DGGE技术及其在食品微生物研究中应用

变性梯度凝胶电泳(DGGE)技术是一种分析微生物区系分子指纹技术,具有可靠性强、重复性好、方便快捷等优点,已被广泛应用于微生物分子生态学领域研究。该文对DGGE技术基本原理及其在食品微生物中多样性分析、食品微生物动态监测、快速检测等应用进行综述,并对该技术局限性和应用前景进行评述。     

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations,underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.     

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 ± 0.07 mol H₂/mol glucose (mean ± S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 ± 0.03 mol H₂/mol glucose, 0.17 ± 0.01 mol H₂/mol glucose, 0.11 ± 0.01 mol H₂/mol glucose and 0.20 ± 0.04 mol H₂/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp.. However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community.     

Effects of organoclays on soil eubacterial community assessed by molecular approaches

The aim of this study was to evaluate the effects of the commercial organoclays, CLOISITE 30B, NANOFIL 804 and DELLITE 26C on soil eubacterial community. An enrichment test was carried out on Nutrient Broth containing the organoclay and the microorganisms previously isolated from soil. Four transfers were made, each after 7 days incubation. The molecular analyses on the eubacterial community were performed before treatment and 7 days after each transfer. DNA was extracted, amplified with eubacterial primers, finally analysed by amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis (DGGE). The profiles of the samples treated with each organoclay showed the absence, the appearance and an increase in the intensity of some bands. These bands were excised from the gels, and the related microorganisms were identified by DNA sequencing, as Pseudomonas putida, Alcaligenes xylosoxidans, Pseudomonas monteilii and Pseudomonas aeruginosa. NAN804 treatment did not have any influence on soil eubacterial community, CLO30B had a slight toxic effect only on P. putida, instead the DEL26C treatment had a stronger toxic effect on P. putida and a slight toxic effect on P. monteilii. Finally, all the tested organoclays stimulated the growth of both A. xylosoxidans and P. aeruginosa.     

Comparison of bacterial community in matured and degenerated pit mud from Chinese Luzhou‐flavour liquor distillery in different regions

The bacterial community in the pit mud of a Luzhou‐flavour liquor distillery in different regions was analysed by combined polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative PCR (qPCR) in order to distinguish a matured and a degenerated pit mud, judged according to sensory and physicochemical characteristics. The phyla Firmicutes, Cloacimonetes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected. Firmicutes predominated in the pit mud. The diversity and homogeneity of the bacterial community in the matured pit mud were superior to those in the degenerated pit mud in the same distillery. There were significant differences in the bacterial community structure between the matured and degenerated pit mud. Moreover, the bacterial community in the degenerated pit mud samples was similar, which indicated that the bacterial community in the degenerated pit mud did not change within the two different regions. However, the bacterial community in matured pit mud samples was different, demonstrating that there were visible differences in the bacterial community between the samples of matured pit mud collected from the Luzhou‐flavour liquor distilleries in the two different regions. Notably, the quantity of Actinobacteria in the matured and the degenerated pit mud was found to be different by quantitative analysis. Potentially, the Actinobacteria could serve as an indicator bacteria to distinguish between matured and degenerated pit muds. Copyright © 2016 The Institute of Brewing & Distilling     

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.     

啤酒废水处理系统中活性污泥微型生物群落结构分析

为了分析啤酒厂废水处理系统活性污泥运行状态,选取了啤酒厂污水处理系统中不同阶段的微型生物群落结构作为研究对象。本文利用16S rDNA及18S rDNA特异性引物作为分子标记,通过PCR扩增污水处理系统中的环境生物群落DNA,以变性梯度凝胶电泳(DGGE)技术分离检测PCR产物获得微生物群落的DNA指纹图谱。研究结果显示,经过活性污泥处理的啤酒厂废水中细菌群落和真核生物群落结构发生了明显的改变。其中在细菌群落DGGE图谱中检测到21条特异性条带,而在真核生物DGGE图谱检测到10条。UPGMA聚类分析显示,进水与活性污泥中的细菌及真核群落结构十分相似(相似性>0.67),而与出水的差异较大(相似性<0.41),这表明了进水对活性污泥生物群落结构的重要影响。     

酸性电解水处理后南美白对虾贮藏过程中肠道微生物的多样性变化

肠道微生物是引起水产品腐败变质的主要污染源之一。酸性电解水(AEW)作为新型环保消毒剂,因其高效广谱杀菌的特点,已逐渐被应用于水产品安全控制方面并且日益受到人们的重视。本研究采用变性梯度凝胶电泳技术,分析了经自来水(TW)与酸性电解水分别处理的南美白对虾(Penaeus vannawei)于0、4、25℃贮藏过程中肠道微生物多样性的变化规律。结果表明,与对照相比,经酸性电解水处理后的样品肠道微生物多样性在贮藏过程中有所减少。因此,酸性电解水能明显改变肠道微生物的多样性,为其保障水产品质量安全提供了理论参考依据。