基于ITS基因文库法研究清香型大曲真菌群落结构

通过构建真菌ITS基因文库、测序和系统发育树分析,分别对汾酒清茬曲、后火曲和红心曲的真菌群落结构进行了比较。研究结果表明,汾酒大曲内的真菌全部分属于酵母目和毛霉菌目,且3种大曲的真菌群落结构存在明显差异,其中清茬曲的优势真菌为毕赤酵母属和根霉属(占全部克隆子的比例分别是66.7%和30%);后火曲的优势真菌为毕赤酵母属(占全部克隆子的比例是80.24%);红心曲的优势真菌为根霉属和横梗霉属(占全部克隆子的比例分别是38.89%和33.33%)。     

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%）。     

高通量测序法对清香大曲真菌群落结构的分析

利用微生物生态学方法客观分析清香大曲真菌群落结构有利于分析大曲功能,同时明确大曲功能菌。利用高通量测序法分析了冷季和热季清香白酒生产用曲真菌群落结构。结果表明,用不同的宏基因组DNA提供试剂盒,以及不同测序区域得到较为一致的结果,清香大曲在热季和冷季的主要真菌类群相同,主要的真菌类群有霉菌和酵母,霉菌有Rhizopus oryzae(米根霉)和Lichtheimia corymbifera(伞枝犁头霉),酵母有Pichia kudriavzevii(库德毕赤酵母)和Saccharomycopsis fibuligera(扣囊复膜酵母),2种霉菌含量明显高于酵母,而且冷季酵母比例较热季明显减少。通过本实验可以确认清香大曲主要真菌类群有米根霉、伞枝犁头霉,酵母菌则比较少,主要有扣囊复膜酵母和库氏毕赤酵母。     

稻花香馫香型白酒酒曲酶活测定与高通量测序分析

以稻花香馫香型白酒生产中所用到的中高温大曲(ZGW),高温大曲(GW)和根霉曲(GM)为例,测定了它们的糖化酶、液化酶、酯化酶和酸性蛋白酶活力;检测了可培养微生物种类及数量(通过稀释平板涂布法);分析了微生物群落结构组成(通过高通量测序)。酶活测定结果表明:中高温大曲(ZGW)的糖化力最高,为189.5 U/g;根霉曲(GM)的液化力、酯化力和酸性蛋白酶活力最高,分别为16.36 U/g、164.69 mg/g和189.32 U/g。传统稀释涂平板结果发现,霉菌、酵母菌、总细菌和芽孢杆菌均以中高温大曲为最高,分别达到9.07×10~2cfu/g、6.02×10~4cfu/g、1.90×10~7cfu/g和1.74×10~7cfu/g。高通量测序结果表明,中高温大曲细菌和真菌的物种多样性最高,而根霉曲的最低。在细菌属水平上,高温大曲的优势菌种有Bacillus(芽孢杆菌属),Kroppenstedtia,Thermoactinomyces(高温放线菌属),Saccharopolyspora(糖多孢菌属),Norank_f_Pseudonocardiaceae,Planifilum,Staphylococcus(葡萄球菌属)和Pediococcus(片球菌属);中高温大曲的优势菌种有Bacillus(芽孢杆菌属),Kroppenstedtia,Thermoactinomyces(高温放线菌属),Weissella(魏斯氏菌属),Lactobacillus(乳杆菌属),Pediococcus(片球菌属),Planifilum和Staphylococcus(葡萄球菌属),其中Bacillus(芽孢杆菌属)丰度最高。在真菌属水平上,高温大曲的优势菌种有Thermoascus(嗜热子囊菌属),Thermomyces(嗜热真菌属)和Aspergillus(曲霉菌属),其中Thermomyces(嗜热真菌属)丰度最高;中高温大曲的优势菌种有Thermoascus(嗜热子囊菌属),Thermomyces(嗜热真菌属),Cutaneotrichosporon,Candida(假丝酵母属),Rhizopus(根霉菌属),Aspergillus(曲霉菌属),Hyphopichia,Trichomonascus和Xeromyces(耐干霉菌属),其中丰度较高的是Thermoascus(嗜热子囊菌属)。而Rhizopus(根霉菌属)作为根霉曲中唯一的优势菌种,丰度可达99.99%。     

基于高通量测序技术分析南方清香型白酒大曲的微生物多样性

利用高通量测序技术对不同香型大曲及南北两地清香型大曲中微生物菌群结构进行分析比较。结果表明，南方清香型白酒大曲的细菌以芽孢杆菌属（Bacillus)为优势菌群，其次以魏斯氏菌属（Weissella)、片球菌属（Pediococcus)及其他乳酸菌（Lactobacillus)为主要菌群；真菌优势菌种为覆膜孢酵母属(Saccharomycopsis)，其次是弯曲假丝酵母(Apiotrichum)、伊萨酵母属(Issatchenkia)、异常威克汉姆酵母属(Wickerhamomyces)等；霉菌以曲霉属(Aspergillus)占比最多。微生物群落由于制曲工艺不同与高温、中高温大曲有较为明显的差别，南北两地清香型白酒大曲的细菌真菌优势菌属均一致，但南方清香型白酒大曲中各类乳酸菌属与酵母属较北方清香型大曲丰富。     

提高汾酒质量和产量的探讨

适当减少大曲中大量糖化力不高的霉菌和发酵力微弱的酵母，增加经过训养后的优良菌种，提高在酒醅发酵中酶活性，就能减少大曲接种量和酒醅中酵母繁殖量，提高了大曲和酒醅淀粉利用率，因而提高产量。在增加酒醅中汉逊酵母数量及提高其活性的同时，增加微生物接种量，提高入缸温度，以缩短繁殖期，提早产酒期，延长酯化期，有利于汾酒增香，提高汾酒质量。     

大曲清香型酒酿造中主要微生物

大曲清香型酒酿造中主要微生物李增胜山西杏花村汾酒厂（０３２２０５）１．汾酒酒醅中酵母菌类以酵母菌属为主，产酒精能力最强，其次是有一定产香和产酒精能力的汉逊酵母和假丝酵母及产酒精不强的拟内孢霉，还有极少量的毕氏酵母和产膜酵母。汉逊酵母在汾酒风味中起重要...     

不同感官特性酱香型大曲理化指标与霉菌群落关联分析

为了探究不同感官特性酱香型大曲理化指标与霉菌群落之间的关联性,以白曲WQ、黄曲YQ和黑曲BQ为研究对象,测定三种大曲的6个理化指标,分离鉴定大曲中霉菌,通过冗余分析法分析三种大曲理化指标和霉菌群落之间的关联性。结果表明,黄曲YQ的液化力较高,发酵力和酯化力较高的为黄曲YQ(1.56 U,22.63 U)和黑曲BQ(1.51 U,22.48 U)。三种大曲共分离鉴定到6个属13种的霉菌78株,其中白曲WQ有7种,分别为Paecilomyces subglobosus、宛氏拟青霉Paecilomyces variotii、伞枝横梗霉Lichtheimia corymbifera,Lichtheimia ornata、缓慢产黄青霉Penicillium tardochrysogenum、产黄青霉Penicillium chrysogenum、Epicoccum endophytica;黄曲YQ有6种,分别为分枝横梗霉Lichtheimia ramosa、伞枝横梗霉Lichtheimia corymbifera、宛氏拟青霉Paecilomyces variotii、 Lichtheimia h...     

西沙野生诺尼种子内生真菌的群落多样性

采用种子表面灭菌、菌种分离与纯化、ITS rDNA和26S rDNA D1/D2区序列测定和系统发育分析方法,对西沙野生诺尼种子内生真菌群落多样性进行了初步研究。从诺尼种子中共分离到内生真菌48株,其中酵母菌39株,霉菌9株;39株酵母菌分属于4个属内的5个种,其中榛针孢酵母(Eremothecium coryli)、Pseudozyma aphidis和浅黄隐球酵母(Cryptococcus flavescens)为优势种,分别占总菌株数的31.25%、27.08%和16.67%;9株霉菌分属于4个属内的4个种,其中Phaeoacremonium sp.和赤霉属(Gibberella sp.)是霉菌中相对优势的种,分别占总菌株数的8.33%和6.25%。研究结果表明,西沙野生诺尼种子中存在大量内生真菌,且种类丰富,其中酵母菌占绝对优势,部分优势种具有潜在的药用价值。     

不同储存期宋河中高温大曲霉菌的分离及鉴定

该研究采用传统分离纯化技术对不同储存期(4～7个月)的宋河中高温大曲中的霉菌进行分离纯化，并通过形态学观察和分子生物学技术对其霉菌进行鉴定，结合其理化指标检测结果确定宋河中高温大曲的最佳储存时间。结果表明，从不同储存期的宋河中高温大曲中共分离得到22株霉菌，其中，储存期为4、5、6个月的宋河中高温大曲分别获得6株、4株、12株霉菌，经鉴定为5个属的14种霉菌，其中，枝孢菌属(Cladosporium)和横梗霉属(Lichtheimia)存在于储存4、5、6个月的宋河中高温大曲中，储存期为6个月的宋河中高温大曲中霉菌种类最多，且糖化力、发酵力和酯化力最高，分别为717.5 mg/(g·h)、1.97 g/(0.5 g·72 h)、13.04 mg/(50 g·7 d)，确定宋河中高温大曲的最佳储存时间为6个月。     

Complex microbiota of a Chinese "Fen" liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR-DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE.     

Use of the selective agar medium CREAD for monitoring the level of airborne spoilage moulds in cheese production

It was investigated if a selective medium for common cheese spoiling moulds (CREAD) could give more relevant information than a general mould medium in hygienic air-sampling in cheese factories. A total of 126 air-samples were taken in six Nordic cheese factories using the general mould medium DG18 and CREAD. The level and genera of air-borne mould was determined. Identification to species-level was performed for a selection of samples. In five cheese factories the mycobiota was dominated by Penicillium spp. and in one cheese factory by Cladosporium spp. The concentration of air-borne moulds varied between the cheese factories ranging from 1 to 270 cfu/m3 on DG18 with a median value of 17. The number of mould colonies was in general lower at CREAD. Identification indicated that CREAD supported growth of common spoilage moulds for cheese, such as Penicillium palitans and P. commune. The mycobiota on DG18 also consisted of moulds not commonly associated with spoilage of cheese, such as Cladosporium spp., P. brevicompactum and P. chrysogenum. Contamination of cheese with mould is periodically a problem in production of semi-hard cheese and the level of air-borne mould is therefore routinely monitored in cheese factories. A clear correlation between the total number of moulds in air and mould growth on products is not always found. The conclusion from the investigation is that it is recommended to use a selective medium for cheese spoilage moulds, such as CREAD in hygienic monitoring.     

Immunofluorescence detection of mould — An aid to the Howard mould counting technique

Antibodies have been raised in rabbits to the soluble and insoluble fractions of a mixture of five moulds representing the major spoilage organisms in tomatoes. Further antibodies were raised to the same moulds after heat treaatment at 100°C for 6 min. Immunofluorescence microscopy has shown that there is little difference between the titres of the antisera from heated and native fungal mixtures, although those raised to the soluble mould fractions produced by far the highest titre. The antibody appears to cross react with both mould species of the same genera, and with moulds of different genera not present in the original cocktail. Little cross reaction was found to occur between the antibody and tomato tissue. The immunofluorescent detection of mould can be used to simplify and reduce errors associated with the Howard Mould counting technique by enabling easy identification and enumeration of mould filaments within the tomato paste matrix.     

Mycobiota and co-occurrence of mycotoxins in Capsicum powder

This study aimed to: (1) determine the mycobiota of Capsicum powder samples, paying a special attention to the mycotoxigenic moulds; (2) evaluate the contamination levels of aflatoxins (AF), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T2 and HT2 toxins in those samples. Thirty-two samples were obtained through the methods of sampling established by the European Union legislation. Aspergillus and Eurotium were the most frequently found genera. Aspergillus section Nigri had the higher relative frequency in the samples, A. niger aggregate being the most representative group of this section. Other potentially mycotoxigenic Aspergillus, Fusarium and Penicillium species were found, but in a lower frequency.Co-occurrence of mycotoxins was confirmed in the 32 Capsicum powder samples. All samples were contaminated with AF and OTA, 27% with ZEA (36% of chilli and 18% of paprika samples), 9% with DON (18% of chilli and 6% of paprika samples), 6% with T2 (18% of chilli samples) and none of the samples contained HT2.Although in the present study the most common genera found (Aspergillus and Eurotium) belong to storage moulds, some field fungi such as Fusarium spp. were also found, and their toxins were sometimes detected. This fact supports the hypothesis that mycotoxin contamination of Capsicum products may occur both in the field and/or during storage.     

Mould and yeast flora in fresh berries, grapes and citrus fruits

Fresh fruits are prone to fungal contamination in the field, during harvest, transport, marketing, and with the consumer. It is important to identify fungal contaminants in fresh fruits because some moulds can grow and produce mycotoxins on these commodities while certain yeasts and moulds can cause infections or allergies. In this study, 251 fresh fruit samples including several varieties of grapes, strawberries, blueberries, raspberries, blackberries, and various citrus fruits were surface-disinfected, incubated at room temperature for up to 14 days without supplemental media, and subsequently examined for mould and yeast growth. The level of contamination (percent of contaminated items/sample) varied depending on the type of fruit. All raspberry and blackberry samples were contaminated at levels ranging from 33% to 100%, whereas 95% of the blueberry samples supported mould growth at levels between 10% and 100% of the tested berries, and 97% of strawberry samples showed fungal growth on 33-100% of tested berries. The most common moulds isolated from these commodities were Botrytis cinerea, Rhizopus (in strawberries), Alternaria, Penicillium, Cladosporium and Fusarium followed by yeasts, Trichoderma and Aureobasidium. Thirty-five percent of the grape samples tested were contaminated and supported fungal growth; the levels of contamination ranged from 9% to 80%. The most common fungi spoiling grapes were Alternaria, B. cinerea and Cladosporium. Eighty-three percent of the citrus fruit samples showed fungal growth at levels ranging from 25% to 100% of tested fruits. The most common fungi in citrus fruits were Alternaria, Cladosporium, Penicillium, Fusarium and yeasts. Less common were Trichoderma, Geotrichum and Rhizopus.     

Moulds and yeasts in fresh and minimally processed vegetables, and sprouts

A limited survey of fresh and minimally processed vegetables, and sprouts was conducted in the Washington, DC area to determine if potentially toxigenic and pathogenic fungi were present in these commodities. Thirty-nine ready-to-eat salads, 29 whole fresh vegetables and 116 sprout samples (bean, alfalfa, broccoli, crunchy, garlic, spicy, onion, clover, lentil and multi-seed sprouts) were purchased from 13 local supermarkets and tested for yeast and mould counts as well as the presence of toxigenic moulds. Yeasts were the most prevalent organisms found in these samples, at levels ranging from less than 100 to 4.0x10(8) cfu/g. Mould counts generally ranged from less than 100 to 4.0x10(4) cfu/g. Two crunchy sprout samples, however, contained unusually high numbers of Penicillium (1.1x10(8) and 1.3x10(8) cfu/g), two alfalfa sprout samples contained Geotrichum populations about 10(6) cfu/g, and two alfalfa sprout samples had Cladosporium counts higher than 2.5x10(5) cfu/g. The most common moulds found in fresh and minimally processed vegetables were Cladosporium, Alternaria and Penicillium; less common was Geotrichum. The most frequently isolated moulds from sprouts were Alternaria, Cladosporium, Penicillium, and Phoma. Phoma was especially common in alfalfa sprouts. Fusarium, Rhizopus, Mucor, and Geotrichum were isolated less often.     

Effect of selected mould strains on lipolysis in dry fermented sausages

 Several batches of fermented sausages were prepared and inoculated with five strains of moulds belonging to the genera Penicillium and Mucor, selected for their enzymatic activity, and a strain of Penicillium nalgiovense obtained as a commercial starter culture. Control batches were manufactured similary, but without inoculation. Sausages were produced with either artificial or natural casings, and very similar results were obtained with both. The evolution of the different lipidic fractions was monitored, paying special attention to the free fatty acid (FFA) fraction. Two strains presented a high lipolytic activity as indicated by the rise in FFA in comparison to control batches. In conclusion, not all the mould strains contributed to the lipolytic process occurring during sausage ripening and, therefore, not all of these had a positive effect on the final quality of the product. If moulds are to be used for this purpose it is, therefore, necessary to carefully select suitable moulds beforehand. Received: 25 September 1998 / Revised version: 4 February 1999     

Fungal colonization of sago starch in Papua New Guinea

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 x 10(3) to 8.3 x 10(7) cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 x 10(2) to 3.0 x 10(6) cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.     

Trichothecenes, ochratoxin A and zearalenone contamination and Fusarium infection in Finnish cereal samples in 1998

The occurrences and concentrations of trichothecenes, ochratoxin A and zearalenone in Finnish cereal samples are presented in this study. Furthermore, infections by moulds, especially Fusarium contamination of grains in the same samples, are reported. In total 68 cereal samples, including 43 rye, 4 wheat, 15 barley and 6 oats samples, were collected after a cool and very rainy growing season in 1998. A gas chromatograph combined with a mass spectrometric detector was used for determination of seven different trichothecenes. A high performance liquid chromatograph with a fluorescence detector was used for ochratoxin A and zearalenone determination. For the identification of moulds, the grain samples were incubated and the moulds were isolated and identified by microscopy. The analytical methods were validated for mycotoxin analysis and they were found to be adequately reliable and sensitive. Heavy rainfalls in the summer and autumn of 1998 caused abundant Fusarium mould infection in Finnish cereals, particularly in rye. Fusarium avenaceum was the most common Fusarium species found in cereals. However, the mycotoxin concentrations were very low and only deoxynivalenol, nivalenol and HT-2 toxin were detected. Deoxynivalenol was detected in 54 samples in the concentration range 5-111 µg/kg. Nivalenol and HT-2 toxin were detected in three and two samples, respectively, in the concentration range 10-20 µg/kg.     

Fungal contamination of Kashar cheese in Turkey.

Fifty random samples of Kashar cheese were collected from shops in different localities in Erzurum, all contained moulds. Mean count of total surface mould was 3.02 x 10(10)/g cheese and that of inner mould was 3.02 x 10(3)/g cheese. The genera Penicillium, Aspergillus, Mucor, Rhizopus and Geotrichum sp. were isolated from cheese samples. Aflatoxins were not detected in cheese samples. Potassium sorbate inhibited mould growth and sporulation in YES broth. The public health importance and economic significance of fungal contamination, and suggested measure for cheese quality are discussed.