Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains.

The use of juvenile Artemia as feed in aquaculture and in the pet shop industry has been getting more attention during the last decade. In this study, the use of selected bacterial strains to improve the nutritional value of dry food for Artemia juveniles and to obtain control of the associated microbial community was examined. Nine bacterial strains were selected based on their positive effects on survival and/or growth of Artemia juveniles under monoxenic culture conditions, while other strains caused no significant effect, significantly lower rates of survival and/or growth, or even total mortality of the Artemia. The nine selected strains were used to preemptively colonize the culture water of Artemia juveniles. Xenic culture of Artemia under suboptimal conditions yielded better survival and/or growth rates when they were grown in the preemptively colonized culture medium than when grown in autoclaved seawater. The preemptive colonization of the culture water had a drastic influence on the microbial communities that developed in the culture water or that were associated with the Artemia, as determined with Biolog GN community-level physiological profiles. Chemotaxonomical characterization based on fatty acid methyl ester analysis of bacterial isolates recovered from the culture tanks was performed, and a comparison with the initially introduced strains was made. Finally, several modes of action for the beneficial effect of the bacterial strains are proposed.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.     

Manipulation of dietary lipids, fatty acids and vitamins in zooplankton cultures

1. A wide range of species that are cultivated in commercial mariculture are planktonic during at least part of their life cycle; for example, the larval stages of shellfish (shrimp and molluscs) and the live feeds (rotifers, brine shrimp, copepods) used in the larviculture of marine fish and shellfish. Over the last decades various techniques have been developed to deliver nutrients to these zooplanktonic organisms either through artificial diets or by manipulating the composition of the live prey fed to the carnivorous stages. This paper reviews the methodology that has allowed aquaculturists to gain knowledge of nutritional requirements and may offer interesting opportunities for ecologists to verify the importance of key nutrients in the natural food chain of marine as well as freshwater ecosystems.
2. Live micro-algae can be replaced partially or completely in the diet of filter-feeders such as rotifers, Artemia , shrimp larvae and bivalves, by various types of preserved algae, micro-encapsulated diets and yeast-based diets, whereas lipid emulsions and liposomes may be utilized to supplement specific lipid-and water-soluble nutrients, respectively. Microbound and micro-encapsulated diets have been designed to supplement live feed in the culture of micro-predators such as fish and shrimp larvae.
3. Live prey organisms, in particular rotifers and Artemia , can be 'bio-encapsulated' with a variety of enrichment diets to manipulate their content in certain nutrients, including ω3 highly unsaturated fatty acids (FA) and the vitamins C, A and E. Nevertheless, the enrichment techniques are not applicable for all nutrients and prey organisms. Phospholipid composition is difficult to manipulate through the diet of live feed and the enrichment of the essential FA docosahexaenoic acid (DHA) is hampered in most Artemia species due to the catabolism of this FA following enrichment.     

Bioencapsulation of Two Different Vibrio Species in Nauplii of the Brine Shrimp (Artemia franciscana)

Two groups of nauplii from the brine shrimp (Artemia franciscana) were enriched with different bacteria, and the dynamics of bacterial uptake by the nauplii were observed. This study showed that the efficiency of Artemia nauplii in bioencapsulating bacteria strongly depends on the type of bacteria used, time of exposure, and status (live or dead) of the bacteria.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Effect of feed on larvae of Macrobrachium malcolmsonii (H. Milne Edwards) reared in synthetic brackishwater

Freshwater prawn, Macrobrachium malcolmsonii, has potential for aquaculture. Larvae of M. malcolmsonii were reared in synthetic brackishwater using commercial salts. Live and artificial feeds were provided to larvae either individually or in combination. Survival was significantly higher when larvae were fed with live feed only. However, larvae fed with artificial feed showed significantly lower survival. Survival was significantly higher when Artemia combined together with other feeds. Despite its limitations, synthetic brackishwater can be used for freshwater prawn seed production.     

The use of probiotics in aquaculture

This study aims to present comprehensive notes for the use of probiotics in aquaculture. Probiotics have been proven to be positive promoters of aquatic animal growth, survival and health. In aquaculture, intestines, gills, the skin mucus of aquatic animals, and habitats or even culture collections and commercial products, can be sources for acquiring appropriate probiotics, which have been identified as bacteria (Gram‐positive and Gram‐negative) and nonbacteria (bacteriophages, microalgae and yeasts). While a bacterium is a pathogen to one aquatic animal, it can bring benefits to another fish species; a screening process plays a significant role in making a probiotic species specific. The administration of probiotics varies from oral/water routine to feed additives, of which the latter is commonly used in aquaculture. Probiotic applications can be either mono or multiple strains, or even in combination with prebiotic, immunostimulants such as synbiotics and synbiotism, and in live or dead forms. Encapsulating probiotics with live feed is a suitable approach to convey probiotics to aquatic animals. Dosage and duration of time are significant factors in providing desired results. Several modes of actions of probiotics are presented, while some others are not fully understood. Suggestions for further studies on the effects of probiotics in aquaculture are proposed.     

Fermented products with probiotic qualities

For several centuries, fermented products derived from plant or animal materials have been an acceptable and essential part of the diet in most parts of the world. Health benefits have also often been associated with them. Probiotics can be defined as fermented food containing specific live microorganisms or a live microbial food or feed supplement, which beneficially effects the human or the host animal by improving its intestinal microbial balance. Nearly all probiotics currently on the market contain Lactobacilli, Streptococci, Enterococci or Bifidobacteria. In contrast to Japan, where freeze-dried microorganisms are consumed by a substantial part of the human population, in Europe, probiotic action towards humans are only claimed for certain fermented dairy products (e.g. yoghurts). Those species that have been extensively studied so far, with several experimental trials on man, are the two yoghurt bacteria Streptocaccus thermophilus and Lactobacillus bulgaricus, L. casei and Bifidobacteria. L. acidophilus has also received important scientific interest, however, only a few human studies have been carried out. From the technological point of view a good probiotic should be stable and viable for long periods under storage, should be able to survive the low pH levels of the stomach, be able to colonise the epithelium of the gastro-intestinal tract of the host, should not be pathogenic and, last but not least, must be capable of exerting a growth promoting effect or a resistance to infectious diseases. The beneficial effects of probiotics are mainly contributed to a direct antagonistic effect against specific groups of microorganisms (Enteropathogenes), by an effect on the metabolism in the gut or by a stimulation of systemic or mucosal immunity. We will present major proven health benefits of milks fermented with those bacterial species and discuss, where possible, the impact of the specific selection and utilisation of particular strains.     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed

In this study, MRS medium supplemented with cysteine hydrochloride and mupirocin, termed Bifidobacterium selective medium (BSM) was found to be elective for bifidobacteria but inhibitory to a wide range of non-bifidobacteria strains commonly included in probiotic animal feed. Bacilli, lactobacilli, lactococci and streptococci failed to form colonies on BSM and enterococci, pediococci and propionibacteria formed colonies <0.5 mm in diameter. Bifidobacteria formed colonies >1 mm in size and could be readily distinguished. The addition of nystatin to BSM further inhibited Saccharomyces cerevisiae. BSM was successfully used to enumerate the bifidobacteria components, confirmed through fructose-6-phophate-phosphoketolase detection, present in two commercial probiotic feeds. The medium is recommended for the enumeration of bifidobacteria from animal feeds especially when not a numerically dominant component.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry

Systematic procedures were used to evaluate the probiotic properties of Lactobacillus fermentum (L. fermentum) strains isolated from swine and poultry. The major properties included their capabilities to adhere to the intestinal epithelium of swine and poultry, the inhibition on pathogenic bacteria, and their tolerance to the gastric juice and bile salts. Results showed that L. fermentum strains from poultry digestive tract showed better adherence to the swine intestine and chicken crop epithelial cells as compared to those strains from the swine origin. In addition, six strains from poultry and one strain from swine showed adhesion specificity to their own intestinal epithelium. Four poultry isolates and one swine isolate were able to adhere to the epithelial cells from both swine and chicken. For gastric juice and bile tolerance, most of the strains isolated from swine or poultry were acid tolerant but less strains were bile intolerant. The spent culture supernatant (SCS) of these L. fermentum strains showed antagonistic effect against the indicator bacteria, such as Escherichia coli, Salmonella spp., Shigella sonnei and some enterotoxigenic Staphylococcus aureus. From the above studies, some L. fermentum strains isolated from poultry were found to have the probiotic properties required for use in animal feed supplement. This study suggested that poultry digestive tract may serve as potential source for the isolation of probiotic lactic acid bacteria.     

Binding of extracellular matrix molecules by probiotic bacteria

AIMS: The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria. METHODS AND RESULTS: ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins. SIGNIFICANCE AND IMPACT OF THE STUDY: Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals.     

Probiotic Formulations: Application and Status as Pharmaceuticals—A Review

The use of probiotics is a new way to control and treat infections in this modern era. Application of beneficial bacteria to protect against detrimental bacteria in the gastrointestinal tract and thus reap a positive health benefit is the basis of probiotic therapy. Probiotics have a long global history of traditional use. They are normally consumed through fermented foods and are currently sold mostly as ingredients in foods or nutritional supplements. They are also supplied as pharma products. Recent research has highlighted the probiotic potential in the treatment or prevention of disease conditions, maintenance of health, improving immunity and in the reduction in the risk of future diseases. But their position in the pharmaceutical industry is still not very clear. Clinical practitioners use probiotic pharma products mostly as supplements. Their status as drugs is still unclear. This review is aimed to analyze probiotics as pharmaceuticals, their current status as dietary supplements and drugs, existing probiotic preparations and future research needs.     

Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens

To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound β-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios.     

The effect of eubiotic feed additives on the performance of growing pigs and the activity of intestinal microflora

The aim of this study was to compare the effect of probiotic bacteria, prebiotics, phytobiotics and their combinations on performance and microbial activity in the digestive tract of growing pigs. The experiment was conducted over 28 d on 48 male pigs of about 12 kg body weight (BW), which were allocated to following treatments.: (1) Control Group (Con) without additive, (2) Group I, addition of a prebiotic (inulin), (3) Group Ph, a phytobiotic (herbal water extracts), (4) Group P, a probiotic composed of four strains of lactic acid bacteria, (5) Group PhP, phytobiotic and probiotic bacteria and (6) Group PhPI, a phytobiotic, probiotic bacteria and a prebiotic. Animal performance was recorded and at d 28 six pigs from each group were euthanised to collect digesta samples. In all groups except for Group I, diarrhoea incidents were observed. Groups Ph and P had significantly higher daily gains and final BW, and Group Ph utilised feed better than other groups. The pH of ileal digesta was significantly lower in Group PhPI. In the caecal digesta of Groups I, P and PhP, the pH level was lower than in the other groups but dry matter contents was significantly higher in Groups Con and I. The short-chain fatty acids and particular acid content differed significantly only in the colonic digesta. The yeast and mould numbers in caecal digesta was highest in Group Con. No treatment effects were observed for the number of lactic acid bacteria, coli group bacteria or Clostridium. However, the observed significantly higher number of total bacteria suggests that a multi-component eubiotic treatment changes the bacterial composition and distribution more effectively. Our findings indicated that all used additives changed the intestinal microflora, but the multi-component eubiotics were not beneficial as feed additives offered separately. Moreover, supplementation of phytobiotics and probiotic bacteria also improved the animal performance significantly.     

Eclossion of cysts of two species of Dendrocephalus (Anostraca: Thamnocephalidae) of potential use as food in aquaculture

The use of Artemia salina nauplii as live food has significantly aided culture of commercial fish and shrimps in recent years. However, reported deficiencies in the nutritional value of some strains originated the study of freshwater fairy shrimps as an alternative to Artemia. This study presents cyst biometry for Dendrocephalus geayi and D. spartaenovae (Anostraca: Thamnocephalidae), and the effects of some physicochemical variables on cyst hatching. The biometric characteristics of D. geayi and D. spartaenovae are within the size range of commercial Artemia strains. Favorable conditions to hatch D. geayi are 30 degrees C and conductivity near that of distilled water (<5 micromhos). For D. spartaenovae, these conditions are 28 degrees C and 280 micromhos. A very low salinity such as 1 per thousand inhibits hatching in both species.     

Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.     

Properties of probiotic bacteria explored by proteomic approaches

The study of health-beneficial effects that probiotic bacteria can exert on humans and animals is at its beginning. Pending scientific questions include the identification of molecular markers of the health-promoting activity of specific strains, which may be used to select novel probiotic strains and to gain understanding of the mechanisms underlying their effects. In that perspective, the role of bacterial proteins must be evaluated, placing proteomics-based approaches at the core of the field. Until now, most proteomic analyses focused on the dynamics of abundant cytoplasmic proteins during adaptation of bacteria to conditions mimicking the gastro-intestinal tract environment. The development of in silico and experimental procedures allowing identification and quantification of surface-exposed and secreted proteins should boost our understanding of bacteria-host crosstalk.