酱香型白酒酵母菌群快速鉴定及发酵性能初探

为了研究酱香型白酒酿造过程中酵母菌群的生物多样性及发酵性能,对前期建立的“国台酒-酵母菌种库”展开研究,利用十二烷基磺酸钠（SDS）热裂解法提取总DNA,进而通过5.8S rDNA-ITS区域限制性片段长度多态性分析（RFLP）的方法结合“Yeast-ID”数据库比对,将酵母菌株鉴定到种的水平,再通过inter-δ PCR的方法将酿酒酵母菌株在种以下水平进一步分类。最后,对每个类型的酵母菌株,进行了发酵性能的初步研究。研究表明,酱香型白酒酿造过程中酵母菌群被分为7种,其中3种表现出较强的产酒功能,1种表现出较强的产乙酸乙酯能力;各菌种之间代谢产酸、产酯和微量物质的能力各不相同。     

Identification of fungi using DNA sequences: an approach to identify Fusarium species isolated from domestic unpolished rice

Molecular approaches are being developed to provide for the rapid and objective identification of fungi. We attempted the identification of Fusarium species by a genetic analysis to validate practically the utility of a molecular approach for fungal identification and to reveal its limitations, and sequenced three regions, the 5' end of the 28S rRNA gene (D2 region) and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions, in the rRNA genes. The DNA sequences of 38 Fusarium strains isolated from domestic unpolished rice were compared for similarity with entries in the GenBank. Based on this comparison, it was estimated that all these three regions, as a minimum, must be compared with the database to identify Fusaria at the species level. According to the combinations of sequences in the three regions, the 38 isolates were classified into 13 groups. Out of the 13 groups, 6 groups (20 isolates in total) could be identified as definite species based only on the sequence data. For the other 6 groups (17 isolates in total), candidate species were limited on the basis of the sequence similarity, and then the isolates were identified at the species level with the aid of morphology. Only one isolate could not be identified. These results verified that DNA sequence comparison with the GenBank database is useful for the identification of Fusarium species.     

ITS序列在酵母批量分子鉴定中的适用性

内部转录间隔区(internal transcribed spacer,ITS)序列是目前最常用的酵母分子鉴定标识之一。该文利用ITS序列同源性分析法对保藏于中国高校工业微生物资源与信息中心的623株酵母分离物进行了鉴定。实验结果显示:581株酵母分离物(93.3%)可通过ITS序列直接鉴定到种一级,40株分离物(6.4%)可鉴定至属一级,仅有2株酵母分离物无法通过ITS序列获得有效鉴定。上述结果表明,ITS序列在大多数酵母种属鉴定中具有很高的敏感性和特异性,可以作为第一分子标识用于大批量酵母分离物的分子鉴定。     

Identification of yeasts associated with milk products using traditional and molecular techniques

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.     

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.     

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.     

YeastIdent-Food/ProleFood, a new system for the identification of food yeasts based on physiological and biochemical tests

A new kit for the identification of yeasts in foods called YeastIdent-Food/ProleFood has been developed. The kit comprises a set of 24 physiological and biochemical tests and computer software (ProleFood) for personal or Macintosh computers that analyses the data obtained from physiological and biochemical tests. Validation of the system in the laboratory revealed that it is very efficient at identifying yeasts from culture collections and those isolated from foods. The results obtained for type yeast strains using YeastIdent-Food coincide with those reported in the literature and were correctly identified by the ProleFood software. In addition, several strains isolated from two types of foods were identified with the new system. Identification was confirmed using low molecular weight RNA profiles that allowed the identification of the yeast species.     

Behaviour of Escherichia coli O157:H7 during the fermentation of Datta and Awaze, traditional Ethiopian fermented condiments, and during product storage at ambient and refrigeration temperatures

The survival of E. coli O157:H7 in fermenting foods and its prolonged survival in refrigerated fermented foods is documented. This prompted the study to evaluate survival of E. coli O157:H7 during the fermentation of Datta and Awaze, traditional Ethiopian condiments. Datta was prepared by wet milling a variety of spices along with green or red chilli and fermenting it by lactic acid bacteria. Awaze is a slurry made of red pepper, garlic and ginger to which various other spices were added and fermented by lactic acid bacteria (LAB) and yeasts. The Datta or Awaze slurry was separately inoculated with three strains of E. coli O157:H7 and the fermentation was allowed to proceed at ambient (20–25°C) temperatures for 7 days. When fermenting Datta or Awaze was initially inoculated at low inoculum level (3 log cfu/g), the test strains were not recovered after 24 h of fermentation. At higher initial inoculum level (6 log cfu/g), however, counts of the test strains in Datta at day 7 were less by about 1.5 log unit than the initial inoculum level. In fermenting Awaze, all test strains were completely eliminated in 7 days. The pH of the fermenting green and red Datta was reduced from 5.2 to 4.4 and that of Awaze dropped from 4.9 to 3.8 during this time. In another experiment, the fermented products were separately inoculated with the E. coli O157:H7 test strains at levels of 6 log cfu/g and incubated at ambient and refrigeration (4°C) temperatures for 7 days. In fermented Datta, two of the three strains were not recovered by enrichment after 6 days of storage at ambient temperatures. In fermented Awaze, all strains were below countable levels at day 5, but could still be recovered by enrichment at day 7. At refrigeration storage, counts of the test strains in Datta and Awaze products were <3 log cfu/g at day 7. The inhibition of our E. coli O157:H7 test strains in Datta and Awaze may be due to the antimicrobial activity of spices and other metabolites produced by LAB which may be effective at low pH.     

新疆塔城地区哈萨克族传统奶酪中酵母菌的分离鉴定

为保护新疆哈萨克族传统奶酪中的优良酵母菌株,从新疆塔城牧区不同牧场采集的10份哈萨克族传统奶酪样品中,分离得到44株酵母菌。采用形态学、生理生化特性鉴定、5.8S rDNA序列同源性分析相结合的方法,对分离菌株进行鉴定。共鉴定出5个种,其中34株库德毕赤酵母（Pichia kudriavzevii）,为优势菌株,6株戴尔有孢圆酵母（Torulaspora delbrueckii）,2株乳酸克鲁维酵母（Kluyve- romyces lactis ）,1株马克思克鲁维酵母（Kluyveromyces marxianus ）,1株发酵毕赤酵母（Pichia fermentans ）。结果表明,哈萨克族传统奶酪制品中所含酵母菌与其他地区的存在差异性,有其独特的酵母菌资源。     

水塔老陈醋大曲酵母的分离鉴定及产乙醇和乙酸乙酯特性

采用传统分离培养方法,从水塔老陈醋大曲中分离纯化出21株酵母菌,利用26S r DNA D1/D2序列分析方法结合形态学特征,对分离纯化的酵母菌进行鉴定,并用其发酵糯米糖化液,用静态顶空-气质联用的方法测定发酵后乙醇和乙酸乙酯的含量。结果发现:21株酵母菌鉴定结果为2株弗比恩酵母（Cyberlindnera fabianii）,8株扣囊复膜酵母（Saccharomycopsis fibuligera）、6株异常威克汉姆酵母（Wickerhamomyces anomalus）、3株葡萄牙棒孢酵母（Clavispora lusitaniae）、2株东方伊萨酵母（Issatchenkia orientalis）。21株菌株发酵后产物乙醇和乙酸乙酯的含量存在较大差别,其中产生乙酸乙酯和乙醇最多的菌株分别为异常威克汉姆酵母m12（含量为4.4415 g/L）和扣囊复膜酵母q25（含量为48.577 g/L）;产生最少的则分别为扣囊复膜酵母q6（含量为0.0037 g/L）和东方伊萨酵母q12（含量为11.4555 g/L）。提示老陈醋大曲中不同种类的酵母菌在发酵中对乙醇和乙酸乙酯具有不同的贡献。      

Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala

Bacillus spp. are the predominant microorganisms in fermented African locust bean called Soumbala in Burkina Faso. Ten strains selected as potential starter cultures were characterised by PCR amplification of the16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), pulsed field gel electrophoresis (PFGE) and sequencing of the 968-1401 region of the 16S rDNA. In previous studies, the isolates were identified by phenotyping as Bacillus subtilis and Bacillus pumilus. The phenotyping was repeated as a reference in the present study.The ITS-PCR and ITS-PCR RLFP allowed a typing at species level. The PFGE was more discriminative and allowed a typing at strain level. Full agreement with the phenotyping was observed in all cases. The sequencing of the 16S rDNA allowed the identification at species level with an identity from 97% to 100% comparing the sequences to those from the GenBank databases. The desired cultures of B. subtilis and B. pumilus from African locust bean fermentation were distinguished by ITS-PCR and ITS-PCR RLFP from Bacillus cereus and Bacillus sphaericus which sometimes occur in the beginning of the fermentation.     

东北酸菜中乳酸菌的分离鉴定与耐酸性菌株的筛选

为筛选出高耐酸性乳酸菌,通过形态学观察和随机扩增多态性DNA标记分析技术,对自然发酵酸菜中分离纯化的乳酸菌菌株进行初步鉴别,随后利用16S rDNA序列同源性分析进行种属鉴定,并筛选在pH 3.0条件下存活率较高的菌株。结果表明,72 株分离乳酸菌包括62 株乳杆菌和10 株乳球菌,其中有21 株菌的指纹图谱不相同,经鉴定,分别为乳肠球菌（Enterococcus lactis）、弯曲乳杆菌（Lactobacillus curvatus）、米曲霉乳杆菌（L. oryzae）、短乳杆菌（L. brevis）、副干酪乳杆菌（L. paracasei）、棒状乳杆菌（L. coryniformis）。pH 3.0条件下存活率在75%以上的菌株有8 株,管家基因rpoA序列同源性分析结果表明高耐酸性的两株菌株为植物乳杆菌（L. plantarum）,为开发功能性乳酸菌食品提供了一定理论支持。     

橙汁腐败酵母菌的分子鉴定及其形态特征分析

目的：结合多种酵母菌鉴定方法,分析腐败橙汁中酵母菌的种类,为快速检测和控制商品橙汁中酵母菌污染奠定基础,也为酵母菌种类的快速鉴定提供参考。方法：以分离自腐败橙汁的8株酵母菌株为材料,观察菌落和细胞形态。用引物ITS1/ITS4扩增菌株5.8S rDNA 及其ITS间隔区(5.8S-ITS),对扩增产物用HhaⅠ、Hae Ⅲ 和 HinfⅠ进行限制性酶切片段多态性(RFLP)分析和测序;用引物NL1/NL4扩增26S rDNA D1/D2区并测序。结果：菌落特征观察将菌株初分为6组,细胞学观察粗分为3组,分子方法都分成4组。用限制性内切酶HhaⅠ、Hae Ⅲ和HinfⅠ对5.8S-ITS区产物进行 RFLP分析,观察到4种不同的图谱类型。5.8S-ITS 区和26S rDNA D1/D2区的序列分析结果相似,8株分离菌与GenBank中的4种酵母菌参考菌株序列一致性达99%以上。分离株与参考菌株以两区序列构建的NJ系统树都分成4枝：Y1与克鲁维毕赤酵母(Pichia kluyveri),Y12-3、Y18-1与发酵毕赤酵母(Pichia fermentans),Y22、Y23、Y26和Y56与Meyerozyma guilliermondii,Y47与Wickerhamomyces anomalus分别聚为一枝。结论：结合形态分析和核酸分析,将8株腐败橙汁分离菌鉴定为P. kluyveri var. kluyveri、P. fermentans、M. guilliermondii和W. anomalus 4个种,其中M. guilliermondii在腐败橙汁中首见报道;ITS- RFLP分析、26S rDNA D1/D2区测序与菌株形态特征结合能有效鉴定酵母菌,核糖体DNA分析可鉴定酵母到生物学种,菌落形态特征可反映种以下的遗传差异,因此,采用分子方法鉴定时不能忽视形态特征分析的重要性。     

Discrimination ofBrettanomyces/Dekkerayeast isolates from wine by using various DNA finger-printing methods

In the recent yearsBrettanomyces/Dekkerayeasts are posing an increasingly severe quality problem in the wine industry. The early and specific detection of these yeasts is therefore needed. Here, we describe the application of genetic techniques in addition to routinely performed physiological tests to identify yeasts isolated from Cabernet Sauvignon wines characterized by wine-makers panels as having `betty' aromas.Brettanomyces/Dekkerareference strains from a type culture collection were included for comparison. A RAPD–PCR assay was developed for species- and strain-specific discrimination of these yeasts. These data were compared to the chromosomal patterns of uncleaved, Sfi 1-digested DNA, and to the physiological behavior of the yeasts. Karyotyping gave clear distinctions, but did not allow for relatedness studies due to the lack of pattern conservation among species and strains. Conversely, RAPD–PCR allowed for species discrimination within the genusBrettanomycesand strain discrimination within the speciesD. bruxellensis.All wine-isolatedBrettanomyces/Dekkerayeasts belonged to the speciesB. bruxellensis.Populations derived from one single clone were found in the 1992 and 1994 vintage wines, but in contrast, the 1989 yeast population differed from these two vintages: two different strains were found instead of a homogenous population. In conclusion, we show that RAPD–PCR can be successfully applied to discriminateBrettanomyces/Dekkerayeasts on the species and strain level, representing an accurate alternative to coventional physiological tests.     

传统四川泡菜中酵母菌的动态变化规律

为研究传统四川泡菜发酵过程中酵母菌的动态变化规律,对传统四川泡菜自然发酵过程中的酵母菌进行分离、鉴定和计数。从自制四川泡菜样品中共分离到5 株不同菌落特点的酵母菌,采用26S rDNA D1/D2区序列分析法鉴定,酿酒酵母（Kazachstania exigua）4 株、膜璞毕赤酵母（Pichia membranefaciens）1 株。结果表明：2 种酵母菌都存在于泡菜发酵前期,初始数量为104～106 CFU/mL,随着发酵进行,泡青菜中2 种酵母菌数量持续减少,泡萝卜中2 种酵母菌数量先增加后减少,泡白菜中没有检测到膜璞毕赤酵母,酿酒酵母数量先增加后减少。2 种酵母菌在泡菜主要发酵时期的第3～5天减少最快,5 d后消失,不同发酵原料和盐水中pH值变化是影响2 种酵母菌动态变化的重要原因。     

Partial 26S rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine fermentations

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.     

贵州黔南地区泡制酸菜中酵母菌的分离与鉴定

采用纯培养的方法从贵州黔南地区20份自然发酵泡制酸菜中分离出115株酵母菌,通过WL培养基观察菌落特征,并结合显微细胞形态及生理生化特性,将115株酵母分为17种类型。从每种类型中挑选一株进行26S rDNA D1/D2区系列测定,通过序列分析及构建系统发育树,进行种属鉴定。结果表明：17类酵母共115株分属于11个属,其中30.43%为Kazachstania酵母、26.96%为毕赤酵母属(Pichia)、17.39%为地霉属(Geotrichum)、4.35%为假丝酵母属(Candida)、3.48%为耶罗威亚酵母属(Yarrowia)、3.48%为丝孢酵母属(Trichosporon)、3.48%为Meyerozyma酵母、2.61%为有孢汉逊酵母属(Hanseniaspora)、2.61%为Wickerhamomyces酵母、2.61%为Apiotrichum酵母、2.61%为棒孢酵母属(Clavispora)。     

DNA条形码COI序列在常见肉类鉴别中的应用研究

为了对常见的4种肉类及相关肉制品进行掺假鉴定,判别与产品标签是否相符,本研究以COI基因为靶基因,建立了4种动物源性食品DNA条形码鉴别技术。分别提取牛、羊、猪、鸭四大物种的基因组DNA为模板,以其COI基因的保守序列区设计6对通用引物,结合文献报道及数据库提供的7对通用引物进行PCR扩增,并将测序结果提交Gen Bank数据库Blast比对,评价不同DNA条形码的检测鉴别能力。筛选出COI-A为最优序列,在4个物种中扩增效率100%。对抽检的20个批次的肉加工品样品进行检测,鉴定结果约有90%的样品与产品标签标示的成分相符。其中1个批次的牛丸制品因肉类成分含量低未扩增成功,1个批次的牛丸制品检出鸭源成分,判定掺假。DNA条形码技术快速有效,本研究筛选的COI-A序列可直接用于牛、羊、猪、鸭及其肉制品的鉴定,并为其它常见动物源性食品的种类鉴定提供一定参考依据。     

Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method

Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks. Furthermore, the primer sets differentiated strains of the target species from strains belonging to other species, even within the genus Brettanomyces/Dekkera. Moreover, the LAMP method with these primer sets could detect about 1 x 10(1) cfu/ml of Brettanomyces/Dekkera yeasts from suspensions in distilled water, wine and beer. This LAMP method with primer sets for the identification of Brettanomyces/Dekkera yeasts is advantageous in terms of specificity, sensitivity and ease of operation compared with standard PCR methods.     

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)₄ and (GAC)₅ was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.