基因型启动子活性略有差别,但未达到显著水平(P0.05)。因此,南阳牛ATP5B基因启动子区域的这3个SNP可考虑作为南阳牛生长性状相关分子标记的候选基因位点,用于南阳牛的分子育种工作,为相关分子育种工作提供参考。     

一株聚磷菌GP44 的筛选、鉴定及其聚磷特性研究

采用纯培养结合蓝白斑筛选法从巢湖和南淝河底泥中分离筛选出能聚磷(P)的 11 株解 P 细菌,好氧培养时菌体吸 P 能力测定结果表明,GP44 的菌体含 P 量达到 11.92%,具有较高的聚 P 能力,对其初步鉴定为鉴定菌株 GP44 属肠杆菌科中的克雷伯氏菌属土生克雷伯氏菌(K.terrigena).GP44 在废水合成培养基上最佳聚 P 温度 30℃、初始 pH 为 7.5、最佳装液量为 120 ml/250 ml 和最适 C 源是葡萄糖,Mg2+、K+ 和 Fe3+ 有利于菌株 GP44 的生物除 P.     

联合固氮菌与根瘤菌协同作用对小麦幼苗的影响

小麦种子分别接种不同组合的联合固N菌和根瘤菌,播种到灭菌蛭石上培养28d后分别测定小麦幼苗F_v/F_m值、鲜物质量、干物质量、含N量及根系固N酶活性结果表明,接种联合固N菌植株均测到鲜根系固N酶活性,最高达386.6(±3.5)nmol/g,且联合固N菌和根瘤菌协同作用时植物干物质量、F_v/F_m值和含N量均略高于其单独作用,尤其是来自干旱半干旱草原羊草根际的联合固N菌——催娩克雷伯氏菌al(Klebsiella oxytoca)和内蒙古草原草木犀根瘤的中华苜蓿根瘤菌(Sinorhizobium,melilot)菌株Ⅱ的组合固N能力更强,对农业生产有一定应用潜力。     

荧光原位杂交技术检测土壤中博德特氏菌探针的设计与应用

根据博德特氏菌(Bordetellasp.)的16S rRNA基因序列,设计荧光原位杂交(FISH)检测博德特氏菌的寡核苷酸探针FW_iso_62和FW_iso_761,在20%～60%甲酰胺均有很强的荧光信号。采用探针FW_iso_62及其竞争探针,结合Nycodenz和DAPI技术,建立定量检测土壤中博德特氏菌的DAPI-FISH方法。该方法可排除土壤颗粒的自动荧光对细菌信号的掩盖,保证图片中有大量微生物供统计分析,还能有效保存微生物的原位信息。应用该方法分析土壤中1,2,4-三氯苯降解菌-博德特氏菌,结果未受氯苯污染的农田土壤中没有检测到博德特氏菌,而氯苯污染土壤中检测到大量的博德特氏菌,每克土壤含3.78×106个。将该污染土壤中分离的博德特氏降解菌及其降解菌群接种至农田土壤中,降解菌的数量均随培养而增加,一个月后分别占DAPI计数的1.7%和3.8%。本研究设计的探针可有效用于复杂环境样品中博德特氏菌的定性与定量检测。     

辐照脱水蔬菜的剂量与灭菌效果研究初报

对几种脱水蔬菜的细菌存活数与辐照剂量的关系进行了研究 ,初步确定了脱水蔬菜的D10 值 ,为脱水蔬菜辐照加工适宜剂量的选择提供了理论依据。     

脱硫混合菌系在传代和硫化氢脱除中的稳定性

为研究由嗜酸氧化亚铁硫杆菌[Acidithiobacillus ferrooxidans(A.ferrooxidans)]和嗜酸氧化硫硫杆菌[Acidithiobacillus thiooxidans(A.thiooxidans)]按数量1∶2配比的一组脱硫混合菌系的硫化氢脱除效果及在传代和硫化氢脱除过程中的稳定性,采用ATP分析法和qRT-PCR分别检测ATP浓度和细菌gyr B基因拷贝数及其比例。结果表明,在传代培养中,ATP含量较为稳定,其值在1.09×10-6~1.54×10-6mol·L~(-1)的小范围内变化,A.ferrooxidans和A.thiooxidans的gyr B基因拷贝数也相对稳定,在最佳脱硫条件下,硫化氢的脱除效率可达99.4%。在脱硫过程中,体系p H值为2.29~2.62。此外,不同时间点的ATP含量和细菌基因拷贝数也相对稳定。在传代培养和硫化氢脱除的过程中,A.ferrooxidans和A.thiooxidans的gyr B基因拷贝数的比例一直维持在1∶2左右。本研究结果为脱硫混合菌系在生物燃气脱除硫化氢中的应用提供了依据。     

Rapid bacteriological screening of cosmetic raw materials by using bioluminescence

Incoming cosmetic raw materials are routinely tested for microbial content. Standard plate count methods require up to 72 h. A rapid, sensitive, and inexpensive raw material screening method was developed that detects the presence of bacteria by means of ATP (bioluminescence). With a 24-h broth enrichment, the minimum bacterial ATP detection threshold of 1 cfu/g sample can be achieved using purified firefly luciferin-luciferase and an ATP releasing reagent. By using this rapid screen, microbiologically free material may be released for production within 24 h, while contaminated material undergoes further quantitative and identification testing. In order for a raw material to be validated for this method it must be evaluated for (1) a potential nonmicrobial light-contributing reaction resulting in a false positive or, (2) degradation of the ATP giving a false negative, and (3) confirmation that the raw material has not overwhelmed the buffering capacity of the enrichment broth. The key criteria for a rapid screen was the sensitivity to detect less than one colony forming unit per g product, the speed to do this within 24 h, and cost efficiency. Bioluminescence meets these criteria. With an enrichment step, it can detect less than one cfu/g sample. After the enrichment step, analysis time per sample is approximately 2 min and the cost for material and reagents is less than one dollar per sample.     

辐照对脱水高丽菜微生物ATP生物发光光谱的影响

以脱水高丽菜为材料,研究辐照对微生物ATP生物发光光谱的影响。结果表明：ATP标准品生物发光光谱的发光区域在490~640nm的范围内,峰时为563nm;辐照后样品中微生物ATP生物发光光谱的发光区域未发生改变;不同剂量辐照处理后,样品中微生物ATP提取液生物发光光谱的峰时没有显著差异;辐照后样品中微生物ATP生物发光光谱峰值高于对照,说明辐照后微生物ATP提取液中ATP的浓度有所增加,而且这种影响在相当长的一段时间内依然存在。     

Modification and application of a soil ATP determination method

Accurate estimation of microbial adenosine 5′-triphosphate (ATP) is a pre-requisite to quantify the impact of varying environments on microbial activity of soil. We investigated the effectiveness of a high efficiency soil ATP determination method (PA) [Webster, J.J., Hampton, G.J., Leach, F.R., 1984. ATP in soil: a new extractant and extraction procedure. Soil Biology & Biochemistry 4, 335-342] in 10 Ontario (Canada) soils collected along a 100 m transect and spanning a textural class gradient ranging from a sandy loam to clay loam with increasing organic matter. Modifications of the method involved using an extract of autoclaved soil to make the standard curve, as it was found that the light emitted by ATP luciferin-luciferase bioluminescence reaction in the pure extractant was different from that in the extracts. Replacing Tricine with Tris buffer in the assay significantly improved the light emission. On an average, the internal standard calibration method (ISM) measured a smaller amount of extracted ATP (1199 ng ATP g⁻¹ soil) and a lower recovery of ATP spike (82.4±7.2%) than did the standard curve method (SCM) (1246 ng ATP g⁻¹ soil and 91.2±4.5%, respectively) (P<0.05 for both comparisons). However, the average total estimated ATP was higher with ISM (1474±102 ng ATP g⁻¹ soil) than with SCM (1373±88 ng ATP g⁻¹ soil) (P<0.07). While the recovery rates determined using SCM were consistent among the soils tested, the rates measured using ISM was negatively correlated with soil clay and organic matter content, implying that the latter assay was affected by the soil properties. Our results confirmed that the recovery rates obtained by the PA method were the highest among those reported, when only SCM was used.     

蔬菜废弃物不同堆制方法对微生物数量的影响

以蔬菜废弃物为材料,采用厌氧覆膜、好氧覆膜、地下式好氧、地下式厌氧、地上式好氧和地上式厌氧6种堆制方法,分别对堆肥40 d后堆肥中的微生物数量进行测定。结果表明:在6种堆制方法的两次试验中,同一处理中细菌、放线菌和真菌在数量上相差一个数量级;好氧覆膜处理的堆肥中细菌、放线菌和真菌数量相对较多,且微生物总数最多,分别为59.9×108和83.9×108cfu/g。因此,好氧覆膜处理的微生物腐解能力最强,操作简单,是处理蔬菜废弃物的最佳堆制方法。     

Luciferase preparation,assay conditions and calculated extraction efficiency as factors in the estimation of soil ATP content

Summary Several factors affecting the measurement of soil adenosine 5-triphosphate (ATP) using a crude (macerated firefly tail) preparation of luciferase and a luminometer were investigated. These factors were luciferase preparation purity (crude or purified), use of Tris(hydroxymethyl)methylamine (Tris) or sodium arsenate (arsenate) buffer for the luciferase preparation and the bioluminescent reaction, storage temperature of luciferase throughout the assay (20°C or 1°C), modes of measuring bioluminescence (peak height or integration of bioluminescence decay) and efficiency of extraction (recovery) of ATP from soil.Crude luciferase produced a linear relationship between bioluminescence and ATP concentration very similar to that produced by purified luciferase and could be stored at 20°C for 3 h without deterioration. Arsenate was, overall, the preferred buffer for the assay. Integrating light output within 15 s of mixing luciferase-luciferin and ATP avoided interference from other reactions in the crude extracts. The procedure used to calculate the ATP concentration and the amount of exogenous ATP added to soil both affected the calculated efficiency of ATP recovery. Thus an assay for soil ATP content using crude preparations has been developed which is as sensitive as those using purified preparations.     

ATP content of soils estimated by two contrasting extraction methods

The ATP content of a series of soils was investigated in relation to various soil properties. Special attention was paid to the discrepancy in ATP, as estimated after extracting with TEA/NRB and TCA. It appears that the first procedure particularly relates to active microbial cells but extracts rather poorly certain types of older microbial biomass. Nevertheless, the TEA/NRB ATP values correlate very significantly with total soil microbial biomass as determined by the CHCl₃ fumigation method. In soils with active growing microbial biomass, the TEA/NRB and the TCA ATP values are about equal. In normal equilibrated soils the TEA/NRB ATP levels average about 40% of the total soil ATP levels. Finally, in densely rooted soils, the TCA ATP levels surpass largely the TEA/NRB levels, but they appear to a major extent to be due to plant cells.     

Specific measurement of soil microbial ATP

A direct procedure to extract and determine microbial adenosine 5'-triphosphate (ATP) in soil has been worked out. The soil is homogenized in cold Tris-EDTA-NaN₃ (TEA) buffer. The ATP content of 100 μl of a 1/1000 suspension is directly determined in a photoncountcr after addition of a detergent (NRB^®) extractant and the firefly luciferin-luciferase system. The method was tested on a wide variety of organisms and soils and compared to several other methods to determine ATP in soils. The method gives ATP recoveries of a minimum of 80% upon addition of cultures to soils.Furthermore, it is rapid, applicable to all soils examined, and most of all strictly specific for microbial biomass.     

堆肥隧道式后发酵技术及效果

该文采用发酵隧道对堆肥进行后发酵处理,并测定发酵过程中的温度、pH值、含氮量、微生物的变化,旨在为集约化生产双孢蘑菇培养料提供理论依据。该技术可以对堆肥进行10 h以上的巴氏灭菌（温度在57～62℃之间）处理和5 d的腐熟处理（温度在45～53℃之间）。处理后发酵堆肥中氮质量分数从1.58%增加至1.85%;pH值从8.7下降到7.5。嗜热细菌的菌落数从5.2×108 cfu/g上升到7.3×108 cfu/g（第3天）,发酵结束时降低为2.88×108 cfu/g;放线菌和嗜热真菌菌落数发酵开始时分别为2.4×105 cfu/g 和3.2×104 cfu/g,发酵结束时分别为19.6×105 cfu/g和10.1×104 cfu/g。试验结果表明,经过隧道式后发酵的堆肥适合于双孢蘑菇生长需要,隧道式后发酵技术可以用于规模化生产优质双孢蘑菇培养料。