Simple and rapid microbiological method for determination of residual antibacterials in meat

A simple and rapid screening method using bioassay for the simultaneous analysis of antibacterials (penicillins, cephalosporins, macrolides, tetracyclines, quinolones, etc.) in meat has been developed. A 5 g sample was homogenized with 5 mL of methanol, and the homogenate was centrifuged for 10 min with 3,000 rpm. The pulp disk method with Bacillus subtilis BGA (Antibiotic Medium 5 (pH 8) and 8 (pH 6)), Micrococcus luteus ATCC 9341 and Geobacillus stearothermophilus as test organisms was employed for the assay of the antibacterials. Typical antibacterials (penicillin G, ampicillin, cefapirin, cefalexin, erythromycin, spiramycin, oxytetracycline, chlortetracycline, streptomycin, dihydrostreptomycin, enrofloxacin and oxolinic acid) were detected at levels of ca. 0.005-2.5 microg/g in meat. Therefore, we recommend this proposed screening method for routine analysis of residual antibacterials in livestock products.     

Evaluation of a modified EC Four Plate Method to detect antimicrobial drugs.

This paper describes a modification of the EC Four Plate Method based on microbial growth inhibition of Bacillus subtilis on agar medium at pH 6.0, 7.2 and 8.0 and Micrococcus luteus at pH 8.0 developed to cope with large numbers of samples. The method's performance was evaluated by determining the Minimum Inhibitory Concentrations of 66 commonly used drugs and determining the between-assay variation of antimicrobial control standards. The modified method proved particularly sensitive for beta-lactams, tetracyclines, quinolones, marcrolides and lincosamides and least sensitive for anticoccidials and nitrofurans. The pH 6.0 and 7.2 plates were more sensitive for 39 of the 66 antimicrobials (59%) whereas the two pH 8.0 plates (B. subtilis, M. luteus) were the most sensitive for 27 (41%). Muscle samples were taken from 1830 routine meat inspection investigations between 1994 and 1996. Of the 38 (2%) positive meat inspection carcasses, the following antimicrobials were confirmed above the MRL: penicillin G (10), oxytetracycline (16), sulphadimidine and sulphadiazine in combination (4) and chlortetracycline (1). The method as described is technically simple, cost effective, robust, suitable for large sample throughput and for frozen, thawed or fresh tissues. When all four plates are used the pattern of inhibition can reduce unnecessary confirmatory assays by indicating the antimicrobial group most likely to be present.     

余甘子果实粗提物的抑菌活性研究

本实验研究并探讨了余甘子果实70%乙醇提取物的抗菌谱和抑菌特性,结果表明：70%的乙醇提取物对供试霉菌几乎没有抑菌活性;对啤酒酵母有一定的抑菌活性;对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、变形杆菌和嗜热脂肪芽孢杆菌有很强的抑菌活性,抑菌活性强弱顺序为：嗜热脂肪芽孢杆菌＞变形杆菌＞金黄色葡萄球菌＞大肠杆菌＞枯草芽孢杆菌,其MIC值分别为：2.0%、2.0%、2.0%、4.0%、4.0%,啤酒酵母的MIC值为8.0%。     

一株具有抗菌活性芽孢杆菌HN-7的分离及抗菌活性研究

研究采用琼脂平板扩散试验,以单核增生李斯特氏菌（Listeria monocytogene）54002为指示菌,从湖南传统发酵豆制品中筛选 出1株对其具有明显抑制作用的芽孢杆菌（Bacillus sp.）HN-7,通过酸碱稳定性、酶敏感性、热稳定性及紫外稳定性试验,可初步推测 该抑菌物质是脂肽。 研究结果表明,菌株HN-7所产抗菌肽抑菌谱较宽,耐热、耐酸性强,对单增李斯特氏菌（Listeria monocytogenes）、 金黄色葡萄球菌（Staphylococcus aureus）、大肠杆菌（Escherichia coli）、藤黄微球菌（Micrococus luteus）,枯草芽孢杆菌（Bacillus subtilis） 都具有抑制作用,并在121 ℃处理20 min、pH 2.0～12.0条件下仍具有抑菌活性。     

不同防腐剂对3 种模式腐败菌抑菌效果的比较

目的：研究食品防腐剂对腐败菌的抑菌效果。方法：采用微量稀释法测定不同防腐剂对藤黄微球菌、枯草芽孢杆菌和大肠杆菌的最小抑菌质量浓度(MIC)。结果：对于藤黄微球菌(G+),抑菌效果最好的是乳酸链球菌素(Nisin)与ε- 聚赖氨酸,MIC 均为12.5μg/mL;对于枯草芽孢杆菌(G+),抑菌效果最好的是丙酸钙、丁二酮、柠檬酸、柠檬醛和D- 异抗坏血酸钠,MIC 均为6.25μg/mL;对于大肠杆菌(G －),抑菌效果最好的是过氧化氢和丁二酮,MIC 均为25μg/mL。结论：不同防腐剂对3 种模式腐败菌的抑制效果有所差异,应复配使用。     

A RAPD-based comparison of thermophilic bacilli from milk powders

The similarity of strains of thermophilic Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Anoxybacillus flavithermus (formerly Bacillus flavothermus), Bacillus licheniformis and Bacillus subtilis isolated from separate milk powder production runs from multiple factories was examined using a random amplified polymorphic DNA (RAPD) protocol. As a result of the analysis of the RAPD fingerprints and data relating to general growth and biochemical tests, over 98% of the 1470 isolates examined (grown at 55 degrees C) were assigned to the species G. stearothermophilus, A. flavithermus, B. licheniformis and B. subtilis. The G. stearothermophilus isolates were identified as being nearly identical to G. stearothermophilus (DSMZ 22; equivalent to ATCC 12980), or G. stearothermophilus var. calidolactis (DSMZ 1550). Three groups of isolates were found to be related to A. flavithermus (DSMZ 2641) by partial small ribosomal subunit (16S) sequence comparisons and shown to be interrelated by RAPD analyses with multiple primer sets. The thermophilic isolates of B. licheniformis were positively identified by comparison with type strains of B. licheniformis DSMZ 13 and DSMZ 8785. All of the B. subtilis strains shared bands in their RAPD profiles and were similar to a common B. subtilis type strain (DSMZ 10 and DSMZ 347). Overall, the most common and prevalent group of strains (group A) was demonstrated to be closely related to G. stearothermophilus (DSMZ 22).     

荸荠英提取物抑菌活性及其应用研究

为了提高荸荠的深加工附加值,本实验从荸荠加工废弃物荸荠皮中提取抑菌物质,研究其抑菌活性及其应用。结果表明,荸荠英提取物对大肠杆菌、金黄色葡萄球菌、藤黄微球菌、枯草芽孢杆菌均有抑制作用,其中对金黄色葡萄球菌的抑制作用最强,且持续性好。荸荠英提取物对大肠杆菌、金黄色葡萄球菌、藤黄微球菌的抑制作用高于1%的山梨酸钾溶液,而对枯草芽孢杆菌的抑制作用与其比较接近;利用荸荠英提取物能有效防止彩色水果番茄贮藏过程中由根霉和链格孢引起的腐烂,与根霉相比,荸荠英提取物对链格孢的抑制效果更好。     

蒲公英不同溶剂提取物体外抑菌效果的研究

采用不同溶剂对蒲公英中的有效成分进行提取,利用平板打孔法对黑曲霉菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌、蜡样芽孢杆菌、纳豆芽孢杆菌、多黏芽孢杆菌等进行体外抑菌实验,研究不同溶剂提取物的抑菌能力,并测定其抑菌直径、最小抑菌浓度和最小杀菌浓度。结果表明,2种提取物对金黄色葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、藤黄微球菌、多黏芽孢杆菌等几种细菌均有不同程度的抑制作用,且表现出蒲公英的醇提液的抑菌能力大于蒲公英水提液。     

枯草芽孢杆菌CCTCC M207209对食源性病原菌的抑制作用

采用杯碟法、对峙培养法和平板对扣法分别检测了枯草芽孢杆菌CCTCC M207209的活菌菌碟、无菌体发酵液、多糖提取液、蛋白质粗提液及其挥发性气体产物对金黄色葡萄球菌、肠球菌、化脓链球菌、大肠杆菌、沙门氏菌等食源性病原菌的抑制作用。结果表明,枯草芽孢杆菌CCTCC M207209的活菌菌碟和发酵液对所有参试病原菌均有显著抑制作用;蛋白质粗提液对除化脓链球菌以外的所有病原菌有显著抑制作用;挥发性气体产物对所有病原菌均无抑制作用;所有抑菌活性物质对金黄色葡萄球菌的抑制作用最强,初步确定其为蛋白类物质。     

一株产抗菌活性物质解淀粉芽孢杆菌的筛选及鉴定

从菜园土壤中分离筛选到1 株产广谱抗菌活性物质的菌株K6,其抗菌活性物质对藤黄微球菌、大肠杆菌、铜绿假单孢菌、枯草芽孢杆菌、蜡样芽孢杆菌、巨大芽孢杆菌、苏云金芽孢杆菌、酿酒酵母、匍枝根霉和白色念珠菌均有抑制作用,其中对革兰氏阳性菌的抑菌作用最强,对霉菌的抑制作用相对较弱。通过形态特征、16SrDNA 序列分析和系统发育分析,菌株K6 与B. amyloliquefaciens 位于同一簇群,同源性达99%,将其初步鉴定为解淀粉芽孢杆菌。     

Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds

A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product. This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators. By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis. While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth. The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E. The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae. The inhibitory compound(s) could possibly serve as a food biopreservative.     

Antimicrobial Activities of Cynara scolymus L. Leaf, Head, and Stem Extracts

ABSTRACT: Successive chloroform, ethanol, and ethyl acetate partitions of extracts from Cynara scolymus L. leaf, head, and stem were tested for their antimicrobial activity against 15 microbial species, including 7 foodborne bacterial pathogens, Bacillus subtilis, Staphylococcus aureus, Agrobacterium tumefaciens, Micrococcus luteus, Escherichia coli, Salmonella typhimurium , and Pseudomonas aeruginosa , 4 yeasts, Candida albicans, Candida lusitaniae, Saccharomyces cerevisiae , and Saccharomyces carlsbergensis , and 4 molds, Aspergillus niger, Penicillium oxalicum, Mucor mucedo , and Cladosporium cucumerinum using the disk diffusion assay technique. The leaf extract was found to be most effective against all of the tested organisms, followed by the head and stem extracts, and the ethanol fraction showed the most significant antimicrobial activity against all of the tests among 3 soluble fractions of extract, followed by the chloroform and ethyl acetate fractions. The minimum inhibitory concentrations (MICs) of extracts determined by the agar and broth dilution method ranged from 1.25 to 10.0 mg/mL. The MIC of ethanol fraction of leaf extracts was the lowest by comparison with the other 2 extracts. The MIC for fungi was at or below 2.5 mg/mL and for bacteria was at or above 2.5 mg/mL.     

不同脱乙酰度蚕蛹壳聚糖抑菌性能的比较

研究了3种不同脱乙酰度蚕蛹壳聚糖对9种供试菌的抑菌效果。结果表明:3种壳聚糖对蜡状芽孢杆菌、鼠伤寒杆菌、枯草芽孢杆菌、白色念珠球菌、绿脓假单胞菌、大肠杆菌和金黄色葡萄球菌有明显的抑制作用,对巨大芽孢杆菌和嗜热脂肪芽孢杆菌的抑制作用不明显。随着壳聚糖溶液浓度的增大,其抑菌能力也增强。3种壳聚糖的抑菌活性为:脱乙酰度为95.96%的壳聚糖>脱乙酰度为86.45%的壳聚糖>脱乙酰度为78.12%的壳聚糖。壳聚糖的抑菌活性呈现随pH降低而增加的趋势,当pH值在pH 4.0～5.0,壳聚糖对所有供试菌均能完全抑制。高脱乙酰度蚕蛹壳聚糖作为热加工食品的防腐剂,可稳定保持其抑菌防腐性能。     

微热协同超高压处理杀灭芽孢杆菌芽孢效果的研究

采用比色法研究了微热协同超高压处理对枯草芽孢杆菌与嗜热脂肪芽孢杆菌芽孢的影响。结果表明,微热协同超高压处理芽孢能够显著提高芽孢2,6-吡啶二羧酸(DPA)的泄漏率(p<0.05)。处理组550、600MPa,50、60、70℃作用于枯草芽孢杆菌与嗜热脂肪芽孢杆菌芽孢,破坏芽孢结构,通透性屏障破坏,导致DPA的泄漏。所泄漏的DPA与灭菌对照组(121℃,30min)相比差异不显著(p>0.05),主要是芽孢质中的DPA。说明微热处理协同超高压杀灭枯草芽孢杆菌与嗜热脂肪芽孢杆菌芽孢的原因可能是其物理结构的破坏。     

广谱抑菌物质产生菌GX-21的筛选及鉴定

本研究在广东省及其周边地区共11个采样地点采集土壤样品156份,分离得到放线菌1026株。土壤预处理采用碳酸钙富集培养法,分离采用平板稀释法,选取高氏一号合成培养基作为分离培养基,添加50 mg/L重铬酸钾作为抑制剂。初筛采用琼脂块法,选择金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希氏菌、白假丝酵母菌、黑曲霉共5种指示菌进行抑菌试验,共得到95株活性菌株。复筛选择初筛的5种指示菌加上副溶血性弧菌、铜绿假单胞菌、变形杆菌共8种指示菌,分两级进行复筛:一级复筛仍采用琼脂块,得到19株活性菌株;二级复筛采用牛津杯定量扩散法,得到5株优良菌株。对优良菌株GX-21进行鉴定,通过形态特征观察、培养特性观察、生理生化试验和16S r DNA序列分析,确定菌株GX-21为多产色链霉菌(Streptomyces polychromogenes)。菌株GX-21具有抑菌谱广的特点,对革兰氏阳性菌、革兰氏阴性菌和真菌均有抑制作用。     

中性蛋白酶酶解羊骨产物抗菌性的研究初探

研究利用中性蛋白酶对羊骨进行酶解，采用五因素五水平正交旋转设计对羊骨进行酶解，以抗菌率为指标，对酶解条件进行优化，并对最优条件下所得产物的抗菌性进行详细对照评定。以大肠杆菌、枯草杆菌和藤黄球菌为供试菌种。结果发现，第21组酶解液（即酶浓度200U／g，底物浓度1：3，时间6h，酶解温度50℃，pH值为7．0）的抗菌性相对较强，但其抗菌性的强度相当于0．015％～0．019％亚硝酸钠溶液的抑菌力。21号酶解液对大肠杆菌、枯草杆菌和藤黄球菌在第4h～10h的生长时间范围内，表现出明显的抑制作用。     

白刺果实提取物的体外抑菌活性研究

探讨白刺果实提取物的体外抑菌活性,以期为白刺果实的综合利用及天然食品防腐剂的开发提供依据。用石油醚、氯仿、乙酸乙酯和正丁醇分别对白刺果实95%乙醇提取物进行萃取,纸片法测定各萃取部分对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、酵母菌、青霉、根霉和黑曲霉7种常见的食源性污染菌的抑菌效果,并通过倍比稀释法测定乙酸乙酯部分对其中3种细菌的最低抑菌浓度。结果表明:白刺果实乙酸乙酯部分、乙醇部分和氯仿部分对3种供试细菌均有抑菌作用,其中乙酸乙酯部分抑菌效果最好,且乙酸乙酯部分对3种细菌的抑制强弱顺序为:枯草芽孢杆菌>大肠杆菌>金黄色葡萄球菌;其中乙酸乙酯部分对枯草芽孢杆菌和金黄色葡萄球菌的最低抑菌浓度(MIC)均为25mg/mL,对大肠杆菌的MIC是50mg/mL。各萃取部分对供试真菌均无明显抑制作用。      

产广谱细菌素乳酸菌的筛选

从四川传统发酵食品、市售酸奶、自制泡菜、香肠中分离出267株乳酸菌,采用平板挖井法从中筛选了64株对大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌、枯草芽孢杆菌有抑制作用的广谱抑菌性乳酸菌菌株,再对这些乳酸菌采用牛津杯抑菌实验,排除酸、过氧化氢干扰后,部分菌株的发酵上清液仍有很强的抑菌作用;进行胰蛋白酶、木瓜蛋白酶处理后,发酵上清液抑菌活性降低,因而确定产生的抑菌物质为蛋白质类细菌素。     

Antimicrobial activity of neutralized extracellular culture filtrates of lactic acid bacteria isolated from a cultured Indian milk product (‘dahi’)

Neutralized extracellular culture filtrate obtained from isolates of Lactobacillus acidophilus, Lactobacillus delbruecki ssp. bulgaricus, Lactobacillus salivarius and Lactococcus lactis ssp. lactis from ‘dahi’ showed weal to moderate inhibition of Staphylococcus aureus, Bacillus cereus, Escherichia coli, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus laterosporus, Bacillus subtilis and Pseudomonas aeruginosa when tested by the diffusion agar well assay method. The effective minimum quantity of lactic culture filtrates required to obtain complete inhibition of an inoculum of 10³ cfu/ml of the bacteria tested was between 20 and 26% (vol/vol), as determined by the agar incorporation method. Neutralized extracellular culture filtrate of these lactic cultures added at a level of 10% in sterile, 10% reconstituted non-fat dry milk was able to either suppress or retard growth of selected bacterial cultures when incubated at 37°C for 24 h. This study indicated the antimicrobial activity of dahi and the potential of using neutralized extracellular culture filtrate of lactic acid bacteria in the biopreservation of foods.     

A rapid assay for detecting sulfonamides in tissues of slaughtered animals

Governments regulate antimicrobial residues in slaughtered animals with surveillance programs for detecting drugs in food-producing animals. Although initial screening bioassay systems are recognized for their sensitivities to antimicrobial drug groups, none are sensitive to sulfonamides at or near the maximum residue levels (MRLs) in the Codex Alimentarious. We have developed a sulfonamide-sensitive rapid assay using Bacillus stearothermophilus inoculated PM indicator agar containing bromcresol purple and trimethoprim, where the end point is a combination of color change in the agar and zone of microbial growth inhibition around the sampling disk. Five sulfonamides, plus 16 other antimicrobial drugs were tested in standard concentrations in water, bovine kidney, and ground beef. Sulfonamides were detected at concentrations near the MRLs, and they were presumptively identified using para-aminobenzoic acid. The rapid assay was extremely sensitive to beta-lactams that were presumptively identified using penase. The system also was sensitive to tetracyclines, aminoglycosides, and macrolides, of which tetracyclines and gentamicin were identified using enzyme-linked immunosorbent assay (ELISA). In trials on slaughterhouse tissues submitted for testing in Ontario's meat surveillance program, the rapid assay identified twofold the number of positive kidneys and threefold the number of positive diaphragm samples compared to a standard microbiological inhibition test (MIT) currently approved. Fifty-three of 471 carcasses were sulfonamide positive with the rapid assay, while no sulfonamides were detected with the MIT. ELISA and thin-layer chromatography were used on selected samples to confirm the rapid assay sulfonamide presumptive results.