Hans Blom

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.

Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4°C

A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments. A consumer acceptance test of products produced with and without the compounds was also performed. Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products. This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products. The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w/w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4-6 weeks. In addition, strict control of temperature during production and storage is very important.

Transformation of Lactobacillus strains used in meat and vegetable fermentations

Abstract For transformation of Lactobacillus plantarum and Lactobacillus sake strains of meat and vegetable origin, electroporation using the electroporation solutions 952 m m sucrose with 3·5 m m MgCl2 or 30% polyethylene glycol (PEG 1500) was found to be efficient. The highest transformation efficiencies for all the meat strains and most of the vegetable strains were obtained using PEG 1500. Although the transformation efficiencies varied, the strains tested showed essentially the same response pattern to different electrical pulses, as illustrated by response surface plots. For screening purposes a setting of 1·5 kV and 400 ohm pulse control resistance is recommended. For optimization of transformation efficiency for specific strains, selected combinations of voltage and pulse control resistance along the optimum shown in the plots can be used. Transformation efficiencies can be further improved by using medium shift from MRS without dextrose to MRS, and by including a magnesium solution in the washing procedure. The combined effect of these pre-treatments has raised the transformation frequency of previously low transforming strains with a factor of 103, giving transformation efficiencies sufficient for most practical applications.

A model based on absorbance data on the growth rate of Listeria monocytogenes and including the effects of pH, NaCl, Na-lactate and Na-acetate.

A mathematical model was developed for predicting the growth of L. monocytogenes at 9 degrees C in the presence of 70 ppm sodium nitrite, and at different levels of pH (5.5-6.5), sodium chloride (1.0-4.0%), sodium lactate (0-0.5%) and sodium acetate (0-0.6%). Collection of the growth data was done using absorbance measurements in broth cultures and the absorbance measurement was evaluated. The model was compared to the Food MicroModel, and against the growth of L. monocytogenes in a vacuum-packed meat product stored at 9 degrees C. A linear relationship was obtained, for the absorbance data on different dilutions of the inoculum, in the absorbance interval studied. There was also a linear relationship between the values of the maximum specific growth rates derived from the absorbance and the ones derived from viable count measurements; and corrections were made accordingly. The statistical evaluation showed that all the main factors, i.e. pH, sodium chloride, sodium lactate and sodium acetate were statistically significant for the growth rate of L. monocytogenes. Comparison to the Food MicroModel (FMM) showed a slight underprediction for the developed model (bias = 0.84). The predictions were, on average, within 20% of the FMM predictions (n = 10). Validation against the observed growth of L. monocytogenes inoculated into an emulsion type of sausage (n = 4) also showed a slight underprediction by the model. The predictions were, on average, 16% below the observed values in the sausage (Bias 0.84, Accuracy 1.26).

A model assay to demonstrate how intrinsic factors affect diffusion of bacteriocins.

A rapid and simple method to elucidate how intrinsic factors in a given food product affect bacteriocin diffusion and efficacy is described. The basic idea of this assay is to help predict which bacteriocin or other inhibitory substance to select for a given product, where increased security towards specific microorganisms is wanted. In an agar plate model system the effect of five factors (number of indicator cells, pH and concentration of NaCl, agar and soy oil) on the diffusion of the bacteriocins sakacin A, sakacin P, pediocin PA-1, piscicolin 61 and nisin was studied. An experimental design permitting simultaneous evaluation of the effect of all factors was used. The results indicated that each bacteriocin has a characteristic intrinsic factor effect profile. However, pH and load of indicator cells affect all the bacteriocins.

Characterization, production, and purification of leucocin H, a two-peptide bacteriocin from Leuconostoc MF215B.

Abstract.Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 105-fold, and the final specific activity was 2 × 107 BU/OD280. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar.

Accelerated production of dry fermented sausage

The scope of this paper is to review work connected with accelerated ripening of dry fermented sausages by addition of proteolytic enzymes. An overview of the following topics is given: practical sausage experiments with addition of various proteinases of bacterial origin, including data from sensory, biochemical and gc/ms analyses; biochemical and genetic characterization of the enzyme shown to be most useful in these experiments, the serine proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151; experiments to transform starter cultures with the genes for production of this proteinase and proposals for future work in this field.

Partial characterization of a thermostable anthocyanin-β-glycosidase from Aspergillus niger

Abstract Anthocyanin-β-glycosidase (anthocyanase) from Aspergillus niger has been partly purified and shown to catalyse a lytic reaction upon the β-glycosidic bond of anthocyanins. The molecular weight as estimated from high performance liquid chromatography and gel chromatography on Ultrogel AcA-34 is in the range (370 ± 30) × 10 3 . SDS electrophoresis indicates a tetrameric composition. Isoelectric point is located at pH 4.

Partial purification and characterisation of a lipase from Lactobacillus plantarum MF32

Abstract A lipase from Lactobacillus plantarum MF32 has been partly purified and characterised. The apparent molecular weight of the lipase was estimated to be approximately 75 000. Isoelectric focusing resulted in two separate bands corresponding to pI values of 7·50 and 7·60, respectively. The enzyme has been shown to contribute to sensory quality and reduced production time of fermented dry sausages. Temperature optimum was 37°C with tributyrin as substrate and 41°C with lard as substrate, the overall activity of the lipase being about three times higher with tributyrin as substrate than with lard. Enzyme activity of the lipase was detectable at pH 4·5 and pH 12, with a pH optimum around 9·3 for both substrates. The enzyme activity was only slightly affected by salt concentrations up to 5% NaCl. The temperature dependence of the enzyme as described by the Arrhenius equation with tributyrin as substrate showed a ΔH2981 (inactivation) of 186 kJ/mol. The activation of the enzyme appears nonlinear with increasing temperature, probably due to changes in availability of the substrate with temperature.

Partial purification and characterization of a cell wall bound proteinase from Lactobacillus casei

The cell wall-bound proteinase from Lactobacillus casei NCDO 151 was partially purified and characterized. Its properties appeared to be different from the two proteolytic components of the same strain described by Ezzat et al. (1988). The proteinase has pH optima at 4·8 using haemoglobin and at 5·6 with casein as substrate, temperature optimum at 35–37° C and is inactivated after 20 min at 50° C. The molecular weight, as estimated from molecular sieve chromatography, is approximately 150 000. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated a tetrameric composition. The isoelectric point is 4·8. Serine proteinase inhibitors strongly inhibit this enzyme, but also metal- chelating compounds affect its activity. The proteinase is thus to be regarded as a serine proteinase. The ions Ca2+ and Co2+ enchance proteinase activity, while Zn2+ and Cu2+ suppress it. The proteinase does not exhibit peptidase activity and it poorly degrades synthetic substrates of other serine proteinases.