Chemical Profiling and Biological Activity of Extracts from Nine Norwegian Medicinal and Aromatic Plants.

There is an increased interest in identifying beneficial compounds of plant origin that can be added to animal diets to improve animal performance and have a health-promoting effect. In the present study, nine herb species of the Norwegian wild flora or which can be cultivated in Norway were selected for phytogenic evaluation (hops, maral root, mint, oregano, purslane, rosemary, roseroot, sweet wormwood, yarrow). Dried herbs were sequentially extracted with dichloromethane (DCM), ethanol (EtOH) and finally water (H2O) by ultrasound-assisted extraction (UAE). The UAE protocol was found to be more rational than conventional Soxhlet with respect to DCM extraction. Total extraction yield was found to be highest for oregano (Origanum vulgare) with 34.4 g 100-1 g dry matter (DM). H2O-extracts gave the highest yields of the three solvents, with up to 25 g 100-1 g DM for purslane (Portulaca oleracea ssp. sativa) and mint (Mentha piperita). EtOH- and H2O-extracts were the most efficient extracts with respect to free radical scavenging capacity (ABTS (=2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and oregano, mint, hops (Humulus lupulus) and maral root-leaves (Leuzea carthamoides) were found to be the most efficient antioxidant sources. Hops (EtOH-extract) contained α- and ß-acids, xanthohumols, chlorogenic acid and the hitherto unreported 3-O-glucosides of kaempferol and quercetin. Maral root-leaves contained among other compounds hexosides of the 6-hydroxy- and 6-methoxy-kaempferol and -quercetin, whereas roseroot (Rosea rhodiola) revealed contents of rosavin, rhodiosin and rhodionin. Sweet wormwood (Artemisia annua) contained chlorogenic acid and several derivatives thereof, scopoletin and poly-methylated flavones (eupatin, casticin, chrysoplenetin). Antimicrobial potential of different plant extracts was demonstrated against Gram-positive and Gram-negative bacteria using the indicator organisms Staphylococcus aureus, and Escherichia coli, and the Atlantic salmon bacterial pathogens Moritella viscosa, Tenacibaculum finnmarkense and Aliivibrio wodanis. DCM extracts possessed the highest activities. Data demonstrate the potential ability of herb extracts as natural antimicrobials. However, future safety studies should be performed to elucidate any compromising effect on fish health.

Effect of Citric Acid Cross Linking on the Mechanical, Rheological and Barrier Properties of Chitosan.

In this study, acetic acid (AA-2% w/v), a combination of acetic acid and citric acid (AA-1% w/v + CA-1% w/w), and three different concentrations of citric acid (CA-2, 4 and 6% w/w) were used to create chitosan solution. The FTIR analysis showed the presence of residual CA in all the CA-containing samples where no trace of AA was observed. The tensile strengths of the CA-containing samples were lower than the AA samples. Whereas the values for the elongation at break of the CA samples were higher than the AA samples, which kept increasing with an increasing CA content due to the plasticizing effect from residual citric acid. The elongation at break values for 4 and 6% CA-containing samples were 98% higher than the AA samples. The samples prepared with CA showed shorter LVE regions that reduced with an increasing CA concentration compared to the AA samples. Different acid concentrations did not have a large effect on the gelation time. However, CA-containing samples showed higher viscosities as compared to the AA-containing solution, which increased with an increasing CA content. The water vapour transmission rates of the CA-containing samples were lower than the others. All the chitosan solutions suppressed the growth of the two test strains, and none of the variants reached an abs 600 nm at 0.2.

Microbiological Food Safety of Seaweeds.

The use of seaweeds in the human diet has a long history in Asia and has now been increasing also in the western world. Concurrent with this trend, there is a corresponding increase in cultivation and harvesting for commercial production. Edible seaweed is a heterogenous product category including species within the green, red, and brown macroalgae. Moreover, the species are utilized on their own or in combinatorial food products, eaten fresh or processed by a variety of technologies. The present review summarizes available literature with respect to microbiological food safety and quality of seaweed food products, including processing and other factors controlling these parameters, and emerging trends to improve on the safety, utilization, quality, and storability of seaweeds. The over- or misuse of antimicrobials and the concurrent development of antimicrobial resistance (AMR) in bacteria is a current worldwide health concern. The role of seaweeds in the development of AMR and the spread of antimicrobial resistance genes is an underexplored field of research and is discussed in that context. Legislation and guidelines relevant to edible seaweed are also discussed.

PLA-Based Materials Containing Bio-Plasticizers and Chitosan Modified with Rosehip Seed Oil for Ecological Packaging.

Several recipes based on PLA, bio-plasticizers, and active agents such as vitamin E and cold-pressed rosehip seed oil encapsulated into chitosan by the emulsion method named here as chitosan modified (CS-M) were elaborated by melt compounding for food packaging applications. Resulted biocomposites have been investigated from the point of view of physical-mechanical, thermal, barrier, antimicrobial, and antioxidant properties to select the formulations with the optimum features to produce food trays and films for packaging applications. The obtained results showed that the elaborated formulations exhibit tensile strength and flexibility dependent on their composition being either rigid or flexible, as well as antimicrobial and antioxidant activity, which will potentially lead to prolonged use for food packaging. The recipe with PLA matrix and 40:60 Lapol®108 as masterbarch/polyethylene glycol (MB/PEG) bio-plasticizers ratio was distinguished by an improvement of over 100 times in terms of flexibility compared with neat PLA, while the highest antioxidant activity (36.27%) was recorded for the sample containing a CS-M and MB/PEG ratio of 60:40. An enhancement of ~50% for the water vapor barrier was recorded for PLA/CS-M_100:0 material. By modulating the MB and PEG bio-plasticizers ratio, the design of new eco-friendly food packaging materials with antimicrobial/antioxidant characteristics by using the existing technologies for processing synthetic polymers (melt mixing, compounding, pressing, thermoforming) has been successfully realized.

Antioxidant/Antibacterial Electrospun Nanocoatings Applied onto PLA Films.

Polylactic acid (PLA) films were coated by coaxial electrospinning with essential and vegetable oils (clove and argan oils) and encapsulated into chitosan, in order to combine the biodegradability and mechanical properties of PLA substrates with the antimicrobial and antioxidant properties of the chitosan⁻oil nanocoatings. It has been established that the morphology of the electrospun nanocoatings mainly depend on the average molecular weight (MW) of chitosan. Oil beads, encapsulated into the main chitosan nanofibers, were obtained using high-MW chitosan (Chit-H). Oil encapsulated in chitosan naoparticles resulted when low-MW chitosan (Chit-L) was used. The coating layer, with a thickness of 100 ± 20 nm, had greater roughness for the samples containing Chit-H compared with the samples containing Chit-L. The coated PLA films had higher antibacterial activity when the nanocoating contained clove oil rather than when argan oil was used, for both types of chitosan. Nanocoatings containing Chit-H had higher antibacterial activity compared with those containing Chit-L, for both types of oil tested, due to the larger surface area of the rougher nanoscaled morphology of the coating layer that contained Chit-L. The chitosan⁻clove oil combination had higher antioxidant activity compared to the simple chitosan nanocoating, which confirmed their synergistic activities. The low activity of systems containing argan oil was explained by big differences between their chemical composition and viscosity.

Comparative Analysis of the Composition and Active Property Evaluation of Certain Essential Oils to Assess their Potential Applications in Active Food Packaging.

The antifungal, antibacterial, and antioxidant activity of four commercial essential oils (EOs) (thyme, clove, rosemary, and tea tree) from Romanian production were studied in order to assess them as bioactive compounds for active food packaging applications. The chemical composition of the oils was determined with the Folin-Ciocâlteu method and gas chromatography coupled with mass spectrometry and flame ionization detectors, and it was found that they respect the AFNOR/ISO standard limits. The EOs were tested against three food spoilage fungi-Fusarium graminearum, Penicillium corylophilum, and Aspergillus brasiliensis-and three potential pathogenic food bacteria-Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes-using the disc diffusion method. It was found that the EOs of thyme, clove, and tea tree can be used as antimicrobial agents against the tested fungi and bacteria, thyme having the highest inhibitory effect. Concerning antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) methods, it has been established that the clove oil exhibits the highest activity because of its high phenolic content. Promising results were obtained by their incorporation into chitosan emulsions and films, which show potential for food packaging. Therefore, these essential oils could be suitable alternatives to chemical additives, satisfying the consumer demand for naturally preserved food products ensuring its safety.

A novel model to assess the efficacy of steam surface pasteurization of cooked surimi gels inoculated with realistic levels of Listeria innocua.

Steam surface pasteurization is a promising decontamination technology for reducing pathogenic bacteria in different stages of food production. The effect of the artificial inoculation type and initial microbial load, however, has not been thoroughly assessed in the context of inactivation studies. In order to optimize the efficacy of the technology, the aim of this study was to design and validate a model system for steam surface pasteurization, assessing different inoculation methods and realistic microbial levels. More specifically, the response of Listeria innocua, a surrogate organism of Listeria monocytogenes, on a model fish product, and the effect of different inoculation levels following treatments with a steam surface pasteurization system was investigated. The variation in the resulting inoculation level on the samples was too large (77%) for the contact inoculation procedure to be further considered. In contrast, the variation of a drop inoculation procedure was 17%. Inoculation with high levels showed a rapid 1-2 log decrease after 3-5 s, and then no further inactivation beyond 20 s. A low level inoculation study was performed by analysing the treated samples using a novel contact plating approach, which can be performed without sample homogenization and dilution. Using logistic regression, results from this method were used to model the binary responses of Listeria on surfaces with realistic inoculation levels. According to this model, a treatment time of 23 s will result in a 1 log reduction (for P = 0.1).

Activation of Bacillus spores at moderately elevated temperatures (30-33 °C).

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30-33 °C, compared to spores stored at 3-8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.

Growth kinetics of listeria isolated from salmon and salmon processing environment: single strains versus cocktails.

The growth dynamics of Listeria monocytogenes strains isolated from salmon or a salmon processing environment and two reference Listeria innocua strains were investigated at refrigerated and close-to-optimal growth temperatures. Estimates for the growth rates and the lag-phase duration at 4, 8, 12, and 30°C were obtained for optical density measurements by using different growth parameter estimation methods, i.e., the serial dilution (SD) method and the relative rate to detection (RRD) method. Both single L. innocua and L. monocytogenes strains and mixtures of L. monocytogenes strains (cocktails) were studied. Both methods show an increase in maximum growth rate (µ(max)) of Listeria with increasing temperatures. Generally, single-strain growth rate estimates were quite similar for both species, although L. monocytogenes showed slightly higher µ(max) estimates at 4°C. The SD method gave the highest estimates for the growth rate, i.e., the estimates from the RRD method were 10 to 20% lower. This should lead to caution when using the latter method for Listeria, particularly at lower temperatures. Overall, the SD method is preferred as this method yields µ(max) estimates close to the biological value and provides estimates for the duration of lag time (λ). For discrimination between different strains, λ appeared to be a more suitable parameter than µ(max). This effect was most prominent for L. innocua. Significant differences were observed between µ(max) and/or λ of L. monocytogenes cocktails and single strains at all temperatures investigated. At 4°C, the average growth rate of cocktails was higher than that of single strains. At 8 and 30°C, this trend was reversed. The average λ of single strains were more than twice as long as those of cocktails at 4°C. At 8 and 30°C, the λ of cocktails were significantly slower than those of single strains, but the variation was considerably less and the differences were less pronounced.

Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores.

BACKGROUND: The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine. RESULTS: In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid. CONCLUSIONS: These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

Promoting Bacillus cereus spore germination for subsequent inactivation by mild heat treatment.

Sublethal heat treatment may activate dormant spores and thereby potentiate the conversion of spores to vegetative cells. As the germinated spore is known to possess lower heat resistance than its dormant counterpart, it has been postulated that double heat treatment, i.e., spore heat activation followed by germination and then by heat inactivation, can be used to control spores in foods. Production of refrigerated processed foods of extended durability often includes more than one heat treatment of the food components. This work simulates conventional heat treatment procedures and evaluates double heat treatment as a method to improve spore control in model food matrixes of meat broth and cream sauce. Bacillus cereus NVH 1230-88 spores were supplemented in food model matrixes and heat activated at 70°C and then heat inactivated at 80 or 90°C. The samples were held at 29 to 30°C for 1 h between primary and secondary heat treatments, to allow spore germination. Nutrients naturally present in the food matrixes, e.g., amino acids and inosine, could act as germinants that induce germination. The levels of germinants could be too low to produce effective germination within 1 h. Following primary heat treatment, some samples were therefore supplemented with a combination of L-alanine and inosine, a germinant mixture known to be effective for B. cereus spores. In both matrixes, a combination of double heat treatment (heat activation, germination, and inactivation) and addition of germinants gave a reduction in spore counts equivalent to or greater than that obtained with a single heat treatment for 12 min at 90°C. Addition of germinants was essential to induce effective germination in cream sauce during 1 h at 29 to 30°C, and germinants were therefore a crucial supplement to obtain an effect of double heat treatment in this matrix. These data will be valuable when setting up temperature-time-germinant combinations for an optimized spore reduction in mild-heat-treated foods.

Microflora assessments using PCR-denaturing gradient gel electrophoresis of ozone-treated and modified-atmosphere-packaged farmed cod fillets.

Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rDNA sequence was used to characterize changes in the microbial flora caused by ozone (O3) treatment of farmed cod (Gadus morhua). Portions of cod were produced under controlled conditions, bathed in fresh water supplemented with 2 ppm of O3 for 30 min, and packaged in modified atmosphere (MA: 60% CO2 and 40% N2) before 4 degrees C storage. Control samples were packaged in MA or air, without prior O3 treatment. Samples were analyzed by PCR-DGGE to determine the predominant bacterial flora and to examine possible differences in the microbial community due to O3 treatment. The DGGE analysis during the storage period showed that the O3 treatment produced no significant difference in the microbial flora compared with the controls. Sequencing of 16S rDNA detected the specific spoilage bacteria Photobacterium phosphoreum, Pseudomonas spp., Shewanella baltica, and Shewanella putrefaciens as the predominant bacteria in all samples. PCR-DGGE results were supported by culture and sensory analyses used in predicting product shelf life. Aerobic plate count, H2S-producing bacteria, and psychrotrophic bacterial counts demonstrated no significant extension of the shelf life of MA-packaged, O3-treated cod fillets.

Characterisation of the bacterial flora of modified atmosphere packaged farmed Atlantic cod (Gadus morhua) by PCR-DGGE of conserved 16S rRNA gene regions.

The present article describes the use of broad-range molecular analyses to characterise the microbial population of farmed Atlantic cod (Gadus morhua) packaged for the retail market. Cod was filleted post rigor, packaged in air or in modified atmosphere (MA) (50% CO(2):50% N(2) or 50% CO(2):50% O(2)) and stored at 0 degrees C for 11 days. To determine the community profiles of the samples the variable V3-region of the bacterial 16S rRNA gene were amplified by PCR, before the PCR products were separated by denaturing gradient gel electrophoresis (DGGE). From sequence analyses Pseudomonas spp. were found to be the predominant bacteria in oxygen enriched atmospheres, whereas the spoilage bacteria Photobacterium sp., Shewanella putrefaciens and Pseudomonas spp. dominated in CO(2):N(2) and air packaged samples. Additional microbial analyses by cultivation methods observed highest bacterial numbers in air stored samples, and both MA mixtures gave growth inhibition when measuring aerobic plate count, psychrotrophic bacteria and H(2)S-producing bacteria. The results show that PCR-DGGE can be applied to examine bacterial diversity and population shifts among different MA-packaged products.

Characterisation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE.

It is not well understood why Atlantic halibut (Hippoglossus hippoglossus) has longer shelf-life than most other white fish species. Our approach was to examine the microbiological diversity of the spoilage microbiota during modified atmosphere (MA) packaging of farmed Atlantic halibut. Portions were packaged with gas mixtures of CO(2):N(2) and CO(2):O(2) (50%:50%) and with air as a reference. The packages were stored at 4 degrees C and samples were taken 6 times during the 23 days of storage. Analyses with molecular techniques (PCR-DGGE) determined profiles of the bacterial populations in the various samples and sequencing detected the bacterial species present. In addition, samples were analysed for microbial, chemical and sensory parameters. The shelf-life was 10-13 days when stored in air and between 13 and 20 days for MA packages, with oxygen-enriched packages suggested as the better gas mixture, based on microbial growth and sensory scores. From sequence analyses of the bacterial population Photobacterium phosphoreum and Pseudomonas spp. were found to dominate in the halibut. Brochothrix thermosphacta was found in most samples at the end of the storage period. Shewanella putrefaciens was found sporadically and in low concentrations based on microbial methods, but not detected by PCR-DGGE.