Tomás G. Villa

Phaffia rhodozyma as an astaxanthin source in salmonid diets.

Abstract The red yeast Phaffia rhodozyma has possible application as a component of diets for use in aquaculture. Its primary value lies in its content of astaxanthin, which is much higher (5–50 times) than that found in crustacean meals. When fed to rainbow trout, the deposition of astaxanthin in the fish flesh was dependent on the proper preparation of yeast cells before their inclusion into the feed. No astaxanthin was nutritionally available from intact yeast. If P. rhodozyma was mechanically ruptured its pigments were transferred to the flesh of rainbow trout, coloring it salmon-pink. The most efficient deposition of astaxanthin in trout occurred when the cell wall of P. rhodozyma was partially removed by enzymatic digestion. The proxiamte composition, amino acid content, fatty acid profile, and astaxanthin content were determined for P. rhodozyma. When grown under the conditions used in this study, P. rhodozyma has a low protein content (∼25%) and a high total lipid content (∼17%) compared with most other microorganisms. Its amino acid profile is well balanced but is deficient in methionine. The predominant fatty acids present in the yeast are oleic, linoleic and palmitic acids.

Changes in Biogenic Amines and Microbiological Analysis in Albacore (Thunnus alalunga) Muscle during Frozen Storage

Albacore specimens of extra quality were analyzed for their biogenic amine contents after 1, 3, 6, and 9 months of frozen storage at -18 degrees C or -25 degrees C. A high-performance liquid chromatography method involving a linear elution gradient was optimized for the identification and determination of putrescine, cadaverine, histamine, spermidine, and spermine in albacore tuna. Putrescine was the biogenic amine that showed the highest increase, reaching concentrations of 59.04 ppm (815% of the initial level) and 68.26 ppm (942% of the initial level) in the white muscle of albacore after 9 months of frozen storage at -18 and -25 degrees C, respectively. Cadaverine, histamine, and spermidine concentrations were below 3, 5, and 11 ppm, respectively, after 9 months of frozen storage, while spermidine underwent a significant decrease at both storage temperatures. Microbiological analysis confirmed the absence of species of Enterobacteriaceae in 75% of the albacore specimens after 9 months of frozen storage; coliforms were always below 3 CFU/g. The survival rate of the psychrotrophic microorganisms after 9 months of frozen storage at -25 degrees C was 4.6%, while 38.9 and 92.1% of the aerobic mesophiles present in the white muscle of albacore before freezing survived 9 months of storage at -18 and -25 degrees C, respectively.

Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga).

Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna.

Cloning of genes related to exo-β-glucanase production in Saccharomyces cerevislae: characterization of an exo-β-glucanase structural gene

Abstract The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain ( exgl -2) with highly reduced extracellular exo-β-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb Hind III- Xho I fragment. Exg + strains and Exg − mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg + wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg + wt strains. The EXG1 gene transformed Schizosaccharomyces pombe , yielding extracellular EXG activity which showed cross-reactivity with anti- S. cerevisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg + strain. Genetic and Southern analysis of several stable Exg − transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exgl -2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae .

Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria.

Antimicrobial peptides (AMPs) are short peptidic molecules produced by most living creatures. They help unicellular organisms to successfully compete for nutrients with other organisms sharing their biological niche, while AMPs form part of the immune system of multicellular creatures. Thus, these molecules represent biological weapons that have evolved over millions of years as a result of an escalating arms race for survival among living organisms. All AMPs share common features, such as a small size, with cationic and hydrophobic sequences within a linear or cyclic structure. AMPs can inhibit or kill bacteria at micromolar concentrations, often by non-specific mechanisms; hence the appearance of resistance to these antimicrobials is rare. Moreover, AMPs can kill antibiotic-resistant bacteria, including insidious microbes such as Acinetobacter baumannii and the methicillin-resistant Staphylococcus aureus. This review gives a detailed insight into a selection of the most prominent and interesting AMPs with antibacterial activity. In the near future AMPs, due to their properties and despite their ancient origin, should represent a novel alternative to antibiotics in the struggle to control pathogenic microorganisms and maintain the current human life expectancy.

Characterization of biogenic amine-producing Stenotrophomonas maltophilia strains isolated from white muscle of fresh and frozen albacore tuna

Abstract Three strains — one of them psychrotrophic and two mesophilic — of Stenotrophomonas maltophilia, an emerging pathogen involved in an increasing number of clinical syndromes, were isolated from fresh and frozen-stored (6 months at −25°C) albacore tuna in a routine screening of histamine-forming bacteria. All the three strains showed histidine decarboxylase activity when assayed in a 2% histidine–0.0005% pyridoxal-HCl containing trypticase soy broth and further analyzed by HPLC. Although all three strains produced less than 25 ppm of histamine, they showed strong lysine-decarboxylating activity, cadaverine being produced at concentrations ranging from 1736 to 4821 ppm after 48 h in 0.0005% pyridoxal-HCl–trypticase soy broth supplemented with 1% lysine. Strong secretion of extracellular lipase and protease was also observed in all three strains. The psychrotrophic strain S. maltophilia 5PC6 was able to produce both extracellular lipase and protease even at 4 and 7°C, respectively. Phenotyping assays — including the investigation of up to 40 biochemical tests and 15 susceptibility assays — revealed slight differences among the three strains. Genotyping experiments — based on RFLP analysis and Southern blot hybridizations — indicated that strains 15MF and 25MC6 were closely related but confirmed that all three strains recovered were different.

Industrial Applications of Hyperthermophilic Enzymes: A Review

Over the past two decades, research scientists have been involved in the investigation of thermophilic and hyperthermophilic microorganisms owing to the unique features of their enzymic systems. Such in-depth investigations are now on their way to mastering the cloning and industrial exploitation of a broad variety of genes encoding enzymes involved in starch hydrolysis, amino acid biosynthesis, protein hydrolysis, etc. In this work, we review the state of the art and future perspectives of industrial applications of enzymes from hyperthermophilic and extreme thermophilic microorganisms, special attention being paid to the biotechnological methods involved in their industrial exploitation.

Mevalonic acid increases trans-astaxanthin and carotenoid biosynthesis in Phaffia rhodozyma

SummaryMevalonic acid has been tested as enhancer of pigment biosynthesis in wild-type Phaffia rhodozyma. The addition of 0.1% mevalonic acid to the culture media stimulated both trans-astaxanthin and total carotenoids biosynthesis, with average increases by ca 400%.

Proteins influencing foam formation in wine and beer: the role of yeast

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa--characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins.

glucanases of the yeast Pichia polymorpha

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl-β-d-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A50 has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different β-glucanases located in the periplasmic space is presented.