Inga Marie Aasen

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687.

Abstract The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l−1. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy.

Interactions of the bacteriocins sakacin P and nisin with food constituents

Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.

Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae.

Two primary selection criteria were used to collect a pool of nearly 500 candidate probiotic bacteria from Atlantic cod (Gadus morhua L.) larvae, i.e. the dominant intestinal bacterial flora and isolates with antagonistic activity against Vibrio anguillarum. Bacteria were isolated from cod larvae from five rearing groups with variable rearing technologies. The bacteria were brought to pure culture and characterized phenotypically. Based on properties such as uniqueness, dominance and fermentative ability, a selection of approximately 10% of the isolates were chosen from the initial pool of bacteria to reduce the number of candidates. These 55 isolates were characterized further in vitro regarding antagonism, adhesion to mucus, growth in mucus, production of extracellular enzymes, fish bile resistance and haemolytic properties. Based on the results of the in vitro tests, the number of probiotic candidates was further reduced to seven isolates. To evaluate the probiotic potential and to assure that the seven isolates were not harmful to the host, yolk sac larvae of cod were exposed to the isolates in a small-scale in vivo experiment. The in vivo experiment excluded two of the candidate bacteria due to increased mortality of cod larvae, whereas three isolates from the dominant (Vibrio and two different strains of Microbacterium) and two from the antagonistic (Ruegeria and Pseudoalteromonas) group improved the survival of larvae compared to the positive control. Thus, a combination of the two screening methods was suited for making multistrain probiotics with complementary modes of action.

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei

Aims: To evaluate the potential of sakacin P and sakacin P‐producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food?

Production of sakacin P by Lactobacillus sakei in a completely defined medium

In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML‐B) with 33 components. Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML‐B. Sakacin P was produced at 4–30 °C, with the highest specific production at low temperatures. More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6·3. Tween‐80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects. The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell‐independent and promoted at high temperature and pH. Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested.

Development of a process for large-scale chromatographic purification of an alginate lyase from Klebsiella pneumoniae

SummaryA process for purification of an alginate lyase, produced extracellularly by fermentation of Klebsiella pneumoniae, has been developed. The process includes two chromatographic steps and is well suited to large-scale operation. By hydrophobic interaction chromatography on Phenyl-Sepharose FF, followed by anion exchange chromatography on Q-Sepharose FF in a negative mode, the specific activity was increased from 0.09 units (U) mg −1 to more than 50 U mg−1. Due to an extremely low product concentration in the fermentation broth, and large amounts of contaminating proteins, the chromatographic adsorbents had low capacities with respect to alginate lyase. By adsorption on the cation exchanger S-Sepharose FF, the capacity was so low that the enzyme could not be concentrated. The binding capacity of Phenyl-Sepharose FF was approximately 20-fold higher, and a three to tenfold concentration was obtained. The first stage of the process, hydrophobic interaction chromatography, has been applied to the isolation of alginate lyase from fermentation batches of 180 l. Several runs have resulted in a purified product with an average quantity of 30 000–35 000 U per fermentation, and an average specific activity of 4.5 U mg−1. Although the raw material employed in this work has been particularly unfavourable, the process developed will also be applicable to raw materials with higher product concentrations.

Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66

Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

Rapid metabolic profiling of developing Pseudomonas aeruginosa biofilms by high-resolution mass spectrometry fingerprinting

Biofilms are single- or multi-species communities of bacteria that are enclosed in an extracellular matrix. These cells generally exhibit phenotypes that are significantly different from those of planktonic cells, yet detailed elucidation of the causality and the exact nature of this metabolic shift remains challenging. Considering the strong correlation of biofilms with pathogenicity and disease in the clinic, as well as the veritable economic impact of biofilms in other areas, a methodology for in-vivo monitoring of biofilm development is necessary. Here, we present high-resolution mass spectrometry fingerprinting as a rapid, sensitive, and generic technique for online, non-invasive monitoring of developing biofilms. The opportunistic pathogen Pseudomonas aeruginosa is used as a model system, and it is demonstrated that strain- and time-dependent changes in biofilm extracellular metabolites are easily detected.

Managing the microbial community of marine fish larvae: a holistic perspective for larviculture

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100–250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.