一株产壳聚糖酶芽孢杆菌的分离及鉴定

采用透明圈法,从黄河三角洲地区的腐败落叶分离出9株产壳聚糖酶的菌株。再经菌株分离纯化、液体发酵复筛及酶活研究,确定其中1株为壳聚糖酶高产菌株,命名为XHT-0903。通过形态学特征、生理生化特征及16S rDNA序列分析进行菌株鉴定,确定XHT-0903为蜡样芽孢杆菌（Bacillus cereus）。其液体发酵酶活力达20 U/mL,具有较好的开发及应用价值。     

产普鲁兰酶蜡样芽孢杆菌的分离鉴定及酶学性质研究

利用选择性培养基从广西南宁淀粉加工厂附近土壤中分离出一株具有产普鲁兰酶能力的野生菌株GXBC-2。该菌株革兰染色为阳性,形成芽孢,菌体细胞杆状,结合生理生化特征和16S rDNA序列分析,初步鉴定该菌株为Bacillus cereus GXBC-2。对该菌所产普鲁兰酶的性质研究表明:酶的最适反应温度为50℃,在30～50℃范围内稳定,最适pH为7.0,稳定pH范围为6～8.5。产物经HPLC分析证明,该酶能水解普鲁兰糖产生麦芽三糖,对可溶性淀粉不起作用,所以该酶属于I型普鲁兰酶。     

Isolation and characterization of an antimicrobial substance from Bacillus subtilis BY08 antagonistic to Bacillus cereus and Listeria monocytogenes

A wild-type bacteria producing an antimicrobial substance was isolated from Korean traditional fermented soybean paste, and identified as Bacillus subtilis. The antimicrobial substance purified by TLC, tentatively named UV254-B, displayed a specific antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes, without inhibiting the growth of soybean-fermenting Bacillus species. The antimicrobial substance was susceptible to proteinase K and lipase but resistant to esterase. Antimicrobial activity was observed over a wide range of pH from 3 to 11, with the maximum activity at pH 9, and thermal stability up to 80°C. The minimum inhibitory concentration of the antimicrobial substance was found to be 64 μg/mL for B. cereus and 128 μg/mL for L. monocytogenes. This antimicrobial substance has a putative molecular weight either at 1,133.6 or 1,700.5, which differs from that of other antimicrobial substances described for B. subtilis such as iturin, surfactin, fengycin, and subtilisin.     

白腐乳中蜡状芽孢杆菌的分离与鉴定

从白腐乳中分离到1株杆菌LX,经生理生化和16S rDNA分子生物技术鉴定,该菌株被鉴定为蜡状芽孢杆菌(Bacillus cereus).采用BCET-RPLA试剂盒对菌株培养液进行了肠毒素检测,结果表明厌氧培养时产肠毒素检测为阴性,而好氧培养时菌株产肠毒素检测为弱阳性.研究表明在白腐乳的生产与保藏中,应注意蜡状芽孢杆菌的存在及其对食用安全性的影响.     

Survival, isolation and characterization of a psychrotrophic Bacillus cereus strain from a mayonnaise-based ready-to-eat vegetable salad

Incidence and population levels of Bacillus cereus in American salad, an industrially manufactured, packaged and refrigerated deli salad containing vegetables and mustard, were determined. Of 12 ready-to-eat samples examined, one (8.3%) was positive for B. cereus at less than 5 x 10(3)cfu g(-1). According to the ISO confirmation procedure, a strain was isolated and further characterized and identified as B. cereus EPSO-35AS by API 50CH/20E phenotypic system, combined with additional tests of motility, oxidase activity and anaerobic growth. This strain produced diarrhoeal enterotoxin in tryptic soy broth culture as detected by BCET-RPLA test, hydrolysed starch and had a low D(90)-value (2.1 min), with an estimated z-value of 6.79 degrees C. After a lengthy lag phase (9-12 days of incubation), the strain was able to grow at 8 degrees C in both nutrient broth and tyndallized carrot broth with specific growth rates from 0.009 to 0.037 h(-1), respectively. In the vegetable substrate, lag time was approximately 3 days (66 h) shorter than in laboratory medium. The effect of temperature abuses on the safety of the product during the time of use or consumption is discussed.     

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible.     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.     

Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores

Spores of Bacillus cereus strains ATCC 7004, ATCC 4342 and ATCC 9818 were produced in four sporulation media (Nutrient Agar supplemented with 1 ppm Mn²⁺, Fortified Nutrient Agar, Angelotti Medium and Milk Agar) and their percentages of sporulation and heat resistance parameters obtained in a wide temperature range were compared. In all conditions studied, high rates of sporulation were obtained. Clear differences among D-values for spores produced in the four media were observed. the medium which yielded the most resistant spores and the magnitude with which the sporulation medium affected D-values was different for each strain. z-Values of the three strains were not influenced by the medium used to obtain spores.     

一株产抑菌物质的蜡样芽胞杆菌的鉴定及其抑菌物质性质

从酱渣中分离到一株产抑菌物质的新菌株,暂命名为菌株B。研究表明,该菌株经碱中和后的无细胞发酵液对革兰氏阳性菌、革兰氏阴性菌和真菌均有抑制作用;其发酵上清液经硫酸铵盐析和透析后,仍然有抑菌活性;双缩脲和茚三酮实验表明,抗菌活性物质为肽类物质。生理生化实验、16S rDNA序列比对及系统发育分析表明,菌株B为蜡样芽胞杆菌,其生长和抑菌最适培养基为NB培养基,在pH 7.0的NB培养液中培养40h其抑菌活性达到最大。     

蜡样芽胞杆菌生长特性及大蒜精油对其抑菌活性的研究

主要研究了37℃条件下3种蜡样芽胞杆菌在营养肉汤中的生长状况,建立了37℃下蜡样芽胞杆菌在营养肉汤中的Boltzmann牛长模犁,3条牛长曲线相关系数鼯均大于0．97;检测了不同培养时间蜡样芽胞杆菌的产芽胞情况,结果表明1号菌株和14号菌株较早产芽胞,培养相同时间,产芽胞数：1号菌株,4号菌株〉标准菌株;采用牛沣杯法和平板计数法研究了大蒜精油对蜡样芽胞杆菌的抑制效果,结果表明浓度为lO。的人蒜精油对3种蜡样芽胞杆菌都有很好的抑制效果,3种菌株中l号菌株最难抑制。     

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.     

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.     

Microbial transformation of aniline derivatives: regioselective biotransformation and detoxification of 2-phenylenediamine by Bacillus cereus strain PDa-1

A bacterial isolate, strain PDa-1, grew well on basal medium supplemented with 2-phenylenediamine, sucrose, and ammonium nitrate and completely transformed 2-phenylenediamine. The isolate was identified as Bacillus cereus. The product formed from 2-phenylenediamine was identified by EI-MS and NMR as 2-aminoacetanilide; whole cells converted 2-phenylenediamine to the product with a 76% molar yield. Whole cells also showed a broad substrate specificity toward 20 of 26 tested arylamines with substituent groups of various size and positions. Especially 2-aminobenzoic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, and 2-aminofluorene were converted completely to the corresponding product with an aminoacetyl group. Cell extracts of strain PDa-1 had a high arylamine N-acetyltransferase activity. The partially purified enzyme converted 2-phenylenediamine to 2-aminoacetanilide. Strain PDa-1 constitutively expressed the enzyme in the absence of 2-phenylenediamine. Effects of 2-phenylenediamine and 2-aminoacetanilide on growth indicated that this enzyme probably plays a role in the detoxification of toxic arylamines in this strain.     

一株Photoboicterium Leiognathi N1发光细菌的分离及鉴定

对从海鱼（鲻鱼）表面分离到的1株发光强度很高的细菌N1进行系统分类鉴定。采用常规方法进行分离培养,以形态学特征、培养特性、生理生化特征以及分子生物学等方法对其进行分类鉴定。结果可知,该菌株为兼性厌氧发酵型革兰氏阴性杆菌,其16SrDNA核苷酸序列测定与Photoboicterium Leiognathi（鰒发光杆菌）的同源性达99.8%,系统进化树也显示,菌株N1与Photobacterium leiognathi构成一个分支;其形态特征与生理生化特性与Photoboicterium Leiognathi（鰒发光杆菌）完全吻合。确定此发光细菌为Photoboicterium Leiognathi（鰒发光杆菌）,命名为Photoboicterium Leiognathi N1。     

Isolation and enumeration of Bacillus cereus from foods on a novel chromogenic plating medium

Bacillus cereus is an aerobic sporeformer commonly found in raw and processed foods. Foodborne illnesses associated with this pathogen are caused primarily by consumption of cooked foods with inadequate refrigeration. Mannitol-egg yolk-polymyxin (MYP) agar is widely used for isolation and enumeration of the pathogen. In the present study, a new selective medium, which contains a chromogenic substrate (5-bromo-4-chloro-3-indoxyl- myo -inositol-1-phosphate) for the detection of phosphatidylinositol-specific phospholipase C in B. cereus/B. thuringiensis organisms, was evaluated and compared to MYP. Twenty B. cereus strains, four B. thuringiensis strains, 95 other Gram-positive and 14 Gram-negative organisms were examined on BCM^®B. cereus/B. thuringiensis (BioSynth) and MYP agar. Only B. cereus and B. thuringiensis formed large (2–7 mm), turquoise, flat colonies±turquoise halos on BCM^®after incubation for 24 h at 35°C, while other Bacillus spp. formed white, 1–2 mm colonies or were inhibited. Some Listeria strains formed <1-mm colonies, white or turquoise, on the medium. Of the Gram-positive non- B. cereus/B. thuringiensis organisms tested, growth of >50% was inhibited on BCM^®, compared to <1% on MYP. No significant difference (P>0·05) was observed on the two media in the recovery of B. cereus strains from foods inoculated or naturally contaminated with the organisms. However, colony enumeration and isolation were easier on BCM^®than on MYP because of higher selectivity and formation of discrete, non-coalescing colonies. Ribotyping (Riboprinter^®Microbial Identification System, Qualicon) of five randomly selected food isolates identified three B. cereus and two B. thuringiensis strains. Agar plates were stable for >3 months at 2–5°C.     

Incidence and characterization of Bacillus cereus isolated from traditional fermented meals in Nigeria

The aim of this study was to examine the presence of Bacillus cereus in fermented meals used in food seasoning in Nigeria. The microbial profiles of iru and ogiri, two Nigerian fermented vegetable proteins, were examined for presence of B. cereus. In the 50 samples tested, B. cereus was detected in all the samples, with the level of detection ranging from log 6.3 to log 8.3 g(-1) sample. Phenotypic characteristics of the B. cereus isolates showed that all of them could not ferment many sugars, most especially mannitol, but they utilized propionate citrate as a source of carbon and grew anaerobically. The isolates do not produce gas from glucose but hydrolyzed starch, casein, and gelatin. API-50CHB combined with API-20E identified the isolates as B. cereus. The diarrheal enterotoxin was detected by a reversed passive latex agglutination test kit. Results showed no significant difference in toxin production between ogiri and iru B. cereus isolated from different sources; all the isolates also demonstrated positive hemolytic activity. The API-ZYM enzyme profile showed that the strains have poor hydrolytic enzyme potential; hence, their possible contributions to the fermentation of vegetable protein is doubtful. This study established the proliferation of B. cereus in fermented protein meal and determined the diarrheal toxin production potential of the organism.     

Prevalence and characterization of toxigenic Bacillus cereus group isolated from low-moisture food products

Food Science and Biotechnology - The present study was conducted to determine the occurrence of B. cereus group members in low-moisture food products by phenotypic and genetic assessment and to...     

产磷脂酶菌株蜡样芽胞杆菌AF-1发酵条件的研究

以蜡样芽胞杆菌(Bacillus cereus) AF-1为发酵菌株,对其发酵产磷脂酶培养基和发酵条件进行优化。结果表明,最佳培养基组成为：葡萄糖2%,蛋白胨3%,磷酸氢二钠1.5%,磷酸二氢钾0.3%,硫酸镁0.06%,氯化钙0.01%;15 L发酵罐发酵条件为发酵时间30 h,装液系数为0.67,接种量为10%,初始pH值为7.5,发酵温度为35 ℃,通气量为0.3 m3/h,在此最佳条件下得到的发酵液磷脂酶活力为41.57 U/mL。