High stocking density alters growth performance, blood biochemical profiles, and hepatic antioxidative capacity in gibel carp (Carassius gibelio).

This study investigated the effect of stocking density on growth performance, blood biochemical profiles, antioxidative capacity, and muscle quality of gibel carp (Carassius gibelio). Gibel carps (initial body weight 57.04 ± 1.89 g) were reared at high stocking density (HSD, 10.85 kg m-3), medium stocking density (MSD, 5.06 kg m-3), and low stocking density (LSD, 1.47 kg m-3) for 60 days. The LSD group exhibited the highest growth rate, while HSD significantly inhibited fish growth. The muscular compositions of crude fat, crude ash, and moisture were significantly changed by stocking density, but crude protein content did not differ significantly. The stocking density altered the muscular texture profiles of gibel carp. Compared to either the HSD group or the MSD group, the highest levels of resilience and springiness occurred in the LSD group. Significant differences were observed in the levels of plasma glucose, alanine aminotransferase, aspartate aminotransferase, cholesterol, and creatinine among three groups. The fish exhibited the highest level of plasma cortisol as well as the lowest levels of triiodothyronine and thyroxine in the HSD group. The fish stocked in the LSD group showed the highest activities of superoxide dismutase, glutathione peroxidase, and catalase as well as the highest content of glutathione in liver. The significant highest total antioxidant capacity occurred in the fish stocked in the LSD group. The results showed that HSD resulted in chronic crowding stress, and exerted negative impact on growth performance, muscle quality, and antioxidative capacity of gibel carp.

Encapsulation of two fermentation agents, Lactobacillus sakei and calcium ions in microspheres.

Alginate microspheres loaded with two fermentation active agents, calcium cations and strain LS0296 identified as Lactobacillus sakei, have been prepared and characterized. The role of calcium cation is twofold, it acts as gelling cation and as fermentation active agent. Encapsulation and the presence of calcium ions in the same compartment do not inhibit the activity of LS0296. Molecular interactions in microspheres are complex, including mainly hydrogen bonds and electrostatic interactions. In vitro calcium cations and strain LS0296 release profiles were fitted to the Korsmeyer-Peppas empirical model. The calcium cation release process is driven at first by Fickian diffusion through microspheres and then by anomalous transport kinetics. The in vitro LS0296 release process is driven by Fickian diffusion through microspheres showing a much slower releasing rate than calcium cations. The release of LS0296 strain is followed by a decrease in the pH value. Results obtained give us a new insight into complex interactions between bacterial cultures and microsphere constituents. Prepared formulations of calcium alginate microspheres loaded with LS0296 could be used as a new promising tool and a model for different starter cultures encapsulation and use in the production of fermented foods.

Characterization of macrolide resistance in bacteria isolated from macrolide-polluted and unpolluted river sediments and clinical sources in Croatia.

Environments polluted with excessively high levels of antibiotics released from manufacturing sites can act as a source of transferable antibiotic resistance (AR) genes to human commensal and pathogenic bacteria. The aim of this study was to evaluate AR of bacteria isolated from the Sava river sediments (Croatia) at the discharge site of effluents from azithromycin production compared to those from the upstream site and isolates collected in Croatian hospitals. A total of 228 environmental strains of azithromycin-resistant bacteria were isolated and identified, with 124 from the discharge site and 104 from the upstream site. In addition, a total of 90 clinical, azithromycin-resistant streptococcal and staphylococcal isolates obtained from the Croatian Reference Center for Antibiotic Resistance Surveillance were analyzed. PCR screening of isolates on 11 relevant macrolide-resistance genes (MRGs) showed that discharge isolates had greater detection frequencies for 4 gene targets (ermB, msrE, mphE and ermF) compared to upstream isolates. Among clinical isolates, the most frequently detected gene was ermB, followed by msrD, mefE and mefC. The discharge site demonstrated a greater abundance of isolates with co-occurrence of two different MRGs (predominantly msrE-mphE) than the upstream site, but a lower abundance than the clinical sources (most commonly msrD-mefE). The simultaneous presence of three or even four MRGs was specific for the discharge and clinical isolates, but not for the upstream isolates. When MRG results were sorted by gene mechanism, the ribosomal methylation (erm) and protection genes (msr) were the most frequently detected among both the discharge and the clinical isolates. Following sequencing, high nucleotide sequence similarity was observed between ermB in the discharge isolates and the clinical streptococcal isolates, suggesting a possible transfer of the ermB gene between bacteria of clinical and environmental origin. Our study highlights the importance of environmental bacterial populations as reservoirs for clinically relevant macrolide-resistance genes.

Sugar transporter genes in grass carp (Ctenopharyngodon idellus): molecular cloning, characterization, and expression in response to different stocking densities.

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m-2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.

The Influence of Meat Batter Composition and SausageDiameter on Microbiota and Sensory Traits of ArtisanalWild Boar Meat Sausages.

In this study, the influence of meat batter composition and sausage diameter on the development of microbiota and sensory traits of traditional, spontaneously fermented wild boar meat sausages are evaluated. This research also demonstrates how principal component analysis (PCA) can be used to relate product sensory properties to particular microbial genotype and to select potential starter or adjunct culture. Generally, similar microbiological results were obtained in all types of products. The undesirable microbiota was either not detected at any sausage production stage or its number decreased below the detection limit in ripened sausages. The low growth rate of lactic acid bacteria (LAB) was consistent with the obtained pH and slow acidification rate. Although no differences in the composition of LAB species were noticed between sausage types (50S=50% wild boar meat in small casing, 50L=50% wild boar meat in large casing, 100S=100% wild boar meat in small casing), a clear separation based on LAB genotypes could be observed. Upon quantitative descriptive analysis, significant differences in sensory attributes between sausage types were established. According to the PCA, the overall acceptability traits of sausages are closely linked to one Leuconostoc mesenteroides genotype (LM_4). Of all tested technological properties, LM_4 strains showed remarkable acidification ability, lowering the pH from pH=5.41 to 3.74, and pronounced proteolytic activity on skimmed milk as well as antagonistic activity against Staphylococcus aureus (DSM 20231) and Brochothrix thermosphacta (LMG 17208). Lipolytic and haemolytic activities were not detected, and all analyzed strains were susceptible to tested antibiotics and possessed no biogenic amine genes.

N2 Gas Flushing Alleviates the Loss of Bacterial Diversity and Inhibits Psychrotrophic Pseudomonas during the Cold Storage of Bovine Raw Milk.

The quality and safety of raw milk still remains a worldwide challenge. Culture-dependent methods indicated that the continuous N2 gas-flushing of raw milk reduced the bacterial growth during cold storage by up to four orders of magnitude, compared to cold storage alone. This study investigated the influence of N2 gas-flushing on bacterial diversity in bovine raw-milk samples, that were either cold stored at 6°C or additionally flushed with pure N2 for up to one week. Next-generation sequencing (NGS) of the V1-V2 hypervariable regions of 16S rRNA genes, derived from amplified cDNA, which was obtained from RNA directly isolated from raw-milk samples, was performed. The reads, which were clustered into 2448 operational taxonomic units (OTUs), were phylogenetically classified. Our data revealed a drastic reduction in the diversity of OTUs in raw milk during cold storage at 6°C at 97% similarity level; but, the N2-flushing treatment alleviated this reduction and substantially limited the loss of bacterial diversity during the same cold-storage period. Compared to cold-stored milk, the initial raw-milk samples contained less Proteobacteria (mainly Pseudomonadaceae, Moraxellaceae and Enterobacteriaceae) but more Firmicutes (mainly Ruminococcaceaea, Lachnospiraceae and Oscillospiraceaea) and Bacteroidetes (mainly Bacteroidales). Significant differences between cold-stored and additionally N2-flushed milk were mainly related to higher levels of Pseudomononadaceae (including the genera Pseudomonas and Acinetobacter) in cold-stored milk samples; furthermore, rare taxa were better preserved by the N2 gas flushing compared to the cold storage alone. No major changes in bacterial composition with time were found regarding the distribution of the major 9 OTUs, that dominated the Pseudomonas genus in N2-flushed or non-flushed milk samples, other than an intriguing predominance of bacteria related to P. veronii. Overall, this study established that neither bacteria causing milk spoilage nor any well-known human pathogen or anaerobe benefited from the N2 gas flushing even though the N2-flushed and non-flushed cold-stored milk differed in bacterial counts by up to 104-fold.

Dynamics of bacterial communities during the ripening process of different Croatian cheese types derived from raw ewe's milk cheeses.

Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe's milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU97 could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening.

Bacterial communities associated with the production of artisanal Istrian cheese.

In this work we report on the main bacterial microflora typical for fermentation and ripening of traditional Istrian cheese. Samples from milk as well as Istrian cheese were analyzed during the ripening process by using culture independent molecular fingerprinting methods as well as culture based approaches. Our results indicate changes in bacterial diversity pattern during the ripening process. Differences in bacterial diversity at the same ripening stage among different farms investigated were comparably low. Sequence analysis of the most prominent bands of denaturing gradient gel electrophoresis fingerprints revealed dominance of Lactococcus lactis subs. lactis in all samples and a strong presence of Enterococcus spp. which was also confirmed by plate count analysis.

Influence of temperature and soil water content on bacterial, archaeal and denitrifying microbial communities in drained fen grassland soil microcosms.

In this study, microcosms were used to investigate the influence of temperature (4 and 28 degrees C) and water content (45% and 90% WHC) on microbial communities and activities in carbon-rich fen soil. Bacterial, archaeal and denitrifier community composition was assessed during incubation of microcosms for 12 weeks using terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA and nitrous oxide reductase (nosZ) genes. In addition, microbial and denitrifier abundance, potential denitrification activity and production of greenhouse gases were measured. No detectable changes were observed in prokaryote or denitrifier abundance. In general, cumulatively after 12 weeks more carbon was respired at the higher temperature (3.7 mg CO(2) g(-1) soil), irrespective of the water content, whereas nitrous oxide production was greater under wet conditions (98-336 microg N(2)O g(-1) soil). After an initial lag phase, methane emissions (963 microg CH(4) g(-1) soil) were observed only under warm and wet conditions. T-RFLP analyses of bacterial 16S rRNA and nosZ genes revealed small or undetectable community changes in response to temperature and water content, suggesting that bacterial and denitrifying microbial communities are stable and do not respond significantly to seasonal changes in soil conditions. In contrast, archaeal microbial community structure was more dynamic and was strongly influenced by temperature.

Quantification of functional genes from procaryotes in soil by PCR.

Controlling turnover processes and fluxes in soils and other environments requires information about the gene pool and possibilities for its in situ induction. Therefore in the recent years there has been a growing interest in genes and transcripts coding for metabolic enzymes. Besides questions addressing redundancy and diversity, more and more attention is given on the abundance of specific DNA and mRNA in the different habitats. This review will describe several PCR techniques that are suitable for quantification of functional genes and transcripts such as MPN-PCR, competitive PCR and real-time PCR. The advantages and disadvantages of the mentioned methods are discussed. In addition, the problems of quantitative extraction of nucleic acid and substances that inhibit polymerase are described. Finally, some examples from recent papers are given to demonstrate the applicability and usefulness of the different approaches.