Bibek Ray

Direct detection of an antimicrobial peptide ofPediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SummaryAn SDS-PAGE technique is described that allows identification of the antimicrobial activity of a peptide secreted by a strain ofPediococcus acidilactici. This peptide has an antimicrobial property against several baeteria associated with food. This technique enables detection of the specific peptide (or protein) band(s) associated with the inhibitory effect which can then be eluted from the gel for further studies.

Sublethal injury makes Gram‐negative and resistant Gram‐positive bacteria sensitive to the bacteriocins, pediocin AcH and nisin

Gram‐negative and some Gram‐positive bacteria that are resistant to bacteriocins of lactic acid bacteria (LAB) were subjected to sublethal stresses and treated with nisin and pediocin AcH. Both bacteriocins reduced the viability of cells surviving sublethal stresses. The results explain the possible mechanisms by which bacteriocins of LAB enter through the walls (or outer membranes) to destabilize the cytoplasmic (or inner) membranes and kill cells of sensitive Gram‐positive and resistant, but injured, Gram‐negative and Gram‐positive bacteria.

Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria.

High hydrostatic pressure, because it can kill microorganisms, is being investigated for potential use as a nonthermal food preservation method. The objective of this study was to determine the hydrostatic pressurization parameters, pressure, time, and temperature, and a bacteriocin that in combination would destroy 7 to 8 log cycles of pathogenic and spoilage bacterial populations. We suspended cells of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Lactobacillus sake, Leuconostoc mesenteroides, Serratia liquefaciens, and Pseudomonas fluorescens in peptone solution and exposed them to the combination of treatments. The combined parameters used were hydrostatic pressure (138 to 345 MPa), time (5 to 15 min), temperature (25 to 50 degrees C), and pediocin AcH (3,000 AU/ml, final concentration). In general, cell death increased as the pressure, time, or temperature increased; however, the cells developed proportionately greater sensitivity as the pressure increased to 276 MPa and higher and the temperature increased above 35 degrees C. Pressurization for longer than 5 min, especially at lower pressure and temperature ranges, had very little added benefit. Among the four gram-negative species, E. coli O157:H7 was the most resistant to pressurization while among the four gram-positive species, L. sake and L. mesenteroides had greater resistance. The death rate at high pressure (345 MPa) and high temperature (50 degrees C) in combination followed first-order kinetics; at lower pressure and temperature combination it showed a late tailing effect. Estimated D value data indicated that even at 345 MPa and 50 degrees C an 8-log-cycle viability loss could not be achieved within 5 min for all eight species. However, when pediocin AcH was included during pressurization this loss was achieved.

Bacteriocins of Lactic Acid Bacteria in Combination Have Greater Antibacterial Activity

Antibacterial efficiency of two bacteriocins from lactic acid bacteria, pediocin AcH, and nisin was tested individually and in combination against several gram-positive bacterial strains including some involved in food spoilage and foodborne diseases. Pediocin AcH and nisin were more antibacterial in combination than when they were used alone. The principles of this greater antibacterial spectrum have been proposed. Bacteriocins in combinations can be used advantageously to design efficient natural food biopreservative(s).

Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H

N‐terminal analysis of purified pediocin AcH produced a partial sequence of 23 amino acids. This sequence matched perfectly with a segment of 23 amino acids in a 62 amino acid molecule generated from the 186 nucleotide sequence open reading frame in a Hind III fragment in pSMB74 encoding pap‐gene (pediocin AcH production). It is suggested that the molecule is translated as inactive prepediocin AcH of 62 amino acids. Then through enzymatic modifications the leader segment of 18 amino acids is removed from the NH2‐terminal. The remaining segment of 44 amino acids is active pediocin AcH of 4628 Mr.

Cellular Damage in Dried Lactobacillus acidophilus

Lactobacillus acidophilus cells surviving freeze drying and vacuum drying became sensitive to oxgall and lysozyme probably from damage to the cell wall. The dried cells also became sensitive to NaCl and permeable to orthonitrophenol β-galactoside from damage to the cytoplasmic membrane. Scanning electron microscopy indicated loss of some surface material from the damaged cells. Transmission electron microscopy (TEM) revealed partial loss of wall and membrane material, but these losses seemed to have resulted from the treatments given during fixation of cells for TEM and as a consequence of damage to the wall and membrane that occurred during drying. A surface protein of 46-kilodalton molecular weight, that is bound to the wall by hydrogen bonding, was also lost from the dried cells. It is postulated that drying adversely affects some weak bonds of the cellular macromolecules probably from the loss of bound water.

Spoilage of vacuum-packaged refrigerated beef by Clostridium

A motile, gram-positive, spore forming, anaerobic, psychrotrophic bacterial species, probably from the genus Clostridium , was involved in spoilage of vacuum-packaged refrigerated fresh beef. The spoilage was associated with accumulation of large quantities of foul smelling gas and purge in the bag and loss of color and texture of the meat. Attempts to grow the organism in several laboratory media were not yet successful; however, inoculation of purge from a spoiled sample into a fresh beef, vacuum-packaging and refrigeration storage facilitated growth of this species and produced characteristic spoilage of beef.

Impact of bacterial injury and repair in food microbiology: its past, present and future

Studies done during the past 25 years revealed that microorganisms present in semipreserved foods can be injured by sublethal treatments. The injured cells, irrespective of differences in sublethal treatments, have similarities in their manifestation of injury and their repair. A simple resuscitation step incorporated into currently recommended isolation procedures would enable detection of these cells. In the future, microbial cell injury studies should be directed to include not only effective detection of index and pathogenic bacteria from foods, but also growth inhibition of spoilage microorganisms and preservation of lactic cultures.

Complete nucleotide sequence of pSMB 74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici

Several Pediococcus acidilactici strains produce a plasmid‐encoded bacteriocin, pediocin AcH. Previous studies have shown that this plasmid, designated as pSMB 74, encodes genes associated with the production of prepediocin, its post‐translation processing to pediocin AcH, transmembrane translocation of these molecules, and immunity of producer cells against pediocin AcH. We report here the complete nucleotide sequence of pSMB 74. The plasmid has a total of 8877 bp. Four genes have been located on pSMB 74. The genes are arranged in a gene cluster of 3500 bp and share a common promoter and rho‐independent stem‐loop terminator. The four genes, each with independent ribosome binding sites (rbs), initiation and termination codons and spacer sequences in between, were designated as pap A, pap B, pap C and pap D and encode respectively for proteins of 62, 112, 174 and 724 amino acids. The results of this study can be useful either to introduce a suitable marker at a unique restriction site in pSMB 74 and use it as a vector or to clone the pap gene cluster in a suitable plasmid and transform desirable strains for pediocin AcH production. The gene sequence has been submitted to Gene Bank (Acc. No. U02482).

Inhibition of Listeria spp. in sterile food systems by pediocin AcH, a bacteriocin produced by Pediococcus acidilactici H

The effectiveness of pediocin AcH, a bacteriocin produced by Pediococcus acidilactici H, in reducing population levels and growth of Listeria monocytogenes strains and Listeria ivanovii in sterile ground beef, sausage mix, cottage cheese, ice cream, and reconstituted dry milk was examined. Predetermined numbers of Listeria cells and concentrations of pediocin AcH were added to the foods suspended in water and the final volumes made to 10 ml. Listeria in the pediocin AcH treated and control samples during storage at 4 and 10°C were enumerated as CFU on tryptic soy agar. Results indicated that: (a) the maximum bactericidal action of pediocin AcH occurred within 1 h and was not interfered with by the foods; (b) Listeria strains differed in sensitivity to pediocin AcH; (c) reduction in CFU was greater with higher levels of pediocin AcH and as the initial cell number of Listeria spp. decreased; and (d) the surviving cells in pediocin AcH treated food samples multiplied in the presence of residual pediocin AcH during storage at 4 and 10°C.