Characterization of some yeasts isolated from foods by traditional and molecular tests

In this study, 22 yeast strains isolated from foods were characterized by traditional and molecular techniques. With the help of traditional identification tests, yeast strains were grouped in 12 species belonging to 11 genera as follows: Candida parapsilosis, Rhodotorula mucilaginosa, Debaryomyces hansenii, Cryptococcus humicolus, Cryptococcus albidus, Aureobasidium spp., Hanseniaspora valbyensis, Metschnikowia pulcherrima, Lachancea thermotolerans, Pichia anomala, Geotrichum candidum and Yarrowia lipolytica. The patterns obtained by the digestion of ITS-18S rRNA gene with MspI and HaeIII restriction endonucleases were similar among strains belonging to the same species. With the help of randomly amplified polymorphic DNA (RAPD) analysis performed within the same species, discrimination of M. pulcherrima strains could be achieved.     

Enumeration and identification of yeasts isolated from Zimbabwean traditional fermented milk

The yeasts in 30 samples of the Zimbabwean traditional fermented milk, amasi, taken from farms, households and milk collection centres were enumerated and identified. The yeast counts ranged from <2 to 8.08 log cfu g⁻¹. Yeast isolates were identified using the API ID 32°C test kits and the simplified identification method (SIM) as well as with reference to the standard taxonomic keys. From the 30 samples, a total of 20 different yeast species were identified. Saccharomyces (S) cerevisiae (22 isolates), Candida (C) lusitaniae (11), C. colliculosa (7) and S. dairenensis (7) were the predominant species identified. Dekera (Dek.) bruxillensis, C. lipolytica and C. tropicalis were identified less often. Seven of the S. cerevisiae isolates were able to assimilatedl-lactate. The strain of C. kefyr isolated could assimilate lactose anddl-lactate, but not citrate. The analysed amasi samples contained a wide variety of yeasts, but only a few species predominated and these could possibly contribute to the characteristics of the fermented milk in the 48 h fermentation.     

三种传统发酵食品中酵母菌的分离鉴定与特性分析

从红茶菌、藏灵菇及酸菜3种传统发酵食品中分离、鉴定酵母菌,并测定其产乙醇能力及生长性能。结果表明,从3种传统发酵食品中分离出9株酵母菌,包括3株马克斯克鲁维酵母（Kluyveromyces marxianus）、1株Starmerella davenportii、3株异常假丝酵母（Candida incommunis）及2株瑟氏哈萨克斯坦酵母（Kazachstania servazzii）。马克斯克鲁维酵母X1-1和X3-3可发酵葡萄糖、蔗糖和乳糖,其他酵母菌株只发酵葡萄糖。3株马克斯克鲁维酵母产乙醇力较弱,分别为29.8%vol、42.6%vol及29.6%vol,但生长速率快且活菌数高,其中菌株X1-2约6 h时进入对数期,约30 h时达到最大生物量（OD600 nm=6.152）,而其他3种酵母则生长缓慢。异常假丝酵母R5-2和瑟氏哈萨克斯坦酵母M2-2产乙醇能力较强,分别为75.8%vol和71.0%vol。     

宁夏御马葡萄酒厂野生酵母菌株的分离筛选及分子鉴定

利用26S rDNA D1/D2区序列分析法,对分离自宁夏御马葡萄园的22株野生酵母菌进行了鉴定,同时构建了22株供试菌株与相关模式菌株的系统发育树,分析了供试菌株与已知酵母菌的亲缘关系及其分类地位.结果表明:供试菌株被鉴定为5属7种,分别是酿酒酵母、巴氏酵母、葡萄汁有孢汉生酵母、克鲁维毕赤酵母、美极梅奇酵母和核果梅奇酵母.     

曲中产香酵母分离及Biolog系统鉴定分析

采用稀释涂布法从8种酿造曲样中分离出7株产香能力强的酵母菌(CX-1-CX-7菌),其碳源利用种类和数量有较大差异,CX-5菌能显著利用D-半乳精和D-木糖/戊醛糖以及D-蜜二糖和D-木糖/戊醛糖作为碳源;麦芽糖、麦芽三糖、蔗糖、α-D-葡萄糖均能被7株菌利用:水苏糖、苦杏仁苷、L-谷氨酸、木糖醇、D-核糖等17种碳源只能分别被CX-5、CX-6、CX-7菌利用;CX-2-CX-7为不同类型的酿酒酵母;CX-1、CX-3、CX-5菌容易培养且生长稳定性好,具有进一步进行产香发酵研究的价值.     

海产品中溶藻弧菌的分离及多种方法鉴定效果的比较

目的了解北京市市售海产品中溶藻弧菌的污染情况,并对使用不同方法鉴定溶藻弧菌的效果进行比较。方法样品经碱性蛋白胨水增菌后,分别于硫代硫酸盐柠檬酸盐胆盐蔗糖(TCBS)琼脂培养基和科玛嘉弧菌显色培养基上划线培养,实时荧光聚合酶链式反应(PCR)法鉴定可疑菌落。以rpoB基因测序方法为参考,比较了实时荧光PCR和VITEK两种方法的鉴定效果。结果对北京市水产品市场随机采集的116份海产品进行了检测,实时荧光PCR法鉴定出溶藻弧菌阳性样品95份,检出率高达82%。使用科玛嘉弧菌显色培养基的检出率高于TCBS培养基,分别为82%(95/116)和72%(83/116)。经rpoB基因核苷酸序列测定确定95株疑似菌株为溶藻弧菌。采用VITEK 2 COMPACT GN鉴定卡对这95株菌株进行鉴定,31株鉴定为溶藻弧菌,其余未能得到准确的鉴定结果。结论北京市市售海产品中溶藻弧菌检出率高。科玛嘉弧菌显色培养基比TCBS琼脂培养基更适于溶藻弧菌的分离,实时荧光PCR法的鉴定效果优于VITEK法。     

腐乳源酵母分离鉴定及其极性脂质分析

从豆腐酸浆及毛坯中鉴定了23个分离菌株,测定了其脂肪酶酶活并利用超高效液相串联质谱对其中7个菌株的脂质进行了分析。结果表明,指纹图谱中包含2种鞘氨醇、16种酰胺、22种酯类和3种脂肪酸。其中,马克斯克鲁维酵母SP-1和挪威毕赤酵母SP-5中植物鞘氨醇、N,N-二甲基鞘氨醇的相对含量最高。此外,嗜酒假丝酵母ATW-1、库德里阿兹威毕赤氏酵母SP-4和阿米塞毕赤氏酵母Y的酯类化合物比较丰富,且其细胞结合型脂肪酶酶活力也比较高,PCA分析进一步表明这3个菌株脂质组成具有较大的相似性,表明酵母菌中丰富的代谢酯类物质可能与细胞结合型脂肪酶之间存在密切联系。     

南疆传统发酵酸奶中酵母菌的分子鉴定及抗氧化活性研究

以开发新疆地区传统发酵乳中的酵母菌为研究目的,对分离的78株酵母菌利用26SrDNA D1/D2区和ITS转录区间分析以及抗氧化活性检测试剂盒进行分子鉴定和抗氧化活性检测。结果表明,78株酵母菌中具有抗氧化活性的菌株有42株,其中NG-40综合抗氧化能力突出,其抗氧化活性为(106.41±3.92)U/mL,抑制羟基自由基能力为(651.24±3.75)U/L,抗超氧阴离子能力为(104.11±3.25)U/mL,而脂质过氧化物含量仅为(1.99±0.65)μmol/L;分子鉴定结果表明,38株为酿酒酵母(Saccharomyces cerevisiae),36株为马克思克鲁维酵母(Kluyveromyces marxianus),4株属于毕赤酵母(Pichia kudriavzevii)。     

Carotenoids of yeasts isolated from the Brazilian ecosystem

The carotenoid composition of pigmented yeasts isolated in Brazil was studied. The yeasts were cultured in yeast malt broth at 200 rpm, 25 °C, for 5 days, without illumination. Open column, thin layer and high performance liquid chromatography were used to separate, identify and quantify the carotenoids. The major pigments found in these yeasts were torulene and β-carotene. β-Carotene predominated in Rhodotorula graminis-125, Rhodotorula glutinis and Sporobolomyces roseus, while torulene was the principal carotenoid in Rhodotorula mucilaginosa. The yeast R. glutinis had the highest total carotenoid production (881 μg/l), followed by R. graminis (594 μg/l), Rhodotorula mucilaginosa-137 (590 μg/l), Rhodotorula mucilaginosa-108 (562 μg/l) and Rhodotorula mucilaginosa-135 (545 μg/l). Rhodotorula minuta and S. roseus had the lowest carotenoid contents (168 and 237 μg/l, respectively). In μg/g of dry cells, R. glutinis had a total carotenoid concentration of 132 μg/g.     

A chromogenic medium for the detection of yeasts with beta-galactosidase and beta-glucosidase activities from intermediate moisture foods

A selective and differential solid medium for the specific detection of some common yeasts frequently causing spoilage in intermediate moisture foods is described. The principle of the method is based on the detection of two enzymes, beta-glucosidase and beta-galactosidase, using the chromogenic substrates salmon-Gluc and X-Gal. Over 140 yeasts and bacteria were tested, and Debaryomyces hansenii and Kluyveromyces marxianus strains produced salmon and dark blue colonies, respectively, thus permitting their clear discrimination from other yeasts common in intermediate moisture foods. The medium was very satisfactory when intermediate moisture foods were tested.     

甘蔗尾中酵母菌的分离与鉴定

以0～90 d打包青贮发酵的甘蔗尾为试验材料,采用传统培养法和平板划线分离法得到7株酵母菌。对纯化后的酵母菌进行形态观察、API 20 C AUX生化鉴定试剂盒鉴定及内转录间隔区（ITS）序列核糖体脱氧核糖核酸（rDNA）分子生物学鉴定,并对其生长性能进行初步探索。结果显示：菌株X1、X7、X9和X10被鉴定为Kazachstania humilis,菌株X3、X4和X6被鉴定为扁平云假丝酵母（Candida humilis）。7个菌株在培养24 h后菌液OD660 nm值达到9.90～12.84,生长性能较好。     

蜂蜜中耐高渗透压酵母菌的分离与鉴定

从我国华北地区采集到不同来源的蜂蜜样品13份,对其中耐高渗透压酵母菌进行分离与鉴定,经形态学特征、生理生化特征和26S rDNA基因测序分析,结果表明,分离到的7株耐高渗酵母菌分别鉴定为:菌株A、B为八孢裂殖酵母(Schizosaccharomyces octosporus);菌株C、D、E为蜂蜜接合酵母(Zygosaccharomyces mellis);菌株F、G为暹罗接合酵母(Zygosaccharomyces siamensis)。      

葡萄表面所得酵母菌的筛选及其鉴定

从市售葡萄表面分离出两株酵母菌,经形态、培养特征、酵母菌假菌丝和酵母菌子囊孢子的观察和生理生化实验及发酵特性的研究,鉴定出这两株酵母菌分别为布鲁塞尔德克酵母(Brettanomyces bruxellensis)和酿酒酵母(Saccharomyces cerevisive).     

A medium for the impedimetric detection of yeasts in foods

A mycological medium, CBAS, was developed for the detection of yeasts in foods using polarization capacitance (Cpol) measurements. The signal obtained due to the growth of yeasts in CBAS provided earlier detections and stronger responses than the signals produced by these yeasts in other common media. Of several impedance components evaluated, Cpol provided the most useful signal for yeasts in CBAS. Further investigation showed that pH change was responsible for approximately 50% of the Cpol signal. Cpol detection times correlated highly with plate counts of a yeast isolated from orange juice when tested in CBAS (r = ?0·99) and in a 1:10 dilution of orange juice in CBAS (r = ?0.94). A concentration of 10² yeast cfu ml⁺¹ in a 1:10 dilution of orange juice in CBAS was detected within 26 h by Cpol in contrast with a 3 to 5 day plate count.     

食源性阪崎肠杆菌耐药性分析及超广谱β-内酰胺酶检测

利用药敏纸片方法,对19株食品来源的阪崎肠杆菌进行16种常规抗生素药敏检测。结果显示,19株阪崎肠杆菌对苯唑西林以及头孢噻吩的耐药率分别为100%和78.95%,而均对其余种类抗生素敏感。采用PCR方法结合纸片扩散法对分离菌株进行分属于四大类β-内酰胺酶中的共10种超广谱β-内酰胺酶检测,结果未发现超广谱β-内酰胺酶阳性菌株。综合以上结果,目前食品中存在的阪崎肠杆菌仍为耐药性较弱的菌种,短时期内将不会对临床治疗带来较大威胁。     

即食食品中单核细胞增生李斯特菌的血清学分型和毒力基因分析

目的掌握即食食品中单核细胞增生李斯特菌(简称单增李斯特菌)的血清型、谱系和感染相关基因的分布。方法以全国食源性致病菌监测网中2007—2009年自即食食品分离的226株单增李斯特菌为研究对象,采用传统的血清学分型技术和等位基因特异性寡核苷酸PCR方法(ASO-PCR)研究其血清学分型,并采用PCR方法检测其与感染相关的基因。结果 226株单增李斯特菌血清学分型结果显示,1/2a、1/2b、1/2c、4b为主要血清型,比例分别为41.59%(94/226)、40.71%(92/226)、10.62%(24/226)和5.31%(12/226)。引起人类疾病的常见血清型1/2a、1/2b和4b菌株占87.61%(198/226)。谱系Ⅰ菌株为105株,谱系Ⅱ菌株为120株,谱系Ⅲ菌株为1株;我国绝大部分即食食品中单增李斯特菌分离株的感染相关基因缺失率较低,只有个别菌株缺失感染相关基因。结论本研究通过对分离自即食食品中的单增李斯特菌进行血清学分型、谱系分析和感染相关基因的检测,提示我国需要加强食品场所卫生管理,降低单增李斯特菌对即食食品的感染风险。     

Characterization of antimicrobial-resistant Salmonella isolated from imported foods

Two-hundred eight Salmonella isolates recovered from over 5,000 imported foods entering the United States in 2001 were tested for antimicrobial susceptibilities and further characterized for quinolone resistance mechanisms, integron carriage, and genetic relatedness. Salmonella Weltevreden (20%), Salmonella Newport (6%), Salmonella Lexington (5%), and Salmonella Thompson (4%) were the four most common serotypes recovered. Twenty-three (11%) isolates were resistant to at least one antimicrobial, and seven (3.4%) to three or more antimicrobials. Resistance was most often observed to tetracycline (9%), followed by sulfamethoxazole (5%), streptomycin (4%), nalidixic acid (3%), and trimethoprim/sulfamethoxazole (2%). One Salmonella Schwarzengrund isolate recovered from squid imported from Taiwan exhibited resistance to eight antimicrobials, including ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Six isolates (Salmonella Bareilly, Salmonella Derby, Salmonella Ohio and three Salmonella Schwarzengrund) contained class 1 integrons, which carried several resistance genes including dhfrI/dhfrXII, aadA, pse-1, and sat1, conferring resistance to trimethoprim/sulfamethoxazole, streptomycin, ampicillin, and streptothricin, respectively. Five of six nalidixic acid-resistant isolates possessed DNA point mutations at either Ser83 or Asp87 in DNA gyrase. One ciprofloxacin-resistant isolate possessed double mutations in DNA gyrase at positions Ser83 and Asp87 as well as a single mutation at Ser80 in parC. The top three serotypes identified, Salmonella Weltevreden (n = 41), Salmonella Newport (n = 13), and Salmonella Lexington (n = 11), were further characterized for genetic relatedness by pulsed-field gel electrophoresis. Fifty-five distinct pulsed-field gel electrophoresis patterns were observed among the 65 isolates, indicating extensive genetic diversity among these Salmonella serotypes contaminating imported foods.     

Characterization of Listeria monocytogenes isolated from retail foods

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.     

食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。     

Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts

Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.