Lactococcus lactis
subsp.
lactis
KT2W2L was used for biopreservation in cooked, peeled and ionized tropical shrimps (CPITS) during storage at 8 °C under modified atmosphere packaging (MAP). The ...neutralized and filtered cell-free supernatant produced by this strain exhibited a broad spectrum of inhibition against 23 strains from 24 indicator strains of food-spoilage bacteria and food-borne pathogens by agar well diffusion assay. The growth of
Brochothrix thermosphacta
CD274,
Carnobacterium maltaromaticum
CD263 and
Listeria innocua
CIP 80.11T was inhibited by
L. lactis
subsp.
lactis
KT2W2L when co-cultivated at 25 °C. However, only
L. innocua
CIP 80.11T was inhibited in a co-cultivation at 8 °C.
L. lactis
subsp.
lactis
KT2W2L was then used for biopreservation in CPITS. Growth of bacteria group inoculated in CPITS was monitored at regular intervals during storage period under MAP at 8 °C by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique.
L. lactis
subsp.
lactis
KT2W2L inhibited the growth of
B. thermosphacta
and
L. innocua
CIP 80.11T batches after 7 days of storage. However, it was inactive against
C. maltaromaticum
batch. The growth of
L. lactis
subsp.
lactis
KT2W2L alone or in the presence of indicators increased to reach about 8–9 log CFU/g within 7 days of storage and remained constant until the end of the experiment. In the batches without
L. lactis
subsp.
lactis
KT2W2L, all bacterial groups grew well on the cooked shrimp matrix, reaching their maximal levels after 2 weeks of storage. The pH of all homogenized suspensions of MAP-CPITS was quite stable at 6.0–6.7. The control batches were under the enumeration threshold (<1.70 ± 0.00 log CFU/g) until 14 days of storage, and then, an increase in growth was detected on agar plates. In addition, TTGE revealed to be an excellent tool to monitor the change of the microbial ecosystem in this product.
This study investigated the sensory quality and physicochemical evolution (pH, glucose,
l-lactic acid, biogenic amine, free amino-acids and volatile compounds) during storage at 8
°C of cooked peeled ...shrimp inoculated with the specific spoilage bacteria
Brochothrix thermosphacta alone or mixed with the protective strain
Lactococcus piscium CNCM I-4031. Growth of both bacteria was monitored at regular intervals during storage by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. Bacterial counts showed that
L.
piscium and
B.
thermosphacta inoculated at 7 log CFU/g and 3 log CFU/g were well adapted to shrimp, reaching a maximum level of 9 log CFU/g after 4
days and 10
days respectively. In mixed culture, the growth of
B.
thermosphacta was reduced by 3.2
±
0.1 log CFU/g. The TTGE technique allowed monitoring the colonisation of the strains on the shrimp matrix and confirming the dominance of
L.
piscium in mixed culture throughout the experiment. Sensory analysis confirmed that
B.
thermosphacta spoiled the product after 11
days, when its cell number attained 8 log CFU/g with the emission of strong butter/caramel off-odours. This sensory profile could be linked to the production of 2,3 butanedione, cyclopentanol, 3-methylbutanol, 3-methylbutanal, 2-methylbutanal, 4-methyl-3-chloro-3-pentanol and ethanol, which were produced in more significant quantities in the
B.
thermosphacta batch than in the batches in which the protective strain was present. On the contrary, TVBN and TMA were not suitable as quality indicators for
B.
thermosphacta spoilage activity. In the products where the protective
L.
piscium strain was present, no adverse effect on sensory quality was noted by the sensory panels. Moreover, biogenic amine assessment did not show any histamine or tyramine production by this strain, underlining its safety profile. Both strains produced lactic acid (1850
mg/kg in
L.
piscium and
B.
thermosphacta batch on days 3 and 10 respectively; 3830
mg/kg on day 7 in mixed culture) and the pH decrease from 6.6
±
0.0 to 5.9
±
0.1 was similar in all batches. Lactic acid production or competition for free amino-acid was not involved in the inhibition mechanism; however rapid glucose consumption by
L.
piscium could partially explain the growth limitation of the spoilage micro-organism. This study demonstrated the spoilage characteristic of
B.
thermosphacta and the usefulness of
L.
piscium as a bioprotective culture for tropical cooked peeled shrimp without any adverse effect on the sensory quality of the product.
►
Brochothrix thermosphacta spoiles cooked/peeled shrimp packed in modified atmosphere. ► Inoculating shrimp by
Lactococcus piscium inhibits growth of
B. thermosphacta. ► This leads to extension of the sensory shelf-life by more that 31 days. ► The inhibition is not due to bacteriocin, lactic acid or competition for amino acids. ►
L. piscium has no adverse effect on shrimp quality and presents safety character.
•Organic substrates (wood and coir fibers) are good substitutes for peat.•The microbial activity in substrates is not sufficient to predict protection.•Growth conditions, substrate type and the ...microbiome impact on the plant protection.
The development of sustainable crop protection is expected by vegetable producers and highly encouraged by authorities. For crops grown in soilless systems, vegetable fibers are relevant for both agronomical and plant protection purposes. This work examines their potential against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-cucumerinum.
Wood fiber, coir fiber and peat were tested over two cucumber cropping periods. Fusarium blight symptoms were monitored on cucumber, and fungal community structure (PCR-TTGE) in substrates. Substrate sterilization and bio-augmentation with antagonistic strains were also studied; they did not modify protection. Compared to the other substrates, wood fiber increased protection at the end of the first assay, but did not during the second assay. Differences in crop season and plant density may have impacted on cucumber physiology and may have indirectly modified rhizosphere fungal community structure.
The sole determination of microbial activity in substrates is not sufficient to predict protection. Growth conditions, substrate type and the microbiome altogether impacted on the protection of cucumber.
To describe a new molecular technique for the assessment of fungal diversity in the air. Air samples were collected every week in a henhouse in France during a 15-week period. After air sampling, the ...collecting membrane was diluted, and the liquid was used for subsequent cultivation and molecular analysis: PCR-temperature temporal gradient electrophoresis (TTGE), which has already been used for the identification of fungal species in air samples and PCR-denaturing high-performance liquid chromatography (D-HPLC), a new technique for the analysis of complex microbial populations. D-HPLC profiles were reproducible from run-to-run, and several fungal organisms could be identified at the species level by sequencing. PCR-D-HPLC enabled the identification of fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The new technique allowed the detection of more fungal species than did the PCR-TTGE technique. However, some fungal species were detected only by PCR-TTGE, suggesting that PCR-D-HPLC and PCR-TTGE are complementary. PCR-D-HPLC represents a considerable saving in time over currently available procedures for detection and identification of fungal organisms in air. However, the fungal diversity detected by PCR-D-HPLC or by PCR-TTGE was lower than that revealed by culture.
Cyanide is the basic component of many industrial processes, among which is gold processing, being very toxic or even lethal. Treatment, with the help of microorganisms, can be used effectively to ...reduce the load of harmful chemicals into the environment. The combination of microbiological methods and molecular tools allowed inferring the presence of a dominant population and the composition varied both in the places of origin and in the method used. The dominant phylogenetic affiliations of the bacteria were determined by sequencing the 16S rRNA gene. The isolates identified, as Bacillus and Enterococcus were capable to degrade 41.9 and 27.5 mg CN- L-1 respectively. This study provides information about the presence of a diverse bacterial community associated with residual effluents from cyanidation processes in Colombia and suggests that their presence could play a role in the biological degradation of cyanide compounds, offering an alternative for mining wastewater treatment.
The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the ...phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 °C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being
Carnobacterium (
C. divergens,
C. maltaromaticum and indiscernible
C. alterfunditum/pleistocenium),
Vagococcus (indiscernible
V. carniphilus/fluvialis) and
Enterococcus (
E. faecalis and
E. faecium). The other groups corresponded to
Brochothrix thermosphacta and
Enterobacteriaceae (
Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (
S. liquefaciens,
B. thermosphacta,
E. faecalis,
C. divergens and
C. maltaromaticum) or identified isolates from culture-dependent analysis (
E. faecium). Other additional informations were provided by fragment cloning (
Psychrobacter sp,
Citrobacter gillenii and
Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products.
The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied ...by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was
Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being
Lactobacillus paracasei, followed by considerably less quantities of
Lb. plantarum and
Lb. brevis. Other lactic-acid bacteria identified were
Pediococcus urinaequi,
Leuconostoc pseudomesenteroides,
Leuc.
mesenteroides,
Lactococcus lactis and even the enterococci
Enterococcus faecium and
E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to
Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to
Lc.
lactis,
Lb.
paracasei,
Lb.
plantarum,
Lb.
brevis,
Lb.
acidophilus and
Enterococcus sp. The non-lactic-acid bacterium detected was identified as
Escherichia coli. All the
Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene
efaAfm. On the basis of their antimicrobial activity against
Listeria monocytogenes we chose two strains of
Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.
In vitro and
in situ anti-listerial properties of 3 strains of Facultative Anaerobic Halophilic and Alkaliphilic (FAHA) species, i.e.
Alkalibacterium kapii ALK 6,
Marinilactibacillus psychrotolerans ...ALK 9 and
Facklamia tabacinasalis ALK 1, were investigated. The 3 strains were isolated from a smear ecosystem originating from a commercial Raclette type cheese and exhibiting strong anti-listerial activity
in situ on cheese surface.
In a first step, strains were tested
in vitro for production of antimicrobial compounds against
Listeria innocua 81000
-1 and
Listeria ivanovii HPB 28.
M. psychrotolerans ALK 9 inhibited both indicator strains in spot-on-the-lawn tests while
A. kapii ALK 6 showed no inhibiting effect.
F. tabacinasalis ALK 1 exerted an
in vitro inhibition on
L. ivanovii HPB 28, but induced the formation of dense ball-shaped microcolonies of
L. innocua 81000
-1 in the soft agar, a typical biofilm microstructure. The extent of the biofilm zone was enhanced when
F. tabacinasalis ALK 1 and
M. psychrotolerans ALK 9 were tested together.
In a second step, different combinations of strains were applied on Raclette cheeses ripened at pilot scale and contaminated with 50
cfu/cm
2
L. innocua at day 7. A control flora of 6 strains, isolated from ecosystem F and corresponding to species commonly found on smear cheeses, was applied on control and test cheeses. In test cheeses, we investigated the impact on
Listeria growth of the addition of the 3 FAHA strains, applied as single or mixed cultures. A 1-log inhibition was obtained at day 15 on cheeses treated with FAHA strains applied either as single or mixed cultures. This 1-log inhibition was correlated with the development of FAHA species that reached their maximal count at day 15.
This study suggests that the development of FAHA species in early ripening likely contributes to the initial part of the
in situ inhibition exerted by the complex cheese surface ecosystem investigated.
► Two of three FAHA species inhibited
Listeria in vitro. ► Growth on cheese surface of each FAHA species was correlated with
Listeria inhibition. ► Synergies between the FAHA species were observed
in vitro but not
in situ.
Unlike nuclear (n)DNA, of which there is one paired copy per cell, there are many copies of mitochondrial (mt)DNA per cell, making PCR amplification of mtDNA easier in samples of limited cellularity. ...The aims of this study were to (i) determine the mutation patterns of breast cancers through a comprehensive screen of mtDNA mutations, and (ii) assess if mutations in the cancers are also detectable in breast nipple aspirate fluid (NAF), a physiologic fluid which contains shed ductal epithelial cells. Fifteen breast cancers, matched benign tissues and NAF were collected. Nine overlapping primer sets were used to sequence the entire mitochondrial genome from tissue samples. For NAF samples, we focused on the 19 nucleotide positions (np) where mutations were found in a 3701 bp region (np 15331 to 2463), which includes the displacement (D)-loop, a mtDNA mutation hot spot. Fourteen of the fifteen (93%) cancer samples had ≥1 somatic mtDNA mutation for a total of 45 at 35 np (9 np reported previously, 26 new). Nine of fifteen tumors had ≥2 mutations. The D-loop contained 17 of 45 (38%) and non-D-loop (coding) regions contained 28 (62%) mutations. Of the 28 mutations in the coding loci, 11 led to an amino acid change. The frequency of mtDNA mutations was higher in the D-loop region (1.5 versus 0.18% of loci). 155 polymorphisms were identified (98 reported previously, 57 new). Sixteen of forty-five (36%) mutations were located at polymorphism sites. Four of nineteen mtDNA mutations in 10 cancers located between np 15331 and 2463 were found in matched NAF (two of eleven mutations in the D-loop and two of eight in non-D-loop regions). No mutations were found in five matched NAF samples from women whose cancers lacked a mutation in the same region. In conclusion, mtDNA mutations in breast cancer occur both within and outside of the D-loop, though the mutation rate in the D-loop is over 7-fold higher than in coding areas. We identified 26 new mutation loci (25 in regions sequenced by others, one in an area not). The high frequency of mtDNA mutations at polymorphic loci requires further investigation. Specific mtDNA mutations can be detected in a subset of NAF samples from women with breast cancer.
The aim of this work was to identify the bacterial biodiversity of traditional Zabady fermented milk using PCR-temporal temperature gel electrophoresis (PCR-TTGE) and PCR-denaturing gradient gel ...electrophoresis (PCR-DGGE). Most of the identified bacterial species in Zabady samples belonged to lactic acid bacteria (LAB), e.g.,
Streptococcus thermophilus,
Lactococcus garvieae,
Lactococcus raffinolactis,
Lactococcus lactis,
Leuconostoc citreum,
Lactobacillus delbrueckii subsp.
bulgaricus and
Lactobacillus johnsonii. Using the culture-dependent and independent methods, the streptococcal and lactococcal groups appeared to be the major bacterial species in Zabady fermented milk, whereas the lactobacilli were the minor ones. The main dominant species was
St. thermophilus followed by
Lc. garvieae. Other molecular tools, e.g., species-specific PCR assay and cloning and sequencing strategy were used to confirm the TTGE and DGGE results.
Lc. garvieae,
Lc. raffinolactis,
Ln. citreum, and
Lb. johnsonii were identified for the first time in this type of Egyptian fermented milk.
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