Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach

In this study, the characterization of the bacterial community present during the fermentation of pulque, a traditional Mexican alcoholic beverage from maguey (Agave), was determined for the first time by a polyphasic approach in which both culture and non-culture dependent methods were utilized. The work included the isolation of lactic acid bacteria (LAB), aerobic mesophiles, and 16S rDNA clone libraries from total DNA extracted from the maguey sap (aguamiel) used as substrate, after inoculation with a sample of previously produced pulque and followed by 6-h fermentation. Microbiological diversity results were correlated with fermentation process parameters such as sucrose, glucose, fructose and fermentation product concentrations. In addition, medium rheological behavior analysis and scanning electron microscopy in aguamiel and during pulque fermentation were also performed. Our results showed that both culture and non-culture dependent approaches allowed the detection of several new and previously reported species within the alpha-, gamma-Proteobacteria and Firmicutes. Bacteria diversity in aguamiel was composed by the heterofermentative Leuconostoc citreum, L. mesenteroides, L. kimchi, the gamma-Proteobacteria Erwinia rhapontici, Enterobacter spp. and Acinetobacter radioresistens. Inoculation with previously fermented pulque incorporated to the system microbiota, homofermentative lactobacilli related to Lactobacillus acidophilus, several alpha-Proteobacteria such as Zymomonas mobilis and Acetobacter malorum, other gamma-Proteobacteria and an important amount of yeasts, creating a starting metabolic diversity composed by homofermentative and heterofermentative LAB, acetic and ethanol producing microorganisms. At the end of the fermentation process, the bacterial diversity was mainly composed by the homofermentative Lactobacillus acidophilus, the heterofermentative L. mesenteroides, Lactococcus lactis subsp. lactis and the alpha-Proteobacteria A. malorum. After a 6-h fermentation, 83.27% of total sugars detected after inoculation were consumed (228.4 mM hexose equivalents) and a carbon (C) recovery of 66.18% in fermentation products was estimated. They were produced 284.4 mM C as ethanol, 71.5 mM C as acetic acid and 19 mM C as lactic acid, demonstrating the presence of homo- and heterofermentative, acetic and alcoholic metabolisms in the final product. It was also found, after hydrolysis, that the exopolysaccharide produced during the fermentation was mainly composed by fructose residues, probably inulin or levan.     

Evaluation of Bacterial Diversity in Palm Wine by 16S rDNA Analysis of Community DNA

Bacterial diversity and fermentation dynamics in palm wine, a traditional alcoholic fermented beverage, collected from upright palm trees from Idiaba community, Abeokuta, Ogun State, Nigeria were evaluated by DNA based method using the 16S rDNA of the microbial community to verify and complement previous reports, improve our understanding and document yet unreported, uncultured microbial diversity associated with palm wine. The 16S rRNA gene fragments were amplified from microbial community and genomic DNA of isolates, by Polymerase Chain Reaction (PCR) using universal primers; and sequenced. The partial sequences were identified by comparison with sequences deposited in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). This analysis revealed that 32 community clones were identified as Lactobacillus sp, Lactobacillus casei strain zhang, Lactobacillusplantarum, Leuconostoc mesenteriodes ssp dextranicum, Leuconcostoc lactis, Pediococcusparvulus strain Bpe-299, Acetobacter pomorum, Acetobacter pasteurianus, Gluconobacter oxydans, Acinetobacter calcoaceticus, Enterobacterium bacterium, Acidovorax sp, Comamonas sp, Bacillus subtilis, Staphylococcuspiscifermentans and uncultured bacteria clone D1-78. The results showed that bacterial diversity in the palm wine sample is dominated by Lactobacillus and Leuconostoc species as reported by previous workers and uncultured bacteria clone D1-78 (1 clone) was detected for the first time in palm wine.     

Microbial diversity and biochemical profile of aguamiel collected from<Emphasis Type="Italic"> Agave salmiana</Emphasis> and<Emphasis Type="Italic"> A. atrovirens</Emphasis> during different seasons of year

Aguamiel is a beverage produced by some Agave species that is consumed in its fresh or fermented form. Despite its uses and popularity, seasonal effects on its microbial and chemical profiles are unknown. In this study, using aguamiel collected from A. salmiana and A. atrovirens during different seasons, we identified microorganisms by sequencing the 16S and 18S rDNA genes and determined their chemical profiles. In total, 49 microbial strains were identified (38 bacteria and 11 yeasts). The highest richness and biodiversity were observed during winter and summer. Different lactic acid bacteria and yeast genera with potential industrial applications were identified, such as Acetobacter, Lactobacillus, Leuconostoc, and Clavispora. The analysis of the chemical profiles indicated the presence of maltooligosaccharides and fructooligosaccharides, which are associated with human health improvements, during spring in Agave aguamiel. Aguamiel can be used in the food industry due to its microbiological and chemical profiles.     

Molecular Identification of Microbial Community in Surface and Undersurface Douchi During Postfermentation

To find the reason for fermentation failure of surface Douchi during postfermentation, the microbial communities in undersurface and surface samples were investigated using cell counting method and denaturing gradient gel electrophoresis (DGGE). The results showed that the microbial biomass in surface Douchi was obviously different from that in undersurface Douchi even sampled from the same fermentation tanks, and a 10‐ to 100‐fold reduction of microbial cell counts in undersurface had been observed. The bacterial DGGE profile and principal component analysis (PCA) results indicated that only Lactococcus lacts subsp. lactis and Bacillus thermoamylovorans were detected from surface Douchi, while Lactococcus lacts subsp. lactis, Staphylococcus lentus and 2 uncultured strains occupied the dominant positions in undersurface Douchi; when amplified using Bacillus‐specific primers, Bacillus thermoamylovorans, Bacillus subtilis, and Enterobacter sp. were found in undersurface Douchi, while only Bacillus thermoamylovorans were detected from surface Douchi; compared to the bacteria and Bacillus, the DGGE profiles and PCA plot of fungi indicated that the fungal community between surface and undersurface Douchi was similar and mainly composed by yeasts. In this study, we detected the microbial biomass and species in postfermentation stage of Douchi, and the various microbial diversity in undersurface and surface samples might be the cause of the fermentation failure in surface fermentation tanks.     

Molecular identification of microbial community in Chinese douchi during post-fermentation process

To track changes in dynamic microbial communities during post-fermentation process, traditional culture method, and denaturing gradient gel electrophoresis (DGGE) were used to study the number and species of dominant microorganisms in douchi. The result of culturedependent method showed that the microbial number changed slightly since the 4^th day while the DGGE indicated that the really steady-state was achieved from the 10^th day. In addition, Lactococcus lactis subsp., Staphylococcus lentus, and 2 uncultured bacterium were identified to occupy the dominant positions in bacterial DGGE pattern, and Bacillus thermoamylovorans, Bacillus subtilis, Enterobacter spp., and Absidia corymbifera, Pichia guilliermondii, Pichia farinose were also detected from Bacillus and fungal DGGE patterns, respectively. In conclusion, some pathogenic microorganisms involving in the douchi fermentation had been detected throughout the post-fermentation process, and the combination of culturedependent and -independent method was proved to be effective in profiling microbial diversity.     

PCR-DGGE技术分析腌制麻竹笋中微生物多样性

采用聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE）技术对盐质量浓度5 g/100 mL和19 g/100 mL腌制麻竹笋的微生物区系进行研究。结果表明,经DNA提取、巢式PCR、DGGE电泳和克隆测序后,从低盐质量浓度（5 g/100 mL）腌制笋中分离出4 条明显的亮带,经鉴定分别为食窦魏斯氏菌（Weissella cibaria）、乳球菌属（Lactococcus sp.）、魏斯氏菌属（Weissella sp.）和乳酸乳球菌（Lactococcus lactis）;从高盐质量浓度（19 g/100 mL）腌制笋中分离出5 条明显的亮带,经鉴定分别为绿色气球菌（Aerococcus viridans）、赖氨酸芽孢杆菌属（Lysinibacillus sp.）、未得到培养的细菌（unculturedbacterium）、厌氧芽孢杆菌属（Anoxybacillus sp.）和芽孢杆菌属（Bacillus sp.）;低盐腌制笋的优势菌多为益生菌,而高盐腌制笋的优势菌则多为抗性较强的菌。基于16S rDNA的PCR-DGGE技术为分析腌制麻竹笋中微生物多样性提供了一条可靠、快速的有效途径。     

Bacterial Community of Koumiss from Mongolia Investigated by Culture and Culture-Independent Methods

The bacterial community of fermented horse milk (koumiss) from Mongolia was studied using three methods: cultivation, direct identification by 16S rRNA clone library and denaturing gradient gel electrophoresis (DGGE). Ninety-eight strains were isolated by traditional cultivation and 61 of those were randomly selected for further identification by 16S rRNA gene sequencing. The strains were dominated by lactic acid bacteria (LAB; six different lactobacilli), Acinetobacter, Bacillus and Psychrobacter. Construction of the clone library analysis revealed that 16S sequences of 220 clones, genus Lactobacillus was dominant, but Streptococcus thermophilus, Acetobacter pasteurianus and uncultured clones were also detected. Ten unique bands were sequenced from the DGGE and revealed: Lactococcus lactis, Lactococcus lactis subsp. lactis, Clostridium acidurici, Acinetobacter johnsonii, Dickeya sp., Enterobacter sp., Pseudomonas sp., Raoultella sp., and Ruminococcus sp.. In vitro growth inhibition of three human pathogens, Escherichia coli, Enterobacter sakazakii and Staphylococcus aureus by 14 culturable bacteria displayed that only three of the isolates tested inhibit growth of E. sakazakii while most of the other bacteria delayed growth of the target bacteria.     

Behavior of Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Shigella flexneri and Shigella sonnei during production of pulque, a traditional Mexican beverage

Pulque is a typical fermented alcoholic beverage of central Mexico, produced from the nectar of maguey agave plants. Production systems are largely artisanal, with inadequate hygiene conditions and exposure to multiple contamination sources. No data exist on pulque microbiological safety and the behavior of pathogenic microorganisms in agave nectar and pulque. An initial trial was done of the behavior of Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Shigella flexneri and Shigella sonnei during fermentation of nectar from a single producer, nectar mixture from different producers, and seed pulque. A second trial simulating artisanal pulque production was done by contaminating fresh nectar with each of the five strains, storing at 22°C for 14 h, adding seed pulque, and fermenting until pulque was formed. During incubation at 16 or 22°C in the first trial, all the pathogenic strains multiplied in both the single producer nectar and the nectar mixture, reaching maximum concentrations at 12 h. Strains concentration then decreased slowly. In the seed pulque, the strains did not multiply and tended to die. In the second trial, all strains increased concentration from 0.7 to 1.6 log at 22°C, and from 0.5 to 1.1 at 16°C in the first 14 h. After addition of seed pulque, they were quickly deactivated until none was detected in the final product. The results suggest that the potential risk to consumers of contracting any of the five tested pathogenic bacterial strains from pulque is low.     

Improvement of growth,fermentative efficiency and ethanol tolerance of Kloeckera africana during the fermentation of Agave tequilana juice by addition of yeast extract

BACKGROUND: The aim of this work was to improve the productivity and yield of tequila fermentation and to propose the use of a recently isolated non‐Saccharomyces yeast in order to obtain a greater diversity of flavour and aroma of the beverage. For that, the effects of the addition of different nitrogen (N) sources to Agave tequilana juice on the growth, fermentative capacity and ethanol tolerance of Kloeckera africana and Saccharomyces cerevisiae were studied and compared. RESULTS: Kloeckera africana K1 and S. cerevisiae S1 were cultured in A. tequilana juice supplemented with ammonium sulfate, diammonium phosphate or yeast extract. Kloeckera africana did not assimilate inorganic N sources, while S. cerevisiae utilised any N source. Yeast extract stimulated the growth, fermentative capacity and alcohol tolerance of K. africana, giving kinetic parameter values similar to those calculated for S. cerevisiae. CONCLUSION: This study revealed the importance of supplementing A. tequilana juice with a convenient N source to achieve fast and complete conversion of sugars in ethanol, particularly in the case of K. africana. This yeast exhibited similar growth and fermentative capacity to S. cerevisiae. The utilisation of K. africana in the tequila industry is promising because of its variety of synthesised aromatic compounds, which would enrich the attributes of this beverage. Copyright © 2009 Society of Chemical Industry     

基于PCR-DGGE技术的四川麸醋固态发酵过程中微生物群落分析

为了解四川麸醋固态发酵过程中微生物群落变化规律,采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对其固态发酵过程中真菌和细菌的多样性进行分析。结果表明,四川麸醋固态发酵过程中优势真菌主要有酿酒酵母(Sac.cerevisiae)、扣囊复膜孢酵母(Sac.fibuligera)、伊萨酵母(Iss.hanoiensis)、黑曲霉(Asp.niger)、草青霉菌(Pen.oxalicum)以及不可培养真菌(Uncultured fungus);主要细菌有嗜酸乳杆菌(Lac.acidophilus)、葡萄糖醋杆菌(Glu.oboediens)、巴氏醋杆菌(Ace.pasteurianus)、甲醇酸单胞菌(Aci.methanolica)、不可培养细菌(Uncultured bacterium)、不可培养芽孢杆菌(Uncultured Bacillus sp.)和不可培养乳杆菌(Uncultured Lactobacillus sp.)。PCR-DGGE技术可鉴别醋醅中多种不可培养微生物,四川麸醋固态发酵过程优势微生物种类多、丰度高,菌系十分复杂,多种微生物交替生长,但整体群落结构变化不明显。     

Dynamics of starter,adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.     

Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives

Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria — LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in cold-fermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives.     

Acid and alcohol tolerance of Escherichia coli O157:H7 in pulque, a typical Mexican beverage

Pulque is a traditional Mexican fermented alcoholic beverage produced from the nectar of maguey agave plants. No data exist on the behavior of Escherichia coli O157:H7 in agave nectar and pulque. An initial trial was done of the behavior of E. coli O157:H7 during fermentation of nectar from a single producer, a nectar mixture from different producers and "seed" pulque. A second trial simulating artisanal pulque production was done by contaminating fresh nectar with a cocktail of three E. coli O157:H7 strains, storing at 16 ° and 22 °C for 14 h, adding seed pulque and fermenting until pulque was formed. A third trial used pulque from the second trial stored at 22 °C as seed to ferment fresh nectar at 22 °C for 48 h (fermentation cycle). This procedure was repeated for an additional two fermentation cycles. During incubation at 16 ° or 22 °C in the first trial, the E. coli O157:H7 strains multiplied in both the single producer nectar and nectar mixture, reaching maximum concentration at 12h. E. coli O157:H7 cell concentration then decreased slowly, although it survived at least 72 h in both fermented nectars. E. coli O157:H7 did not multiply in the seed pulque but did survive at least 72 h. In the second trial, the numbers of E. coli O157:H7 increased approximately 1.5 log CFU/ml at 22 °C and 1.2 log CFU/ml at 16 °C after 14 h. After seed pulque was added, E. coli O157:H7 concentration decreased to approximately 2 log CFU/ml, and then remained constant until pulque was produced. In the third trial, the E. coli O157:H7 cells multiplied and survived during at least three nectar fermentation cycles. The results suggest that E. coli O157:H7 can develop acid and alcohol tolerance in pulque, and constitutes a public health risk for pulque consumers.     

Physico-chemical characterisation and tensile mechanical properties of Agave americanaL. fibres

In this paper, the physico-chemical and mechanical properties of Agave americana L. fibre are studied to explore the possibilities of using this fibre in textile industry. Three different processings for extracting fibres from the leaf of Agave americana L. plant were investigated: (i) raw fibres manually extracted, (ii) fibres extracted by retting leaves in seawater and (iii) fibres extracted after hydrolysis treatment of the leaves in distilled water. Chemical composition (cellulose, lignin and hemicellulose contents), physical properties (density, fineness and crystallinity) as well as mechanical properties of the resultant fibres were measured. The chemical composition reveals that the Agave americana L. fibre has a cellulose content at the order of 62% which is similar to that of other lignocellulosic fibres. The distilled water extracted fibres developed the highest cellulose content. Lignin content of Agave americana L. fibre is low compared to other natural fibres (2.4%) and seawater-extracted fibres generated the lowest lignin content (2.12%). Fourier transform infrared spectra (FTIR) and x-ray diffractometry of all extracted fibres revealed the same chain conformation. X-ray diffractograms showed that cellulose I is the main crystalline constituent. Concerning physical and mechanical properties, raw fibres were characterised by the lowest density (0.9) and the highest crystallinity (51.2%), they are also stronger and less extensible than the other two fibres. In this paper, we attempt to study the effect of extraction in water on the mechanical and physical properties of Agave americana L. fibres.     

Study of the Mechanical Properties of Fibers Extracted from Tunisian Agave americana L.

Agave americana L. fibers, the most abundant variety in Tunisia, have a quite important textile potential. This potential is demonstrated by studying the extraction of these fibers from leaves, their physical properties such us fineness or density and their mechanical behavior in tensile tests. In this work, results of a mechanical behavior study of fibers extracted from the Agave americana L. plant are presented. These results deal with the principal and mechanical characteristics of these fibers which are the strain at break, the elasticity modulus and the rupture facture. These results permitted to situate these fibers, compared to the other textile fibers, as materials that can be used in technical applications such as reinforcing composites or geotextile. In order to understand the mechanical properties of these fibers, a correlation study between the properties already cited and the fine structure was done. The obtained results showed that the mechanical properties of Agave americana L. fibers are closely related to the individual fibers deformations and to the natural matrix (lignin and gums) that are linked to these elementary fibers.     

PCR-DGGE分析木瓜酵素自然发酵过程中微生物的多样性

为探究木瓜酵素自然发酵过程中的微生物多样性及变化规律,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术分析木瓜酵素自然发酵过程中细菌和酵母的多样性及变化规律。结果表明,木瓜酵素自然发酵过程中主要细菌有植物乳杆菌(Lactobacillus plantarum)、假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、类肠膜魏斯氏菌(Weissella paramesenteroides)及不可培养的丙酸菌(Uncultured Propionibacterium sp.),其中植物乳杆菌为主要优势细菌;主要酵母有:酿酒酵母(Saccharomyces cerevisiae)、假丝酵母(Candida xestobii、Candida intermedia)、毕赤酵母(Pichia guilliermondii、Komagataella phaffii、Pichia punctispora、Pichia galeiformis)、棒孢酵母(Clavispora sp.)及Cyberlindnera fabianii,其中酿酒酵母和毕赤酵母为主要优势酵母;Lac plantarum、Pic galeiformis分别与Uncultured Propionibacterium sp.、Cyb fabianii之间的亲缘性较高,与其他菌之间的亲缘性较小。木瓜酵素自然发酵过程中菌群交替生长,有一定的亲缘性,菌落结构变化较小,分布较为均匀,这为进一步的研究提供理论基础。     

Predictive models for bacterial growth in sea bass (Dicentrarchus labrax) stored in ice

The purpose of this paper was to estimate microbial growth through predictive modelling as a key element in determining the quantitative microbiological contamination of sea bass stored in ice and cultivated in different seasons of the year. In the present study, two different statistical models were used to analyse changes in microbial growth in whole, ungutted sea bass (Dicentrarchus labrax) stored in ice. The total counts of aerobic mesophilic and psychrotrophic bacteria, Pseudomonas sp., Aeromonas sp., Shewanella putrefaciens, Enterobacteriaceae, sulphide‐reducing Clostridium and Photobacterium phosphoreum were determined in muscle, skin and gills over an 18‐day period using traditional methods and evaluating the seasonal effect. The results showed that specific spoilage bacteria (SSB) were dominant in all tissues analysed, but were mainly found in the gills. Predictive modelling showed a seasonal effect among the fish analysed. The application of these models can contribute to the improvement of food safety control by improving knowledge of the microorganisms responsible for the spoilage and deterioration of sea bass.     

冷却牦牛肉贮藏过程中优势菌的 PCR-变性梯度凝胶电泳分析

应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究托盘保鲜膜包装的冷却牦牛肉在4℃贮藏过程中的微生物多样性及其动态变化.直接从样品中提取细菌总的DNA,采用降落PCR扩增16S rDNA的V3可变区序列,再通过DGGE得到动态变化的指纹图谱,并对主要条带进行测序分析.结果表明:检测到的优势腐败菌为Pseudomonas sp.(假单胞菌)、Lactococcus sp.(乳球菌)、Acinetobacter sp.(不动杆菌)、Brochothrix thermosphacta(热死环丝菌)、Enterobacteriaceae bacterium(肠杆菌科细菌),此外还检测到Uncultured Citrobacter sp.(非培养的柠檬酸杆菌)和Staphylococcus sp.(葡萄球菌)     

Study of the Lactobacillus sakei protective effect towards spoilage bacteria in vacuum packed cooked ham analyzed by PCR-DGGE

The effectiveness of Lactobacillus sakei B-2 inoculated as a protective culture on the inhibition of spoilage bacteria on sliced vacuum packed cooked ham was investigated by using culture-dependent and -independent approaches. Total microbial DNA was directly extracted from both control and treatment samples, and subjected to a nested PCR protocol, PCR–DGGE analysis was used to identify and monitor the dynamic changes in the microbial population, followed by partial 16S rDNA sequencing. The DGGE profile demonstrated that the protective culture effectively suppressed growth of predominant spoilage bacteria L. sakei, Lactobacillus curvatus and Leuconostoc mesenteroides in cooked ham during storage at 4 °C, however, growth of uncultured Leuconostoc was not inhibited. The shelf-life of this product inoculated with L. sakei B-2, at levels of 5.91 ± 0.04 log₁₀ CFU g⁻¹ was 35 days, compared to 15 days of control samples, when the ham was stored at 4 °C.     

Application of denaturing gradient gel electrophoresis to microbial diversity analysis in Chinese Douchi

BACKGROUND: Douchi is a traditional Chinese soybean food which has been consumed for thousands years as an important protein source and flavouring ingredient. Studies have rarely been carried out to investigate its microbial composition and these are urgently required for the commercial labels and safety considerations. RESULTS: Microbial counts were statistically significant different among Douchi samples. Although the maximum diversity indexes of bacterial, bacillus and fungal polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) patterns were only 79%, 70% and 64%, some microorganisms, e.g. Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas sp., Saccharomyces cerevisiae and Pichia farinose, were found to share dominant positions in most Douchi samples. In addition, some pathogens, e.g. Staphylococcus saprophyticus, Pantoea sp., Staphylococcus sciuri, Enterobacter sp. and Staphylococcus sp., were also identified. CONCLUSION: The PCR‐DGGE technique was used for the first time as an effective method to assess the microbial communities in different Chinese Douchi samples. This information may be useful in improving the product quality, reformatting production methods, extending shelf life and scaling up the fermentation process. Copyright © 2012 Society of Chemical Industry