Microbial biodiversity of raw milk collected from Yangzhou and the heterogeneous biofilm-forming ability of Pseudomonas

This study aimed to reveal microbial populations in Chinese raw milk using both 16S rRNA sequencing and high-throughput sequence (HTS) methods. Psychrotrophic isolates were clustered into 23 genera and 57 species, and the abundant genera in raw milk were Pseudomonas, Acinetobacter and Lactococcus. Genetic diversity and biofilm-forming capacity of Pseudomonas isolates were analysed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Heterogeneous biofilm-forming ability of Pseudomonas isolates with the same RAPD patterns was observed. HTS analysis revealed a 33-fold higher diversity at the genus level, and a different bacterial population was predominated by Flavobacterium, Pseudomonas and Acinetobacter. The results provide insight into the microbial contamination of raw milk.     

Metagenomic reveals succession in the bacterial community and predicts changes in raw milk during refrigeration

In this study, metagenomics was used to analyze microbial succession and predict changes in raw milk during 6 days of storage at 4°C, aimed at determining how microorganisms drive the deterioration of refrigerated raw milk. The microbial community in raw milk changed significantly with an extension of refrigeration time (p < .01). The dominant bacterial genera gradually evolved from Acinetobacter, Streptococcus, and Anaplasma to Flavobacterium, Pseudomonas, and Lactococcus. KEGG annotation results indicated that the main role of the bacterial community included amino acid biosynthesis, carbon metabolism, and functioning as an ABC transporter. Additionally, lipid, amino acid, and carbohydrate metabolism pathways were significantly expressed at the beginning, middle, and end of refrigeration, respectively. Refrigeration time is an important factor affecting the composition of the microbial flora in raw milk. The results of this study illustrate the role of microorganisms in the deterioration of refrigerated raw milk.     

Bacterial microflora of carp (Cyprinus carpio) and its shelf-life extension by essential oil compounds

The microflora of common carp (Cyprinus carpio) skin, gill and intestine were analysed and the antimicrobial activities of garlic oil and nine constituents of essential oils (allyl isothiocyanate, carvacrol, cinnamaldehyde, citral, cuminnaldehyde, eugenol, isoeugenol, linalool and thymol) against the carp isolates were studied to identify compounds that might extend the shelf-life of carp fillet. A total of 90 isolated strains were identified to belong to seven genera: Acinetobacter (6), Alcaligenes (2), Bacillus (2), Flavobacterium (20), Micrococcus (2), Moraxella (6) and Pseudomonas (4), and two families Enterobacteriaceae (14) and Vibrionaceae (34). The dominant micro-organisms of carp were found to be Flavobacterium (37%) and Vibrionaceae (33%) in skin, Flavobacterium (33%) in gill and Vibrionaceae (63%) and Flavobacterium (37%) in intestine. Against these isolates, thymol, carvcarol and cinnamaldehyde had the strongest antimicrobial activities, followed by isoeugenol, eugenol, garlic oil, and then citral. The antimicrobial properties of the other constituents tested (cuminnaldehyde, linalool and allyl isothiocyanate) were low. In tests of mixed compounds, a combination of carvacrol and thymol had the highest antimicrobial activity. Moraxella, Flavobacterium and Vibrionaceae were more sensitive to the compounds, whereas Alcaligenes strains were resistant. Dipping carp fillets in a solution of 0.5% carvacrol and 0.5% thymol before storage at 5°C and 10°C reduced both the total microbial load by about 100-fold and the Volatile Bases Nitrogen (VB-N), as compared with controls. In addition, dipping treatment delayed bacterial growth and extended the shelf-life of the fillets from 4 to 12 days at low temperature (5°C). However, the treated and control fillets showed little difference during storage at 10°C. Data from sensory evaluation showed that dipped fillets in 1% (carvacrol+thymol) extended the shelf-life of carp fillets by 8 and 4 days at 5° and 10°C, respectively. Thus, carvacrol and thymol dipping can improve the microbial stability of fish fillets by removing bacteria and by inhibiting bacterial growth.     

Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis

Investigation of the initial and spoilage microbial diversity of iced stored sea bream was carried out. Culture dependent methods were used for bacterial enumeration and phenotypic identification of bacterial isolates, while culture independent methods, using bacterial 16S rRNA gene amplification, cloning and sequencing of DNA extracted directly from the flesh were also employed. The culture dependent approach revealed that the initial microbiota was dominated by Acinetobacter, Shewanella, Pseudomonas and Flavobacterium, while at the end of shelf-life determined by sensory analysis (16 days), the predominant microbiota was Pseudomonas and Shewanella. Culture independent approach showed that initially the sea bream flesh was strongly dominated by Acinetobacter, while Pseudomonas, Aeromonas salmonicida and Shewanella were the predominant phylotypes at the end of shelf-life. Initial and spoilage microbiota comprised of phylotypes previously identified by others using traditional or molecular techniques. However, Aeromonas has not been reported as part of the dominant microbiota of sea bream at the time of spoilage. Combination of classical and molecular methodologies better reveals the microbiota during storage by revealing bacteria that escape standard approaches and, thus, provides valuable complementary information regarding microbiological spoilage.     

Microbial diversity analysis of vineyards in the Xinjiang region using high‐throughput sequencing

The microbial diversity of soil, grape leaves and grape fruits from three wine‐growing areas in Xinjiang, China were analysed using high‐throughput sequencing. Results show that five fungal phyla and 237 fungal genera, as well as eight bacterial phyla and 314 bacterial genera were detected in 27 samples. For both fungi and bacteria, the microbial diversity was most complex in the soil, followed by grape leaves and fruit. In the soil samples, Ascomycota, Sordariales, Tetracladium and Geomyces were the dominant fungal genus, while Arthrobacter, Kaistobacter and Skermanella were the dominant bacterial genus. With regard grape leaves and fruit, Aureobasidium, Cryptococcus, Aspergillus and Pleosporaceae were the dominant fungal genus, while Pseudomonas, Sphingomonas and Adhaeribacter were the dominant bacterial genus. Different geographical locations were associated with differences in microbial diversity. The complex microflora of vineyards provides a basis for further microbial screening and its potential utilization in different regions. © 2018 The Institute of Brewing & Distilling     

Microflora of brown shrimp (Penaeus aztecus) from Georgia coastal waters

Initial aerobic plate counts of shrimp harvested under controlled conditions were approximately 2 logs lower than counts obtained from commercially harvested shrimp and remained lower throughout 18 days of storage on ice. Commercial shrimp microflora capable of proteolysis gradually shifted to 100% by day 18. Shrimp obtained from fish markets averaged bacterial counts of 2·0 × 10⁵ on day of purchase, 45% of those were capable of proteolysis. The natural spoilage microflora of freshly caught Georgia coast shrimp were predominantly Acinetobacter, Enterobacter, and Flavobacterium species, while that of commercial shrimp was predominantly Acinetobacter, Moraxella, and Corynebacterium. After 18 days of ice storage, Acinetobacter and Flavobacterium species predominated on both fresh and commercial shrimp.     

Investigation of donkey milk bacterial diversity by 16S rDNA high-throughput sequencing on a Cyprus donkey farm

The interest in milk originating from donkeys is growing worldwide due to its claimed functional and nutritional properties, especially for sensitive population groups, such as infants with cow milk protein allergy. The current study aimed to assess the microbiological quality of donkey milk produced in a donkey farm in Cyprus using culture-based and high-throughput sequencing techniques. The culture-based microbiological analysis showed very low microbial counts, whereas important food-borne pathogens were not detected in any sample. In addition, high-throughput sequencing was applied to characterize the bacterial communities of donkey milk samples. Donkey milk mostly composed of gram-negative Proteobacteria, including Sphingomonas, Pseudomonas, Mesorhizobium, and Acinetobacter; lactic acid bacteria, including Lactobacillus and Streptococcus; the endospores forming Clostridium; and the environmental genera Flavobacterium and Ralstonia, detected in lower relative abundances. The results of the study support existing findings that donkey milk contains mostly gram-negative bacteria. Moreover, it raises questions regarding the contribution of (1) antimicrobial agents (i.e., lysozyme, peptides) in shaping the microbial communities and (2) bacterial microbiota to the functional value of donkey milk.     

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4 °C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.     

Microorganisms on Commercially Processed Alaskan Finfish

Whole and processed Alaskan fish were examined for aerobic plate counts, total coliforms, and Escherichia coli. Washing whole salmon and halibut reduced skin microbial counts from 10³/cm² to 10²/cm². Whole and dressed fish had coliform counts less than 13/cm² and E. coli counts less than 0.3/cm². Alaska pollock and Pacific cod fillets had microbial counts between 10³/g and 10⁶/g, coliform counts less than 70/g, and E. coli counts less than 4/g. Whole fish contained microbial flora predominated by Moraxella species whereas dressed fish and fillets had microbial flora consisting of Arthrobacter/Corynebacterium, Flavobacterium, and Pseudomonas species. Microorganisms on conveyor belts and other contact surfaces may have contributed contaminants to fillets.     

Physicochemical and microbiological changes in postmortem crayfish (Procambarus clarkii) stored at 4 °C and 25 °C

The physicochemical and microbiological parameters of postmortem crayfish stored at 4 °C and 25 °C were evaluated in order to reduce safety risks of crayfish from temperature abuse during transportation and storage. Results showed that hepatopancreas of postmortem crayfish had significantly higher contents of total volatile basic nitrogen, biogenic amines (BAs) and higher microbial loads than tail meat, regardless of storage temperature. Enterobacteriaceae and Pseudomonas counts reached log 6.41 log₁₀ CFU g⁻¹ and 6.31 log₁₀ CFU g⁻¹ in hepatopancreas of crayfish at 25 ℃ for 6 h. Putrescine and cadaverine were the main BAs in tail meat with levels of 28.18 ± 0.73 mg kg⁻¹ and 187.32 ± 3.57 mg kg⁻¹, respectively, whilst cadaverine, spermidine and spermine were the major BAs in hepatopancreas, reaching 283.45 ± 3.95 mg kg⁻¹, 62.87 ± 9.02 mg kg⁻¹ and 155.31 ± 4.55 mg kg⁻¹, respectively, after postmortem storage at 25 °C for 12 h. With time increasing, Acinetobacter, Flavobacterium, Aeromonas and Chryseobacterium at 25 °C and Acinetobacter, Flavobacterium, Psychrobacter at 4 °C in tail meat as well as Bacteroides and Muribaculaceae at 25 °C, and Acinetobacter, Psychrobacter at 4 °C in hepatopancreas, gradually became the major genus at the end of storage. Based on the results of spoilage microorganisms and biogenic amines, crayfish stored at 4 °C and 25 °C could be edible within 24 h and 6 h, respectively.     

Effect of fermentation with mixed starter cultures on biogenic amines in bighead carp surimi

The effects of one group mixed starter cultures, combined with Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen, and Monascus anka, and a batch without starter as control on biogenic amines accumulation in the bighead carp surimi during fermentation were investigated. Determination of five different biogenic amines was carried out by reverse‐phase high‐performance liquid chromatography with UV detection. Results showed that mixed starter cultures decreased the pH quickly from initial pH value of 6.4 to 5.2, inhibited the growth of contaminant microorganisms, such as Enterobacteriaceae and Pseudomonas, present in the raw materials, and suppressed the accumulation of histamine, tyramines, spermine and spermidine. Fermentation inoculated with combination of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen, and Monascus anka with negative‐decarboxylase activity may prevent biogenic amine formation in the fermented ssurimi and improve hygienic quality of fermented surimi.     

Monitoring the microbial community during solid-state acetic acid fermentation of Zhenjiang aromatic vinegar

Zhenjiang aromatic vinegar is one of the most famous Chinese traditional vinegars. In this study, change of the microbial community during its fermentation process was investigated. DGGE results showed that microbial community was comparatively stable, and the diversity has a disciplinary series of changes during the fermentation process. It was suggested that domestication of microbes and unique cycle-inoculation style used in the fermentation of Zhenjiang aromatic vinegar were responsible for comparatively stable of the microbial community. Furthermore, two clone libraries were constructed. The results showed that bacteria presented in the fermentation belonged to genus Lactobacillus, Acetobacter, Gluconacetobacter, Staphylococcus, Enterobacter, Pseudomonas, Flavobacterium and Sinorhizobium, while the fungi were genus Saccharomyces. DGGE combined with clone library analysis was an effective and credible technique for analyzing the microbial community during the fermentation process of Zhenjiang aromatic vinegar. Real-time PCR results suggested that the biomass showed a “system microbes self-domestication” process in the first 5 days, then reached a higher level at the 7th day before gradually decreasing until the fermentation ended at the 20th day. This is the first report to study the changes of microbial community during fermentation process of Chinese traditional solid-state fermentation of vinegar.     

基于16S rRNA基因测序分析烟叶发酵过程中表面微生物的多样性

雪茄烟叶经过发酵处理才能制成风味独特的雪茄产品,研究烟叶发酵过程中微生物的多样性对提高国产雪茄烟叶品质有重要意义。作者采集了来自云南省8个地区初步晾晒的雪茄烟叶和7种来自多米尼加共和国各地的优质雪茄烟叶,基于16S rRNA基因测序技术鉴定各个样品表面微生物种类,通过OTU聚类分析、Alpha多样性分析、物种组成分析等方法进行微生物多样性分析,获得晾晒烟叶表面的优势菌。结果显示,云南省雪茄烟叶的优势菌属为葡萄球菌属,其他常见菌属有假单胞菌属、无色杆菌属、棒状杆菌属、泛菌属等;多米尼加共和国雪茄样品中微生物物种分布较中国雪茄样品更均匀,微生物的多样性整体高于中国雪茄样品,其优势菌属多为葡萄球菌属、棒状杆菌属、四联球菌属;云南省临沧市雪茄烟叶优势菌属与多米尼加雪茄相似,而且它的物种多样性和均匀度高于中国其他地区,具有较大的发展潜力。     

Characterization of the microbial community in three types of fermentation starters used for Chinese liquor production

This study examined and compared the microbial community in three typical fermentation starters (called as Daqu, Xiaoqu, and Fuqu in China) used for liquor production by analysing the 16S and 18S rRNA gene clone library. The results show that the microbial diversity in the three types of fermentation starters (JiuQu) differs significantly. The bacterial species in Daqu and Fuqu were mainly thermophilic or thermotolerant. In Daqu, the dominant bacterial species were Thermoactinomyces sanguinis (53.85%) and Pantoea agglomerans (19.23%), followed by uncultured bacteria (15.39%). The lactic acid bacterium Weissella cibaria (50%) and a member of Enterobacteriaceae, Enterobacter ludwigii (10%), were the dominant bacterial species in Xiaoqu. Low abundances of other bacteria, including Deinococcus radiodurans, Corynebacterium variabile and Acinetobacter baumannii, were reported for Xiaoqu. Enterococcus faecium, Clostridium beijerinckii and Bacillus cereus were observed in Fuqu and accounted for 46.67, 23.33 and 16.67% of the total bacteria identified, respectively. Fungal diversity was high in Daqu and consisted exclusively of thermophilic moulds, such as Aspergillus glaucus (62.5%), Thermomyces lanuginosus (12.5%) and Thermoascus crustaceus (12.5%). Only two fungal species were reported for Fuqu and Xiaoqu and both contained the mould Rhizopus oryzae. Saccharomyces cerevisiae and the non‐Saccharomyces yeast (Saccharomycopsis fibuligera) were also identified in Fuqu and Xiaoqu, respectively. This finding suggests that microbial community structure in JiuQu starters is the key factor to determine the variety of flavours. Copyright © 2015 The Institute of Brewing & Distilling     

Survival of amine-forming bacteria during the ice storage of fish and shrimp

Survival of amine-forming bacteria during the ice storage of fish and shrimp was investigated up to 14 days of storage. On iced storage the total bacterial load was reduced to one log from an initial load of 10⁵ cfu g⁻¹ in fresh fish/shrimp due to cold shock. The total incidence of biogenic amine-forming bacteria was found to be 74·63% in fish and the same was recorded as 56·05% in shrimp. The amine-forming bacteria recorded were cadaverine- and putrescine-forming bacteria in fish/shrimp, and no histamine former was detected. Gram-negative, non-fermentative rods, viz. Alcaligenes. Flavobacterium, Acinetobacter. Shewanella andPseudomonas , were the predominant amine-forming bacteria during the ice storage of fish and shrimp, in addition to the only Gram-positive genus Micrococcus. The genera Aeromonas and Photobacterium also survived ice storage to a certain extent and may also be responsible for the formation of amines in fish and shrimp.     

Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE

In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR–TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO₂/50% N₂) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium divergens, Lactococcus piscium, Lactobacillus fuchuensis, Vagococcus carniphilus, Leuconostoc gasicomitatum and Brochothrix thermosphacta. The PCR–TTGE profiles and band identification enabled a shift of the dominant populations during the storage to be visualised for all the batches, probably due to the temperature change and the packaging. At the beginning of storage, Pseudomonas sp. dominated the raw salmon microbiota while in the following days (7 and 10), P. phosphoreum and L. piscium were identified as the main bacterial groups. This study enhances the knowledge of MAP and vacuum-packed raw salmon spoilage microbiota.     

Microbial Flora and Storage Quality of Partially Processed Lettuce

Storage of partially processed lettuce resulted in an increase in microbial population, pH, and change in package atmosphere composition (increasing CO₂ and decreasing O₂). Microbial populations varied in size with variations in processing and packaging. Typical initial log₁₀ counts per g were: bacteria 3.4–5.1, yeasts 2.5–3.2; molds were infrequent. Commercial packaging inhibited bacterial growth and retarded browning. Although Gram-negative bacteria were numerically dominant, a large yeast population was also found. Species in the genera Pseudomonas, Erwinia, and Serratia were the most frequently isolated bacteria. Cryptococcus, Pichia, Torulaspora and Trichosporon spp. were the most common yeasts.     

Effect of washing with citric acid and antioxidants on the colour and microbiological quality of whole mushrooms (Agaricus bisporus L.)

The effect of washing with citric acid followed by anti‐browning treatment involving rinsing with sodium d ‐isoascorbate or sodium l ‐ascorbate on the colour, microbiological quality and bacterial blotch of whole mushrooms when stored at 5 °C for up to 13 days at 80% RH was evaluated. Washing with 1% citric acid reduced Pseudomonas counts avoiding bacterial blotch, but caused an important deterioration of colour. When washing with citric acid was followed by an anti‐browning treatment, mushrooms colour was improved, at the same time as bacterial blotch was reduced, although the decrease of Pseudomonas counts was smaller than in mushrooms washed only with citric acid. The most effective treatment was washing with 1% citric acid followed by anti‐browning treatment with 1.5% sodium l ‐ascorbate.     

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.     

Microbiological quality and safety of fresh cultivated and wild mushrooms commercialized in Spain

402 samples of 22 species of cultivated and wild fresh mushrooms sold in retail markets and supermarkets in Zaragoza (Spain) were studied to quantify their microbial load (mesophilic aerobic microorganisms, Pseudomonas genus, Enterobacteriaceae, lactic acid bacteria, total and thermotolerant coliform bacteria, Escherichia coli, yeasts and moulds) and to investigate the presence of E. coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Yersinia enterocolitica. The total microbial counts ranged from 4.4 to 9.4 log cfu/g, the genus Pseudomonas being the most prevalent with counts from 3.7 to 9.3 log cfu/g and Auricularia auricula-judae the species with the highest microbial load (9.4 log cfu/g). No significant differences (p > 0.05) were detected between mean counts of wild and cultivated species in all the microbial groups studied. The microbiological safety level of the cultivated mushrooms was excellent since no pathogens were isolated, and the microbial counts of indicator microorganisms were low, being detected in only half of the species. Salmonella spp, E. coli O157:H7 and S. aureus were not isolated from any sample, Y. enterocolitica was detected in only four samples of wild mushrooms whereas twenty-six (6.5%) were positive for L. monocytogenes, their occurrence being relatively high in Calocybe gambosa (40%), Hygrophorus limacinus (40%) and Tuber indicum (100%). These results suggest that a strategy to reduce bacterial populations, and to improve the microbiological safety of some species of fresh mushrooms, should be investigated.