Improving the shelf life of minced beef by Cystoseira compressa polysaccharide during storage.

A polysaccharide extracted from the brown alga Cystoseira compressa (CCPS) was evaluated as a food additive to extend the shelf-life of raw beef meat. The antioxidant potential of CCPS was demonstrated by its inhibition of ß-carotene bleaching (64.28 %), superoxide radicals (70.12 %), and hydroxyl radicals (93 %) at a concentration of 10 mg/ml. The polysaccharide also showed antibacterial activity with MIC values between 6.25 mg/ml and 50 mg/ml against five foodborne pathogenic bacteria. Furthermore, CCPS exhibited excellent functional, foaming, and emulsifying properties. Furthermore, microbiological and chemical effects of CCPS at concentrations equivalent to 1 MIC (CCPS-1), 2 MIC (CCPS-2), and 4 MIC (CCPS-3) were conducted. Chemical analyses showed that treated beef had significantly reduced TBARS levels below 2 mg MDA/kg at day 14. The treatment also decreased carbonyl groups, improved heme iron transformation, inhibited microbial growth (p < 0.05), and kept MetMb levels below 40 % by day 14. Moreover, two multivariate approaches, principal component analysis (PCA) and hierarchical cluster analysis (HCA), were effectively used to analyze the results characterizing the main attributes of the stored meat samples. In conclusion, these findings demonstrated that CCPS could be employed as a functional and bioactive component in the meat industry.

Development of a duplex q-PCR for the simultaneous detection of Parachlamydia acanthamoebae and Simkania negevensis in environmental and clinical samples.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

A new TaqMan real-time PCR assay to detect Parachlamydia acanthamoebae and to monitor its co-existence with SARS-COV-2 among COVID-19 patients.

Human respiratory infections caused by a large variety of microbial pathogens are the most common diseases responsible for hospitalization, morbidity and mortality. Parachlamydia acanthamoebae, a Chlamydia-related bacterium, has been found to be potentially associated with these diseases. An early and accurate diagnosis of this pathogen could be useful to avoid the potential respiratory complications linked especially to COVID-19 patients and to set suitable outbreak control measures. A TaqMan-PCR assay was developed to detect and quantify Parachlamydia acanthamoebae in environmental and clinical samples from patients of all ages with COVID-19. The selected hydrolysis probe displayed no cross-reaction with the closely related Chlamydia or the other tested pathogens. This q-PCR achieved good reproducibility and repeatability with a detection limit of about 5 DNA copies per reaction. Using this q-PCR assay, Parachlamydia acanthamoebae was detected in 2/78 respiratory specimens and 9/47 water samples. Only one case (1.3%) of Parachlamydia acanthamoebae and SARS-COV-2 co-infection was noticed. To our knowledge, the combination of these two respiratory pathogens has not been described yet. This new TaqMan-PCR assay represents an efficient diagnostic tool to survey Parachlamydia acanthamoebae on a large-scale screening programs and also during outbreaks.

Microalgae as feedstock for bioactive polysaccharides.

Due to the increase in industrial demand for new biosourced molecules (notably bioactive exopolysaccharides (EPS)), microalgae are gaining popularity because of their nutraceutical potential and benefits health. Such health effects are delivered by specific secondary metabolites, e.g., pigments, exopolysaccharides, polyunsaturated fatty acids, proteins, and glycolipids. These are suitable for the subsequent uses in cosmetic, nutraceutical, pharmaceutical, biofuels, biological waste treatment, animal feed and food fields. In this regard, a special focus has been given in this review to describe the various methods used for extraction and purification of polysaccharides. The second part of the review provides an up-to-date and comprehensive summary of parameters affecting the microalgae growth and insights to maximize the metabolic output by understanding the intricacies of algal development and polysaccharides production. In the ultimate part, the health and nutraceutical claims associated with marine algal bioactive polysaccharides, explaining their noticeable potential for biotechnological applications, are summarized and comprehensively discussed.

Structural Characterization and Rheological and Antioxidant Properties of Novel Polysaccharide from Calcareous Red Seaweed.

A novel sulfated xylogalactan (JASX) was extracted and purified from the rhodophyceae Jania adhaerens. JASX was characterized by chromatography (GC/MS-EI and SEC/MALLS) and spectroscopy (ATR-FTIR and 1H/13C NMR) techniques. Results showed that JASX was constituted by repeating units of (→3)-ß-d-Galp-(1,4)-3,6-α-l-AnGalp-(1→)n and (→3)-ß-d-Galp-(1,4)-α-l-Galp-(1→)n substituted on O-2 and O-3 of the α-(1,4)-l-Galp units by methoxy and/or sulfate groups but also on O-6 of the ß-(1,3)-d-Galp mainly by ß-xylosyl side chains and less by methoxy and/or sulfate groups. The Mw, Mn, D, [η] and C* of JASX were respectively 600 and 160 kDa, 3.7, 102 mL.g-1 and 7.0 g.L-1. JASX exhibited pseudoplastic behavior influenced by temperature and monovalent salts and highly correlated to the power-law model and the Arrhenius relationship. JASX presented thixotropic characteristics, a gel-like viscoelastic behavior and a great viscoelasticity character. JASX showed important antioxidant activities, outlining its potential as a natural additive to produce functional foods.

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

Development of a novel TaqMan qPCR assay for rapid detection and quantification of Gymnodinium catenatum for application to harmful algal bloom monitoring in coastal areas of Tunisia.

Gymnodinium catenatum is a dinoflagellate known to cause paralytic shellfish poisoning (PSP), commonly associated with human muscular paralysis, neurological symptoms, and, in extreme cases, death. In the present work, we developed a real-time PCR-based assay for the rapid detection of the toxic microalgal species, G. catenatum, in environmental bivalve mollusc samples as well as seawater samples. G. catenatum-specific primers and probe were designed on the ITS1-5.8S-ITS2 rDNA region. Hydrolysis probe qPCR assay was optimized. ITS1-5.8S-ITS2 rDNA region copy numbers per G. catenatum cell genome were estimated to be 122.73 ± 5.54 copies/cell, allowing cell quantification. The application of the optimized qPCR assay for G. catenatum detection and quantification in field samples has been conducted, revealing high sensitivity (detection of around 1.3105 cells/L of seawater samples. Thus, the designed hydrolysis probe qPCR assay could be considered an efficient tool for phytoplankton monitoring whilst ensuring accuracy and sensitivity and providing cost and time savings.

Microalgae Xanthophylls: From Biosynthesis Pathway and Production Techniques to Encapsulation Development.

In the last 20 years, xanthophylls from microalgae have gained increased scientific and industrial interests. This review highlights the essential issues that concern this class of high value compounds. Firstly, their chemical diversity as the producer microorganisms was detailed. Then, the use of conventional and innovative extraction techniques was discussed. Upgraded knowledge on the biosynthetic pathway of the main xanthophylls produced by photosynthetic microorganisms was reviewed in depth, providing new insightful ideas, clarifying the function of these active biomolecules. In addition, the recent advances in encapsulation techniques of astaxanthin and fucoxanthin, such as spray and freeze drying, gelation, emulsification and coacervation were updated. Providing information about these topics and their applications and advances could be a help to students and young researchers who are interested in chemical and metabolic engineering, chemistry and natural products communities to approach the complex thematic of xanthophylls.

Sulfated exopolysaccharides from Porphyridium cruentum: A useful strategy to extend the shelf life of minced beef meat.

Sulfated exopolysaccharides (EPS) from Porphyridium cruentum strain were extracted and their antioxidant and anti-bacterial potentials were evaluated based on DPPH free radical, ABTS•+ radical cation and DNA nicking assays, and against four foodborne pathogenic bacteria, respectively. They showed also interesting functional, foaming and emulsion properties. Moreover, microbiological and chemical effects of EPS at 0.5, 1 and 2% on refrigerated minced beef meat were undertaken. Chemical analyses revealed that the treated meat underwent significant decrease (P < 0.05) of primary and secondary lipid oxidation. By the end of the storage period, exopolysaccharides at 2% reduced the metmyoglobin and carbonyl group accumulation compared to control samples and were more efficient (P < 0.05) against microflora proliferation. Furthermore, two multivariate exploratory techniques namely Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were applied successfully to all obtained data describing the main characteristics attributed to refrigerated meat samples. Overall, these findings indicated that EPS from P. cruentum are worthy being developed as functional and bioactive components for the meat industry.

q-PCR-based assay for the toxic dinoflagellate Karenia selliformis monitoring along the Tunisian coasts.

Karenia selliformis is a marine dinoflagellate responsible for fish-kill events. Its presence has been reported along the Tunisian coasts (south-eastern Mediterranean Sea) since the 1990s. In the present study, a quantitative-PCR assay, based on the internal transcribed spacer (ITS) molecular marker, was developed to detect and quantify K. selliformis in environmental bivalve mollusk samples and in seawater samples. The assay was optimized, and its specificity was confirmed using cross-reactivity experiments against microalgal species commonly found on the Tunisian coasts and/or closely related to K. selliformis. Calibration curves were performed by tenfold dilutions of plasmid DNA harboring target sequence and genomic DNA, attaining a limit of detection of around 5 copies of target DNA per reaction, far below one K. selliformis cell per reaction. The field application of the developed assay showed a powerful detection capability. Thus, the designed assay could contribute to the deployment of in-field diagnostic tools for K. selliformis blooms monitoring.

Development and application of a real-time PCR assay for the sensitive detection of diarrheic toxin producer Prorocentrum lima.

Prorocentrum lima (P. lima) is a widely spread dinoflagellate in the Mediterranean Sea and it has become increasingly involved in harmful algal blooms. The purpose of this study is to develop a probe-based real-time polymerase chain reaction (PCR) targeting the ITS1-5.8S-ITS2 region for the detection and absolute quantification of P. lima based on linear and circular DNA standards. The results have shown that the quantitative PCR (q-PCR), using circular plasmid as a template, gave a threshold cycle number 1.79-5.6 greater than equimolar linear standards. When microalgae, commonly found in aquatic samples were tested, no cross-amplification was observed. The q-PCR brought about a good intra and inter-run reproducibility and a detection limit of 5 copies of linear plasmid per reaction. A quantitative relationship between the cell numbers and their corresponding plasmid copy numbers was attained. Afterwards, the effectiveness of the developed protocol was tested with 130 aquatic samples taken from 19 Tunisian sampling sites. The developed q-PCR had a detection sensitivity of up to 1 cell. All the positive samples were taken from three sampling sites of Medenine Governorate with cell abundances that ranged from 22 to 156,000 cells L-1 of seawater. The q-PCR assay revealed a high sensitivity in monitoring the aquatic samples in which the low concentrations of P. lima were not accurately detected by light microscopy. Indeed, this approach is at the same time rapid, specific and sensitive than the traditional microscopy techniques and it represents a great potential for the monitoring of P. lima blooms.

Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Quantitative PCR assay for the simultaneous identification and enumeration of multiple Karenia species.

Quantitative PCR (qPCR) is the method of choice for specific detection and quantification of harmful algal bloom (HAB) species. Development of qPCR assay for simultaneous enumeration of species that frequently co-exist in HABs is required. A high sensitivity TaqMan qPCR assay, using probe and primers, located at ITS1-5.8S-ITS2 rDNA region, detecting, specifically, Karenia selliformis, K. bidigitata, and K. mikimotoi, was designed. ITS1-5.8S-ITS2 rDNA region copy numbers per Karenia cell genome were estimated to 217.697 ± 67.904, allowing cell quantification. An application of the designed methodology in field samples has been conducted, and it showed high sensitivity (detection of around 10-1 cell/100 mg of bivalve mollusk tissue, equivalent to about 20 copies of the target sequence). We suggest that the optimized method could contribute to early detection of three closely related Karenia species in seafood cultivating areas to promote control quality, guarantee a fast and effective intervention, and improve public health prevention.

Protective Role of Spirulina platensis Against Bifenthrin-Induced Reprotoxicity in Adult Male Mice by Reversing Expression of Altered Histological, Biochemical, and Molecular Markers Including MicroRNAs.

: The potential reprotoxicity of bifenthrin remains unclear if only the common clinical indicators of reproductive disease are examined. The present study aimed to investigate the efficacy of Spirulina platensis, a microalga rich in antioxidant compounds, against bifenthrin-induced testicular oxidative damage in male mice. At the first, we demonstrate that administration of bifenthrin resulted in a decline of testosterone level and in deterioration of sperm quality that was correlated with significant transcription changes of some specific mRNA and microRNA involved in cholesterol transport, testosterone synthesis, and spermatogenesis. At the biochemical level, we found that oxidative stress was obvious in the bifenthrin group, as evidenced by increase in malondialdehyde (MDA), protein carbonyls (PCO), reactive oxygen species (ROS), and nitrite oxide (NO) that was correlated with activation of genes related to mitochondrial apoptotic signal pathways. We then brought, for the first time to our knowledge, solid and complete experimental evidences that administration of mice with Spirulina extract was sufficient to protect against deleterious effects BF in testicular tissues by abrogating the change in antioxidant enzyme activities; the increase in MDA, PCO, and NO concentrations; and the altered expression level of miRNA and mRNA involved in spermatogenesis. We finally demonstrate that Spirulina restores the production of testosterone in mice as well as epididymal sperm viability and motility. These results suggest a potential antitoxic activity of Tunisian Spirulina deserving further attention.

Deciphering the Biological Activities of Dunaliella sp. Aqueous Extract from Stressed Conditions on Breast Cancer: from in Vitro to in Vivo Investigations.

In order to harness local resources to improve well-being and human health, we aim in this study to investigate if the microalgae Dunaliella sp. isolated from the Tunisian coastal zone possesses any anticancer activity. Dunaliella sp. was cultured under normal (DSC) or stressed (DSS) conditions and extracted using different procedures. The biological activity assessment was performed on the Triple Negative Breast Cancer (TNBC) using 4T1 murine cells as a model. Results indicate that: (i) aqueous extract was the most cytotoxic compared to ethanolic and hydroalcoholic extracts; (ii) DSS activity was superior to that of DSC. DSS extracts induced apoptosis rather than necrosis, as evidenced by DNA fragmentation, PARP-1 cleavage and caspase-3 activation. Evaluation in an orthotopic TNBC model validated the anticancer activity in vivo. Intratumoral injection of DSS extract resulted in reduced tumor growth and an enhanced immune system activation. On the transcriptional side, the expression level of the immunosuppressive enzyme Arg-1 was decreased, as well as those of NOS-2 and COX-2 genes. These results suggest a potential anticancer activity of Tunisian Dunaliella sp. deserving further attention.

Quality Characteristics and Functional and Antioxidant Capacities of Algae-Fortified Fish Burgers Prepared from Common Barbel (Barbus barbus).

INTRODUCTION: Algae have been used as natural ingredients to produce new canned fish burgers prepared from minced flesh of common barbel. In this research, the impact of the addition of Cystoseira compressa and Jania adhaerens at concentrations of 0.5, 1, and 1.5% w/v on the texture and sensory characteristics of fish burgers were investigated. RESULTS: Compared to controls, fish burgers containing 1% algae had better texture and sensory properties (P < 0.05). Also, these burger formulations had higher water and oil holding capacities as well as swelling ability, due to the important polysaccharides and dietary fibers contents of algae. In addition, algae-supplemented burgers were characterized as having low L⁎, a⁎, and b⁎ values, which made the color appear to be paler. Thanks to their high richness in pigments (chlorophylls and carotenoids) and polysaccharides, algae considerably enhance the antioxidant activities of the new ready-to-eat fish burgers. So, Cystoseira compressa and Jania adhaerens could be used as nutritious additives to produce new fish-based products.

Serological and molecular evidence of coxiellosis and risk factors in sheep flocks in central-eastern Tunisia.

In this study, we conducted an investigation to determine the true prevalence of coxiellosis in sheep in central-eastern Tunisia. A total of 492 veterinary samples taken from 110 flocks were screened for coxiellosis using IS1111-based real-time PCR assay. Sheep sera were tested using an indirect enzyme-linked immunosorbent assay. Based on molecular and serological results, the true adjusted animal and herd-level prevalence of coxiellosis were 11.8% and 20.21%, respectively. Bacterial excretion was observed in 17 flocks, and 19 females showed evidence of Coxiella burnetii shedding (100%). In addition, a statistically significant association was found between vaginal and milk shedding for sheep. Multivariable logistic regression analysis at the animal-population level indicated that strata and vaccination variables were found to be associated with coxiellosis. Besides, it was shown that this infection increased when the intensive farm was exposed to carnivores and when the cleaning practices were not respected, while it decreased when a suitable quarantine was introduced for any introduction of a new animal. Good hygiene and sanitation practices on-farm should be handled as strategies to deal with this zoonotic pathogen in herds.

Optimization of lipids' ultrasonic extraction and production from Chlorella sp. using response-surface methodology.

BACKGROUND: Three steps are very important in order to produce microalgal lipids: (1) controlling microalgae cultivation via experimental and modeling investigations, (2) optimizing culture conditions to maximize lipids production and to determine the fatty acid profile the most appropriate for biodiesel synthesis, and (3) optimizing the extraction of the lipids accumulated in the microalgal cells. METHODS: Firstly, three kinetics models, namely logistic, logistic-with-lag and modified Gompertz, were tested to fit the experimental kinetics of the Chlorella sp. microalga culture established on standard conditions. Secondly, the response-surface methodology was used for two optimizations in this study. The first optimization was established for lipids production from Chlorella sp. culture under different culture conditions. In fact, different levels of nitrate concentrations, salinities and light intensities were applied to the culture medium in order to study their influences on lipids production and determine their fatty acid profile. The second optimization was concerned with the lipids extraction factors: ultrasonic's time and temperature, and chloroform-methanol solvent ratio. RESULTS: All models (logistic, logistic-with-lag and modified Gompertz) applied for the experimental kinetics of Chlorella sp. show a very interesting fitting quality. The logistic model was chosen to describe the Chlorella sp. kinetics, since it yielded the most important statistical criteria: coefficient of determination of the order of 94.36%; adjusted coefficient of determination equal to 93.79% and root mean square error reaching 3.685 cells · ml- 1. Nitrate concentration and the two interactions involving the light intensity (Nitrate concentration × light intensity, and salinities × light intensity) showed a very significant influence on lipids production in the first optimization (p < 0.05). Yet, only the quadratic term of chloroform-methanol solvent ratio showed a significant influence on lipids extraction relative to the second step of optimization (p < 0.05). The two most abundant fatty acid methyl esters (≈72%) derived from the Chlorella sp. microalga cultured in the determined optimal conditions are: palmitic acid (C16:0) and oleic acid (C18:1) with the corresponding yields of 51.69% and 20.55% of total fatty acids, respectively. CONCLUSIONS: Only the nitrate deficiency and the high intensity of light can influence the microalgal lipids production. The corresponding fatty acid methyl esters composition is very suitable for biodiesel production. Lipids extraction is efficient only over long periods of time when using a solvent with a 2/1 chloroform/methanol ratio.

Modelling Tetraselmis sp. growth-kinetics and optimizing bioactive-compound production through environmental conditions.

The aim of this study is to predict Tetraselmis cells growth-kinetic and to induce the synthesis of bioactive compounds (chlorophylls, carotenoids and starch) with high potential for biotechnological applications. Using the statistical criteria, the Baranyi-Roberts model has been selected to estimate the microalgae growth-kinetic values. The simultaneous effects of salinity, light intensity and pH of culture medium were investigated to maximize the production of total chlorophylls, carotenoids and starch. The optimal culture conditions for the production of these compounds were found using Box-Behnken Design. Results have shown that total chlorophyll and carotenoids were attained 21.6mg·g-1DW and 0.042mg·g-1DW, respectively. In addition, the highest starch content of 0.624g·g-1DW has been obtained at neutral pH with high irradiance (182µmolphotonsm-2 s-1) and low salinity (20). A highly correlation (R2 = 0.884) has been found between the gravimetric and flow cytometric measurements of chlorophyll content.

Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production.

The green microalgae Dunaliella genus is known for the production of high added value molecules. In this study, strain AL-1 was isolated from the Sebkha of Sidi El Hani (Sousse, Tunisia). This isolate was identified both morphologically and genetically via 18S rRNA gene sequence as a member of the genus Dunaliella. Strain AL-1 was found to be closely related to Dunaliella salina, Dunaliella quartolecta and Dunaliella polymorpha with more than 97% similarity. Response surface methodology was used to maximize carotenoid production by strain AL-1 by optimizing its growth conditions. The highest carotenoid content was obtained at salinity: 51, light intensity: 189.89 µmol photons·m-2·s-1, and nitrogen: 60 mg·L-1. Proteomic profiling, using two-dimensional gel electrophoresis, was performed from standard and optimized cultures. We detected 127 protein spots which were significantly differentially expressed between standard and optimized cultures. Among them 16 protein spots were identified with mass spectrometry and grouped into different functional categories using KEGG (Kyoto Encyclopedia of Genes and Genomes) such as photosynthetic Calvin cycle, regulation/defense, energy metabolism, glycolysis, and cellular processes. The current study could be of great interest in providing information on the effect of stressful conditions in microalgae carotenoid production.