Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization

The microbial community developing during the spontaneous fermentation of sour cassava starch was investigated by cultivation-independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant organisms were all lactic acid bacteria (LAB), mainly close relatives of Bifidobacterium minimum, Lactococcus lactis, Streptococcus sp., Enterococcus saccharolyticus and Lactobacillus plantarum., Close relatives of Lb. panis, Leuconostoc mesenteroides and Ln. citreum were also found. A complementary analysis using hybridization of 16S rRNA with phylogenetic probes was necessary to detect the presence of the recently discovered species Lb. manihotivorans. Although it represented up to 13% of the total lactic acid bacteria of sour cassava starch, this species could not be detected by DGGE as the PCR product migrated to the same position as Lc. lactis. In addition, it was shown that a strong pH decrease in the time course of fermentation was most probably responsible for the competitive selection of acid-resistant LAB vs. both homo and heterofermentative acid-sensitive LAB.     

Diversity of lactic acid bacteria in fermented brines used to make stinky tofu

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.     

Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation

Natural populations of lactic acid bacteria (LAB) and silage fermentation of vegetable residues were studied. Fifty-two strains of LAB isolated from cabbage, Chinese cabbage, and lettuce residues were identified and characterized. The LAB strains were gram-positive and catalase-negative bacteria, which were divided into 6 groups (A to F) according to morphological and biochemical characteristics. The strains in group A were rods that did not produce gas from glucose and formed the d and l isomers of lactate. Groups B and C were homofermentative cocci that formed l-lactic acid. Groups D, E, and F were heterofermentative cocci that formed d-lactic acid. Based on 16S rDNA gene sequence analysis, group A to F strains were identified as Lactobacillus plantarum, Lactococcus piscium, Lactococcus lactis, Leuconostoc citreum, Weissella soli and Leuconostoc gelidum, respectively. The prevalent LAB, predominantly homofermentative lactobacilli, consisted of Lactobacillus plantarum (34.6%), Weissella soli (19.2%), Leuconostoc gelidum (15.4%), Leuconostoc citreum (13.5%), Lactococcus lactis (9.6%), and Lactococcus piscium (7.7%). Lactobacillus plantarum was the dominant member of the LAB population in 3 types of vegetable residues. These vegetable residues contained a high level of crude protein (20.2 to 28.4% of dry matter). These silages prepared by using a small-scale fermentation system were well preserved, with low pH and a relatively high content of lactate. This study suggests that the vegetable residues contain abundant LAB species and nutrients, and that they could be well preserved by making silage, which is a potentially good vegetable protein source for livestock diets.     

豆酱不同发酵阶段细菌群落多样性及动态变化分析

以东北传统自然发酵豆酱为研究对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reactiondenaturing gradient gel electrophoresis,PCR-DGGE)技术结合高通量测序方法分析豆酱发酵过程中细菌的多样性及动态变化。结果显示,细菌的可操作分类单元数值在发酵过程中上下波动,并在后发酵第21天时达到最大值,此时细菌数目最多;整理分析PCR-DGGE图谱鉴定结果以及细菌在属水平上的分布得出,明串珠菌属、肠球菌属、四联球菌属和乳杆菌属为豆酱样品不同发酵阶段的优势细菌菌属,其中四联球菌属波动性较大,易受内外环境的影响;屎肠球菌、明串珠菌属、绿色魏斯菌随着发酵时间的延长在减少,说明这些细菌主要参与豆酱发酵初期产酶分解物质过程,并在整个发酵过程中起到调整发酵内环境等作用。     

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.     

新疆酸驼乳微生物种群结构的PCR-DGGE 分析

采用PCR-DGGE 技术,比较新疆传统发酵酸驼乳中微生物种群结构及图谱相似性。结果表明：不同来源的酸驼乳中微生物组分存在差异,9 份样品间细菌种群结构的相似性为78%～84%,5 份样品间酵母菌种群结构的相似性为80%～92%。对酸驼乳细菌和酵母菌DGGE 图谱上主要条带的DNA 进行测序和序列分析。结果表明：酸驼乳中细菌组成包括巨型球菌(Macrococcus caseolyticus)、瑞士乳杆菌(Lactobacillus helveticus)、短乳杆菌(Lactobacillus brevis)、希腊魏氏菌(Weissella hellenica)、清酒乳杆菌(Lactobacillus sakei)、肠球菌(Enterococcusdurans)、屎肠球菌(Enterococcus faecium)及乳酸明串珠菌(Leuconostoc lactis)等;酵母菌主要包括巨大克洛维酵母(Kluyveromyces marxianus)、单孢酿酒酵母(Kazachstania unispora)、嗜酒假丝酵母(Candida ethanolica)及一种地霉菌(Geotrichum sp.)。     

A culture-dependent and -independent approach for the identification of lactic acid bacteria associated with the production of nem chua,a Vietnamese fermented meat product

In this study, the diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented meat, was described using a combination of culture-dependent and culture-independent methods. A total of two hundred seventy-three LAB isolates were subjected to a polyphasic identification approach combining (GTG)₅-PCR fingerprinting and phenylalanyl-tRNA synthase α subunit (pheS) and RNA polymerase α subunit (rpoA) gene sequence analysis. LAB associated with nem chua were identified as Lactobacillus pentosus (21%), Lactobacillus plantarum (29.7%), Lactobacillus brevis (5%), Lactobacillus paracasei (0.4%), Lactobacillus fermentum (0.7%), Lactobacillus acidipiscis (0.4%), Lactobacillus farciminis (23%), Lactobacillus rossiae (0.4%), Lactobacillus fuchuensis (0.7%), Lactobacillus namurensis (0.4%), Lactococcus lactis (0.4%), Leuconostoc citreum (9.5%), Leuconostoc fallax (1%), Pediococcus acidilactici (1%), Pediococcus pentosaceus (4%), Pediococcus stilesii (1%), Weissella cibaria (0.7%) and Weissella paramesenteroides (0.7%). Furthermore, PCR-DGGE was also applied as a culture-independent method in this study. Results indicated the presence of species of which no isolates were recovered, i.e. Lactobacillus helveticus/crispatus, Lactococcus garvieae and Vagococcus sp. Conversely, not all isolated bacteria were detected by PCR-DGGE. Principal component and discriminant analysis disclosed correlations between the different production locations and certain isolated LAB species and strains and/or DGGE bands suggesting possible influences of locally prevailing production practices on the nem chua LAB microbiota.     

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.     

Analysis of kimchi microflora using denaturing gradient gel electrophoresis

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.     

六堡茶乳酸菌多样性及其降胆固醇特性分析

探究六堡茶渥堆发酵过程中乳酸菌多样性并筛选具有降胆固醇活性菌株，旨在评价其作为潜在益生菌的体外降胆固醇能力，预防人体因饮食不均衡导致的高血脂症。采用高通量测序、纯培养法分析不同渥堆发酵时期六堡茶中乳酸菌多样性，并对其体外降胆固醇能力、胃肠耐受性进行研究。结果表明，从25 个样品中共检测到乳酸菌4 个属（乳杆菌属（Lactobacillus）、魏斯氏菌属（Weissella）、乳球菌属（Lactococcus）和肠球菌属（Enterococcus））5 个种，其中乳杆菌属为优势菌。共分离得到76 株乳酸菌，包括55 株戊糖乳杆菌（Lactobacillus pentosus）、3 株铅黄肠球菌（Enterococcus casseliflavus）、5 株类肠膜魏斯氏菌（Weissella paramesenteroides）、8 株希腊魏斯氏菌（Weissella hellenica）、5 株台湾乳球菌（Lactococcus taiwanensis）。通过乳酸菌体外降胆固醇实验，发现胆固醇去除率为0%～30%，其中戊糖乳杆菌L131的去除胆固醇率最高，为（29.56±0.37）%；在pH值分别约为3.0、8.0的人工胃液、人工胰液中，L131有最高存活率，分别为（92.70±0.71）%和（77.54±4.81）%，显著高于其他菌株（P＜0.05）。本研究探明六堡茶渥堆发酵过程中乳酸菌的多样性，且发现具有降胆固醇特性的优良乳酸菌株，为健康黑茶产品和茶源益生菌的开发提供理论基础。     

水牛乳中可培养乳酸菌多样性分析

采用传统分离培养方法,从三品杂交生水牛奶混合样品中,分离出105株乳酸菌,通过形态、生理生化、API细菌鉴定系统及16S rDNA基因序列分析方法对各菌株属种进行鉴定。16S rRNA序列分析结果显示,105株菌共分为5个属8个种,呈现较为丰富的乳酸菌多样性,具体数量分布为乳酸乳球菌21株,植物乳杆菌19株,格氏乳球菌17株,乳明串珠菌13株,食窦魏斯氏菌11株,肠膜明串珠菌8株,类肠膜魏斯氏菌6株,嗜热链球菌5株,糊精乳杆菌5株。由此可知,水牛乳中可培养乳酸菌优势菌群的主次关系为：乳酸乳球菌（Lactococcus lactis）＞植物乳杆菌（Lactobacillus plantaru）＞格氏乳球菌（Lactococcus garvieae）＞乳明串珠菌（Leuconostoc lactis）＞食窦魏斯氏菌（Weissella cibaria）,此为后续开发水牛乳中优势乳酸菌资源提供了良好的理论基础。     

酱油发酵过程中细菌群落结构的动态变化

本文采用16S r DNA PCR-DGGE(变性梯度凝胶电泳)指纹图谱和系统发育分析方法,以高盐稀醪发酵工艺生产的广东酱油为研究对象,揭示了酱油生产发酵过程中细菌群落结构的多样性及动态变化。从样品中提取总细菌DNA,用降落PCR扩增16S r DNA V3片段序列,再通过分析DGGE图谱选择特异性条带,进行割胶回收、测序及Blast分析。DGGE图谱表明,发酵初期样品具有丰富的微生物群落,但之后只有少数种类细菌存活,整个酱油发酵过程微生物群落结构的演变规律是由复杂到简单,这也说明酱油发酵环境具有抑制微生物生长的作用。测序结果表明,代表最相似菌为魏斯菌属(Weissella cibaria)和非培养的肠杆菌属(Uncultured Enterobacter sp.),其次是嗜盐四联球菌(Tetragenococcus halophilus)、类肠膜魏斯氏菌(Weissella paramesenteroides)、弗氏柠檬酸杆菌(Citrobacter freundii)、产气肠杆菌(Enterobacter aerogenes)和肠杆菌属(Enterobacter sp.BF1-8)。     

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.     

山西老陈醋和怀仁醋酒精发酵阶段细菌菌群多样性分析

为了探明山西老陈醋酿造工艺和怀仁醋酿造工艺对细菌菌群的影响,该研究利用高通量测序技术对两种工艺酒精发酵阶段样品（分别编号为S和X）的细菌菌群多样性进行分析。结果表明,S样品的细菌菌群多样性更高。两种工艺酒精发酵阶段样品的细菌菌群组成差别较大,与S样品相比,X样品中相对丰度较高的乳杆菌属（Lactobacillus）、魏斯氏菌属（Weissella）、乳球菌属（Lactococcus）均增多,片球菌属（Pediococcus）、明串珠菌属（Leuconostoc）、泛菌属（Pantoea）、链霉菌属（Streptomyces）、短状杆菌属（Brachybacterium）、肠杆菌属（Enterobacter）和非培养细菌（uncultured bacterium）均减少。导致两种工艺酒精发酵阶段样品有显著差异的细菌在S样品中为片球菌属、明串珠菌属、链霉菌属和泛菌属,在X样品中则为乳杆菌属、魏斯氏菌属和乳球菌属。相关性分析结果表明,乳杆菌属、泛菌属、魏斯氏菌属和片球菌属与其他物种联系紧密。     

A polyphasic approach in order to identify dominant lactic acid bacteria during pasta manufacturing

Using a polyphasic approach, we have isolated and identified, lactic acid bacteria (LAB) in samples directly collected from an artisanal pasta-making manufactory located in Puglia (South Italy). Samples were collected during several steps of pasta processing and LAB were isolated on MRS and M17 plates. Furthermore, strains were grouped in a total of eight species by means of randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing and 16S rDNA sequencing. The majority of strains were identified as belonging to Pediococcus pentosaceus and Enterococcus faecium species. The remaining strains were characterized and assigned to Weissella confusa, Pediococcus acidilactici, Leuconostoc mesenteroides, Leuconostoc citreum, Lactobacillus fermentum and Lactobacillus plantarum species. Strains were identified from all the analysed samples, and growth of LAB was monitored from extrusion to pre-heating steps.     

Inactivation of food spoilage bacteria by high pressure processing: Evaluation with conventional media and PCR–DGGE analysis

Microbial diversity and dynamic changes of sliced vacuum-packed cooked ham during refrigerated storage (0–90 days) after high pressure processing (400 MPa at 22 °C for 10 min) was investigated by using culture-dependent and culture-independent approaches. Isolation of genome DNA and total RNA directly from meat samples, followed by PCR–denaturing gradient gel electrophoresis (DGGE) and RT-PCR–DGGE on 16S rDNA V3 region, was performed to describe the structure of the bacterial community and active species in pressurized sliced cooked ham. The DGGE profile showed that most spoilage bacteria including Lactococcus garvieae, Weissella cibaria, Lactobacillus sakei, Lactobacillus curvatus, Weissella paramesenteroides, Leuconostoc carnosum and Lactococcus lactis subsp. lactis were completely inactivated after high pressure processing (HPP), whereas Weissella viridescens and Weissella minor survived HPP and induced the final spoilage. The microbial diversity of HPP samples during the whole refrigerated storage period was extremely simple. Our results clearly indicated that HPP was an efficient method for avoiding the growth of the major spoilage bacteria and could be used to prolong the shelf-life of sliced vacuum-packed cooked ham.     

Isolation and Selection of Lactic Acid Bacteria as Biocontrol Agents for Nonacidified, Refrigerated Pickles

ABSTRACT: A nonacidified, deli-type pickle product was used as a model system to study the potential use of biocontrol as a means to prevent the growth of pathogens in minimally processed fruits and vegetables (MPFV). Fresh pickling cucumbers were blanched and brined with sterile spices and garlic oil. The product was stored at 5 °C for 3 wk and then transferred to various abuse temperatures (16 °C, 25 °C, 30 °C). Lactic acid bacteria (LAB) were isolated and characterized as potential biocontrol agents, and the isolates were tested for bacteriocin-like activity. A total of 118 LAB isolates were obtained. Among the LAB identified were species of Lactococcus, Leuconostoc, Lactobacillus, Weissella , and Enterococcus . Three isolates showed transient bacteriocin activity against— Listeria monocytogenes , and 7 isolates ( Lactococcus ) had bacteriocin-like activity against other LAB. Although it did not produce a bacteriocin, a Lactobacillus curvatus isolate (LR55) was found to have desirable characteristics for use as a biocontrol (competitive exclusion) culture to enhance the safety of nonacidified deli-type pickles.     

白腐乳发酵过程中细菌群落演替规律研究

为解析白腐乳发酵过程中细菌的多样性,应用高通量测序和培养组学相结合的方法分析了前酵和后熟过程中细菌的变化规律,通过高通量测序分析白腐乳发酵过程中细菌16S rDNA V3-V4区基因序列。结果表明,白腐乳从前酵到后熟过程中细菌群落有较大变化,豆腐坯中的优势菌为乳杆菌（Lactobacillus）,毛坯中的优势菌为不动杆菌（Acinetobacter）,后熟45 d腐乳中的优势菌为明串珠菌（Leuconostoc）,后熟180 d腐乳中的优势菌为四联球菌（Tetragenococcus）;利用培养分离方法从腐乳发酵过程中部分样品分离鉴定出75株可培养细菌,包括融合魏斯氏菌（Weissella confuse）、蜡样芽孢杆菌（Bacillus cereus）、肉葡萄球菌（Staphylococcus carnosus）、乳酸乳球菌（Lactococcus lactis）、嗜盐四联球菌（Tetragenococcus halophilus）、粪肠球菌（Enterococcus faecalis）和耐久肠球菌（Enterococcus durans）等。     

不同盐质量分数泡白菜发酵过程中乳酸菌群落结构的变化

设计3种不同盐质量分数的泡白菜,包括低盐(2%)、中盐(5%)、高盐(8%),利用可培养方法结合多种分子生物学手段研究其发酵过程中乳酸菌的群落结构。结果表明:盐质量分数越低,乳酸菌增长速率越快。本研究从3种不同盐质量分数泡白菜发酵过程中分离筛选得到了563株乳酸菌,并采用16S rDNA测序、多重聚合酶链式反应、聚合酶链反应-限制性片段长度多态性、API 50CH等多种不同的鉴定方法进行鉴定,鉴定结果表明这563株乳酸菌属于5个属11个种。结果表明,低盐泡白菜发酵前期的优势菌为Lactococcus lactis、Lactobacillus pentosus和Leuconostoc,发酵后期则由Lactobacillus pentosus主导;中高盐泡白菜发酵前期由Lactobacillus pentosus和Weissella主导,发酵后期主要由Lactobacillus pentosus完成。     

PCR-DGGE技术对中华开菲尔微生物菌群的分析

为了解中华开菲尔微生物菌群的结构特征,本论文运用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对开菲尔菌株发酵过程中微生物菌群的结构变化进行了实验分析,结果表明:细菌菌群DGGE图谱上出现有三种不同迁移位置的斑带,而酵母菌群DGGE图谱上只有一条斑带;经过DNA序列的对比分析可知:细菌菌群分别为肠膜明串珠菌(Leuconostoc mesenteroides)、马乳样乳杆菌(Lactobacillus kefiranofaciens)和开菲尔乳杆菌(Lactobacillus kefir),它们的序列同源性都达到100%;酵母菌群为德尔布有孢圆酵母(Torulaspora delbrueckii),其序列同源性为99%。本论文首次报道了德尔布有孢圆酵母在开菲尔菌落中的存在。