Antimicrobial Activity of Nisin and Natamycin Incorporated Sodium Caseinate Extrusion‐Blown Films: A Comparative Study with Heat‐Pressed/Solution Cast Films

Antimicrobial edible films based on sodium caseinate, glycerol, and 2 food preservatives (nisin or natamycin) were prepared by classical thermomechanical processes. Food preservatives were compounded (at 65 °C for 2.5 min) with sodium caseinate in a twin‐screw extruder. Anti‐Listeria activity assays revealed a partial inactivation of nisin following compounding. Thermoplastic pellets containing food preservatives were then used to manufacture films either by blown‐film extrusion process or by heat‐press. After 24 h of incubation on agar plates, the diameters of K. rhizophila growth inhibition zones around nisin‐incorporated films prepared by solution casting (control), extrusion blowing or heat pressing at 80 °C for 7 min of nisin‐containing pellets were 15.5 ± 0.9, 9.8 ± 0.2, and 8.6 ± 1.0 mm, respectively. Since heat‐pressing for 7 min at 80 °C of nisin‐incorporated pellets did not further inactivate nisin, this indicates that nisin inactivation during extrusion‐blowing was limited. Moreover, the lower diameter of the K. rhizophila growth inhibition zone around films prepared with nisin‐containing pellets compared to that observed around films directly prepared by solution casting confirms that nisin inactivation mainly occurred during the compounding step. Natamycin‐containing thermoplastic films inhibited Aspergillus niger growth; however, by contrast with nisin‐containing films, heat‐pressed films had higher inhibition zone diameters than blown films, therefore suggesting a partial inactivation of natamycin during extrusion‐blowing.     

Poly(p-tert-butoxycarbonyloxystyrene): a convenient precursor to p-hydroxystyrene resins

An efficient synthetic route to pure, high molecular weight poly(p-hydroxystyrene) is reported. The route involves synthesis of a new monomer, p-tert-butoxycarbonyloxystyrene, polymerization by radical initiation or by cationic initiation in liquid SO₂, followed by thermolysis or acidolysis of the tert-butoxycarbonyl protecting group. Porous, crosslinked resin beads containing the nucleophilic, phenol pendant group have been prepared in a similar fashion from the precursor terpolymer of p-tert-butoxycarbonyloxystyrene, styrene and divinylbenzene. The utility of this resin for solid-phase synthesis has been demonstrated.     

A data base management system for industrial process control

Present-day systems for monitoring energy distribution must be capable of handling continuous changes in the structure and the extent of the data. Only a data base system which satisfies the special requirements of process control can cope with this. The process real-time information management System for on-line Control (PRIMO) data base system described here is implemented on DEC PDP-11 computers from $\text{11}\text{34}$ upwards and is used for process control in Brown Boveri energy control software systems.     

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.     

Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.     

Growth and germination of proteolytic Clostridium botulinum in vegetable-based media

The growth of proteolytic Clostridium botulinum from spore inocula and changes in spore counts in mushroom, broccoli, and potato purées were monitored. Four strains of proteolytic C. botulinum types A and B were inoculated separately at approximately 10(4) spores per ml in nutrient broth and vegetable purées incubated at 15, 20, and 30 degrees C for up to 52 days. The times for the cell populations to increase 1,000-fold (T1,000) in the tested vegetables (1 to 5 days at 30 degrees C, 3 to 16 days at 20 degrees C, 7 to > 52 days at 15 degrees C) were similar to those for meat or fish. Only temperature significantly influenced growth rate. In contrast, the lag phase depended on the strains and media tested, in addition to temperature. Lag times and T1,000S for proteolytic C. botulinum were longer for potato and broccoli purées than for mushroom purée. These differences were not related to different pHs or redox potentials. The germination level, evaluated as the decrease in the spore count, was low. The addition of a germinant mixture (L-cysteine, L-alanine, and sodium lactate) to some strains inoculated in vegetable purées resulted in an increase in germination, suggesting a lack of germination-triggering agents in the vegetable purées.     

Quality control of Aspergillus flavus and A. parasiticus agar and comparison with dichloran 18% glycerol agar: a collaborative study

AFPA culture medium, which is used for recognition of Aspergillus flavus and A. parasiticus, has been validated in a collaborative study including nine laboratories located in Australia, Brazil, Denmark, The Netherlands, Sweden and United Kingdom. Three freeze-dried fungal mixtures, containing A. flavus/A. parasiticus and background fungi, were produced and checked for homogeneity. The coefficients of variance were low, ranging from 0.81% to 1.09% for total fungal counts and between 2.50% and 2.72% for counts of A. flavus/A. parasiticus. The laboratories analysed the contents of two vials of each mixture on commercial A. flavus and A. parasiticus agar (AFPA), in-house-made AFPA, and on a standard media, dichloran 18% glycerol agar (DG18). Reproducibility values for counts of A. flavus/A. parasiticus indicated no differences between the commercial AFPA and the in-house-made AFPA. Variation between laboratories was low, indicating that the medium was effective in use. Reproducibility values for DG18 were higher. There were no differences in counts of A. flavus/A. parasiticus on AFPA and DG18. However, DG18 gave slightly higher total fungal counts compared to AFPA.     

Formation of toxic 2-nonyl-p-benzoquinones from α-tertiary 4-nonylphenol isomers during microbial metabolism of technical nonylphenol

In many environmental compartments, microbial degradation of α-quaternary nonylphenols proceeds along an ipso-substitution pathway. It has been reported that technical nonylphenol contains, besides α-quaternary nonylphenols, minor amounts of various α-H, α-methyl substituted tertiary isomers. Here, we show that potentially toxic metabolites of such minor components are formed during ipso-degradation of technical nonylphenol by Sphingobium xenophagum Bayram, a strain isolated from activated sewage sludge. Small but significant amounts of nonylphenols were converted to the corresponding nonylhydroquinones, which in the presence of air oxygen oxidized to the corresponding nonyl-p-benzoquinones-yielding a complex mixture of potentially toxic metabolites. Through reduction with ascorbic acid and subsequent analysis by gas chromatography-mass spectrometry, we were able to characterize this unique metabolic fingerprint and to show that its components originated for the most part from α-tertiary nonylphenol isomers. Furthermore, our results indicate that the metabolites mixture also contained several α, β-dehydrogenated derivatives of nonyl-p-benzoquinones that originated by hydroxylation induced rearrangement, and subsequent ring and side chain oxidation from α-tertiary nonylphenol isomers. We predict that in nonylphenol polluted natural systems, in which microbial ipso-degradation is prominent, 2-alkylquinone metabolites will be produced and will contribute to the overall toxicity of the remaining material.