贮藏玉米中优势腐败霉菌M13的分离与鉴定

贮粮的安全关系到国计民生,对其中的腐败微生物进行分析研究具有重要意义。从贮藏玉米中分离到一株优势腐败霉菌M13,通过形态特征观察和ITS序列分析,将该菌株初步鉴定为米曲霉（Aspergillus oryzae）,并构建了其系统发育树,更是提供了一种准确可靠且简单易行的粮食真菌鉴定技术。     

凉果中优势腐败霉菌的分离鉴定

凉果加工与贮藏中霉菌污染直接影响凉果的品质。为明确凉果贮藏中优势腐败霉菌的种类,该研究对在常温贮存4个月后霉变的葡萄干、话梅、杏脯中腐败霉菌进行了分离纯化,得到7种典型腐败霉菌。将分离出的腐败霉菌回接至无菌的葡萄干、话梅、杏脯表面,进行回接侵染实验,通过凉果霉变时间确定出4株菌株为优势腐败霉菌。通过菌落形态特征、菌丝和分生孢子显微结构观察,结合核糖体内转录间隔区（rDNA-ITS）序列分析,构建系统发育,鉴定优势霉菌种属。结果表明：葡萄干的优势霉菌为芽枝状枝孢霉（Cladosporium velox）和暗黄青霉（Penicillium citreonigrum）,话梅的优势腐败菌为细极链格孢（Alternaria tenuissima）,杏脯的优势腐败霉菌为菌核青霉（Penicillium sclerotiorum）。该研究结果可为凉果腐败霉菌的分离鉴定提供可靠的方法,也可为后续凉果杀菌方法的选择,贮藏条件的优化提供依据。     

鲜剥大蒜中腐败微生物的分离纯化及鉴定

对鲜剥大蒜霉变部分的腐败微生物进行分离纯化,观察待测菌株的菌落形态特征、菌丝显微结构,并进行分子生物学分析。结果表明,经过分离纯化后一共得到4株菌,通过对其菌落形态特征和菌丝显微结构观察,初步判定为青霉、根霉、赤霉及曲霉;通过聚合酶链式反应（PCR）扩增、产物测序和序列分析,最终鉴定4株菌分别为：桔青霉（Penicillium citrinum）、匍枝根霉（Rhizopus stolonifer）、赤霉（Gibberella intermedia）、赭曲霉（Aspergillus ochraceus）。     

蚕茧霉变菌株的分离、纯化及鉴定

为鉴定蚕茧霉变菌株种类和防止霉菌危害的发生,从霉变蚕茧中分离纯化得到3株霉菌.采用点植法观察霉菌在察氏培养基上的生长特性、菌落形态及显微结构特征,结合ITS序列测定等分子生物学方法,对蚕茧霉变菌株进行鉴定.结果表明:3株霉菌分别属于曲霉属(Aspergillus),节孢霉属(Arthrinium),黑孢霉属(Nigro...     

茯砖茶优势菌株分离鉴定及发酵低级绿茶分析

对湖南白沙溪茯砖茶中的优势菌株进行分离、纯化,并通过形态学分析和ITS序列比对进行鉴定.以云南大叶种低级绿茶为原料进行纯菌发酵,分析发酵液主要成分含量的变化.结果表明:从湖南白沙溪茯砖茶中分离得到的4株优势菌株,其中J1被鉴定为谢瓦曲霉(Aspergillus chevalieri);J2、J3和J4均被鉴定为冠突曲霉...     

年糕中微生物的分离纯化和鉴定

通过对发生腐败变质的真空包装年糕中微生物进行分离、纯化和反证得到2株细菌、3株酵母菌、1株霉菌。经过形态观察、生理生化、微生物鉴定系统等方法,初步鉴定2株细菌分属于枯草芽孢杆菌(Bacillus subtilis)、不动杆菌属(Acinetobacter sp.),3株酵母菌都属于假丝酵母属(Candida spp.),1株霉菌属于构巢曲霉(Aspergillus nidulans)。     

贮藏玉米中1株优势腐败霉菌M14的分离与鉴定

贮粮的安全关系到国计民生,对其中的腐败微生物进行分析,具有重要意义。从贮藏玉米中分离到1株优势腐败霉菌M14,通过形态特征观察和ITS序列分析,该菌株初步被鉴定为球毛壳菌,构建了其系统发育树。本试验结果提供了1种准确、可靠且简单易行的粮食真菌鉴定技术。     

贮藏稻谷中一株优势腐败霉菌的分离与鉴定

贮粮的安全关系到国计民生,对其中的腐败微生物进行分析研究,具有重要意义。从贮藏稻谷中分离到一株优势腐败霉菌M33,通过形态特征观察和ITS序列分析,将该菌株初步鉴定为菌核曲霉(A.sclerotiorum),并构建了其系统发育树。     

赊店老酒大曲中可培养真菌的分离和鉴定

该研究采用传统分离培养方法,对赊店老酒大曲中的真菌进行分离纯化并通过形态学观察及分子生物学鉴定确定所获菌株的分类地位。结果表明,老酒大曲样品中共计分离得到45株酵母菌和103株霉菌。共鉴定出6株酵母菌,分别为酿酒酵母（Saccharomyces cerevisiae）、异常威克汉姆酵母（Wickerhamomyces anomalus）、扣囊复膜酵母（Saccharomycopsis fibuligera）、发酵毕赤酵母（Pichia fermentans）、胶红酵母（Rhodotorula mucilaginosa）及光滑假丝酵母（Candida glabrata）;酵母的优势菌属为酿酒酵母（Saccharomyces cerevisiae）（相对含量71%）、光滑假丝酵母属（Candida glabrata）（相对含量11%）。共鉴定出4个霉菌属,分别为根霉属（Rhizopus）、横梗霉属（Lichtheimia）、曲霉属（Aspergillus）和毛霉属（Mucor）;霉菌的优势菌属为根霉属（Rhizopus）（相对含量37%）、横梗霉属（Lichtheimia）（相对含量33%）。     

黑豆酱醅中霉菌的分离筛选及初步鉴定

对黑豆酱醅中的霉菌进行分离纯化,分离获得28株菌。28株菌经过酪素培养基平板初筛得到12株菌的H/C值较大,然后接种到PDA斜面培养基培养48h及72h时测定孢子数,并接种麸皮固体培养基测定中性蛋白酶活力,筛选出蛋白酶活力高的菌株分别命名为LM-7、LM-15、LM-20。对3株霉菌进行初步鉴定,确定LM-7、LM-20为米曲霉,LM-15为黑曲霉。     

高大平房仓稻谷粮层霉菌量与优势霉菌的差异性研究

采用传统培养法对湖南与湖北两省粮库中的稻谷进行研究,对高大平房仓粮仓上中下三层的稻谷霉菌量及优势霉菌进行研究。研究结果表明:湖南省储藏一年稻谷与新入库稻谷中层霉菌数分别为6.4×10~3 CFU/g、1.3×10~3 CFU/g,相比上、下层,中层最多;湖北省储藏一年稻谷下层霉菌数为1.4×10~4 CFU/g,相比上、中层,下层最多,湖北省新入库稻谷上层霉菌数为1.4×10~4 CFU/g,相比中、下层,上层最多。通过传统的菌落培养及菌丝、孢子观察,初步判断上中下三层的优势霉菌,并结合分子生物学的方法对其ITS序列进行分析,通过PCR扩增,将扩增出来的基因序列,在GenBank进行BLAST,最终鉴定优势菌株为黄曲霉、白曲霉、聚多曲霉、内生真菌、黑曲霉。     

Development of a PCR protocol to detect aflatoxigenic molds in food products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.     

THE AFLATOXIN CONTAMINATION OF FIG FRUITS IN AYDIN CITY (TURKEY)

The mold flora of 50 dried fig samples consumed in Turkey was examined and the aflatoxigenic ones were determined. Among 127 fungi isolated, 74 were Aspergillus, 24 were Trichoderma, 16 were Fusarium and 13 were Acremonium. Of the isolates, 17 were aflatoxigenic and four of them were capable to produce aflatoxin, three of which were characterized as A. flavus and one as A. parasiticus. Aflatoxin production of four strains was confirmed by high pressure liquid chromotography. The effect of UV irradiation on mold count and aflatoxin quantity was also tested. It was found that UV irradiation led to a decrease in the mold count and aflatoxin quantity.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of aflatoxins may exceed the determined limits in dried figs. Its presence can be a potential threat to the health of consumers. Dried figs are one of the major agricultural export products of Turkey ( Senyuva et al. 2005 ). The effects of UV irradiation on mold flora of dried figs and aflatoxins have been examined. The Aspergillus flavus and parasiticus agar (AFPA) medium is used for detection of aflatoxigenic species, and coconut agar medium (CAM) is used to detect the aflatoxin-producing ability of aflatoxigenic strains. It was found that the reproduction of the molds in dried figs, consequently the aflatoxigenic mold strains, can be depressed by UV irradiation. It was found that increasing time of UV irradiation led to a decrease in the mold count in dried figs. In addition, a UV irradiation applied for 90 min, was found to decrease the aflatoxin quantity in dried figs in an amount of 25%. Because of inexpensiveness and easiness of the application it was concluded that the UV irradiation can be used as a practical application.     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

Mold and Aflatoxin Reduction by Gamma Radiation of Packed Hot Peppers and Their Evolution during Storage

The effect of gamma radiation on moisture content, total mold counts, Aspergillus counts, and aflatoxins of three hot pepper hybrids (Sky Red, Maha, and Wonder King) was investigated. Whole dried peppers packed in polyethylene bags were gamma irradiated at 0 (control), 2, 4, and 6 kGy and stored at 25°C for 90 days. Gamma radiation proved to be effective in reducing total mold and Aspergillus counts in a dose-dependent relationship. Total mold counts in irradiated peppers immediately after treatments were significantly lowered compared with those in nonirradiated samples, achieving 90 and 99% reduction at 2- and 4-kGy doses, respectively. Aspergillus counts were significantly reduced, by 93 and 97%, immediately after irradiation at doses of 2 and 4 kGy, respectively. A radiation dose of 6 kGy completely eliminated the population of total molds and Aspergillus fungi. The evolution of total molds in control and irradiated samples indicated no further fungal proliferation during 3 months of storage at 25°C. Aflatoxin levels were slightly affected by radiation doses of 2 and 4 kGy and showed a nonsignificant reduction of 6% at the highest radiation dose of 6 kGy. The distinct effectiveness of gamma radiation in molds and aflatoxins can be explained by the target theory of food irradiation, which states that the likelihood of a microorganism or a molecule being inactivated by gamma rays increases as its size increases.     

宋河浓香型白酒酒醅纯培养霉菌群落的组成及功能酶筛选

为探究宋河浓香型白酒酒醅纯培养霉菌群落组成,检测纯培养菌株与酒醅发酵过程相关的功能酶酶活特征,为白酒相关功能菌株发掘与利用提供依据,本实验采用纯培养方法,对不同发酵时间、不同位置酒醅样品中的霉菌进行分离;根据形态特征去重复后进行18S rRNA基因克隆测序,初步判断其分类地位;采用点接法进行酒醅发酵相关5种功能酶筛选。结果表明,宋河浓香型白酒酒醅中蕴含丰富霉菌类群,共获得纯培养霉菌63株,形态去重复后共计15株,归属于：草本枝孢（Cladosporium herbarum）、黑曲霉（Aspergillus niger）、沃特曼篮状菌（Talaromyces wortmannii）、嗜松篮状菌（Talaromyces pinophilus）、杂色曲霉（Aspergillus versicolor）、米曲菌（Aspergillus oryzae）、毛栓菌（Trametes hirsuta）、桔青霉（Penicillium citrinum）、菲律宾青霉（Penicillium freii）、雪白丝衣霉（Byssochlamys nivea）及两个潜在新分类单元;霉菌的纯培养检出率在宋河浓香型白酒酒醅发酵3~15 d较高,之后随发酵时间增加逐渐降低;酒醅中的纯培养霉菌菌株具有丰富的功能酶酶活特性,淀粉酶、纤维素酶、果胶酶、蛋白酶、酯化酶的酶活筛选率分别为：100%、93.3%、33.3%、73.3%、46.7%,为后续进一步研究菌株的功能及开发利用奠定基础。     

中国南方稻谷主产区黑曲霉菌群的流行特征及产毒能力研究

本研究从我国南方十省份代表性粮库中采集稻谷样品,根据形态学特征筛选初步鉴别出黑曲霉群(Aspergillus niger aggregate)菌株,进一步依据ITS和CaM序列特征在种水平上进行鉴定;采用ELISA、HPLC-FLD方法,结合OTA生物合成基因鉴定方法联合确证黑曲霉群中分离株的产OTA能力。结果表明,从稻谷样品中共筛选出107株,划分为4个种：黑曲霉(A.niger)、百岁兰曲霉(A.welwitschiae)、塔宾曲霉(A.tubingensis)和新黑曲霉(A.neoniger)。其中百岁兰曲霉和黑曲霉在广西和广东地区流行率较高,塔宾曲霉的流行率随纬度的升高而升高,说明地理环境因素对我国南方地区仓储稻谷中黑曲霉群流行特征的影响较大。虽然黑曲霉群菌株产OTA测定中ELISA检测出7株菌假阳性,但进一步采用HPLC-FLD结合OTA生物合成基因鉴定方法确证107株黑曲霉群菌株均不产生OTA。本研究为我国稻谷的安全储藏和霉菌针对性防控提供了数据支撑。     

微波干燥对高水分稻谷中优势霉菌致死率的研究

研究了高水分稻谷中分离纯化的优势霉菌,以及探讨了微波工艺对染单一优势霉菌稻谷的菌落活性及后期储藏期间菌落数的影响。研究发现,从高水分稻谷中分离纯化分析得出优势霉菌是:链格孢霉、雪腐镰刀菌、白曲霉、灰绿曲霉、产黄青霉。然后在微波功率485、927和1349 W对染单一优势菌稻谷进行灭菌处理,发现稻谷表面霉菌降低约3 lg CFU/g,内部霉菌致死率约95%,同时随着微波时间的延长,受优势菌侵染的稻谷表面及内部霉菌数量均降低,致死率上升。此外通过微波工艺对杀菌后的稻谷侵染单一优势菌,以常规热风处理为对照组进行储藏,发现微波处理后稻谷霉菌生长量明显低于常规热风储藏的稻谷,有益于粮食长时间储藏。因此本文研究了微波工艺对高水分稻谷优势霉菌致死率及后期储藏的影响,为高水分粮食安全储藏提供一定的理论基础。     

粮油食品防霉微生物筛选及其活性物质初步研究

为开发新型防霉剂,降低粮油食品储藏、运输、流通等过程中霉变对其品质和食用安全的影响,研究筛选获得具有强防霉活性的微生物。采用抑菌圈法,以禾谷镰刀菌（Fusarium graminearum）为指示菌筛选具有防霉活性的菌株。利用形态学观察、生化特征鉴定和16S rDNA基因序列比对进行菌株鉴定;通过不同温度、pH和蛋白酶K处理对防霉活性物质进行初步分析。结果显示：通过初筛获得33株对禾谷镰刀菌具有强抑制活性的菌株,复筛后选择的7株菌归属为芽孢杆菌属（Bacillus）;活性物质分析发现7株菌的活性物质均不耐高温和强酸碱,同时蛋白酶K处理对其防霉效果没有明显影响,初步推断为肽类;通过抑菌谱测定发现菌株ASAG 62对常见霉菌（产黄青霉Penicillium flavum、黑曲霉Aspergillus Niger、赭曲霉Aspergillus ochre、黄曲霉Aspergillus flavus、禾谷镰刀菌F. graminearum）均具有良好的抑制效果。