Mark A. Daeschel

Nisin resistance of foodborne bacteria and the specific resistance responses of Listeria monocytogenes Scott A

Eight foodborne pathogenic and spoilage type gram-positive bacteria were evaluated for their spontaneous resistance frequencies to the peptide antimicrobial nisin. In brain heart infusion medium, spontaneous nisin resistance frequencies were in the range of 10-6 to 10-8 when exposed to nisin at concentrations 2 to 8 times the minimal inhibitory concentrations. A resistant mutant of Listeria monocytogenes Scott A (2000 U nisin per ml) was obtained by increasing stepwise exposure to nisin and was subsequently characterized. Nisin was not inactivated after exposure to mutant or parent cells growing in brain heart medium. Membrane fatty acid composition, phase transition temperature profiles (by differential scanning calorimetry), and specific growth rates of the resistant mutant and parent strain were compared. The resistant mutant had a higher phase transition temperature, higher percentage of straight-chain fatty acids, and a lower percentage of branched chain-fatty acids. The specific growth rate (k) of the resistant mutant was significantly decreased at the suboptimal temperature of 20°C where (k) was 40.9% of the parent strain k. In contrast, at 37°C, the mutant (k) was 87.6% of the parent (k). Collectively, these observations indicated that as a resistance response to nisin, fundamental changes occurred in bacterial membrane structure and function as opposed to a resistance response involving nisin degradation.

Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics

Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwalpha (comprising 29 residues) and Plwbeta (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwalpha, and Plwbeta contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.

FISH SAUCE PRODUCTS AND MANUFACTURING: A REVIEW

Fish sauce, due to its characteristic flavor and taste, is a popular condiment for cooking and dipping. Biochemically, fish sauce is salt-soluble protein in the form of amino acids and peptides. It is developed microbiologically with halophilic bacteria, which are principally responsible for flavor and aroma. This review article covers the manufacturing methods of fish sauce, factors affecting the quality of fish sauce, nutritional values of fish sauce, microorganisms involved with fermentation, and flavor. In addition, rapid fermentation to reduce time and new parameters to estimate the quality of fish sauce are reviewed. Along with a new approach for estimating the quality of fish sauce, the quantitative analysis of degradation compounds from ATP and other specific protein compounds in fish sauce are discussed.

Antimicrobial activity of nisin adsorbed to hydrophilic and hydrophobic silicon surfaces

The antimicrobial activity of nisin was studied after its adsorption to hydrophilic and hydrophobic silicon surfaces. Adsorption was allowed to occur from buffered nisin solutions under static conditions, and the adsorbed mass of nisin was calculated from the resultant film thickness and refractive index, determined using ellipsometry. Once adsorbed, nisin was observed to be stable to buffer rinsing; the amount of nisin adsorbed onto each type of surface was determined to be of a quantity sufficent for inhibition of susceptible bacteria. Antimicrobial activity was maintained both upon silicon surface contact with microbial media and after nisin desorption induced by surfactant displacement.

Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides.

A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed alpha and beta, which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the alpha and beta peptides. From the N-terminal end, 21 and 22 amino acid residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, alpha and beta may be encoded by the same gene, since alpha appeared to be a truncated form of beta. Alanine, the first amino acid residue at the N-terminal end of beta was not present at this position in alpha. Otherwise the sequences of alpha and beta appeared to be identical. The calculated molecular masses of the sequenced part of alpha and beta were 2426 and 2497 Da, respectively. The molecular masses of alpha and beta as determined by mass spectroscopy were 2687 +/- 30 and 2758 +/- 30 Da, respectively, indicating that (i) the only difference between alpha and beta was the presence of the N-terminal alanine residue in beta, and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the alpha and beta peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of Cellular Phospholipid Content with Nisin Resistance of Listeria monocytogenes Scott A

A nisin-resistant mutant of Listeria monocytogenes Scott A has been characterized by comparing its phospholipid composition with the nisin-sensitive parental strain. The total phospholipids of resistant cells were significantly (P < 0.001) decreased compared to the parental strain. The types of phospholipids isolated from nisin-resistant and sensitive cells were identical, but there was a significant decrease (P < 0.01) in the amount of three individual phospholipids. Nisin-resistant cells were found to bind less nisin and release less phospholipids than sensitive cells when treated with same concentrations of nisin. The cell surface of resistant cells was less hydrophobic compared to sensitive cells, which also may have contributed to the observed nisin resistance. The results suggest that fundamental changes occurred in the membrane structure and function of the resistant mutant as a response to nisin.

Edible Coatings for Enhancing Microbial Safety and Extending Shelf Life of Hard-Boiled Eggs

Hard-boiled eggs were coated with chitosan-lysozyme (CL), whey protein isolate (WPI), or Bake sheen (BS), inoculated with Listeria monocytogenes or Salmonella enterica Ser. Enteritidis at 10(4) CFU/g, and stored for 4 wk at 10 degrees C. Microbial populations were enumerated weekly. Two nonchallenge studies were also conducted with hard-boiled eggs: coated shell-on eggs packaged in plastic containers or peeled eggs coated and vacuum-packaged. Total plate counts (TPC), coliforms, yeasts, molds, weight loss, pH, and color of eggs were determined during the 10-wk storage at 10 degrees C. Uncoated eggs served as controls. All the coatings were not effective in inhibiting the growth of L. monocytogenes, but CL coating controlled the growth of S. Enteritidis. At the end of 4-wk storage, the numbers of S. enteritidis on CL-coated eggs were about 4-log(10) CFU/g less than that of the controls. Coatings reduced (P < 0.05) the populations of coliforms and TPC, and completely inhibited mold growth during the 10-wk storage. Coatings also reduced (P < 0.05) the weight loss of eggs, 4.1% to 4.8% on coated eggs compared with 7.5% in uncoated ones at the end of 10-wk storage. The pH of CL-coated eggs remained stable throughout the storage period, while the control eggs increased from 7.6 to 8.6. Color changes in CL- and WPI-coated eggshells were less (P < 0.05) than those of BS-coated and the control. The CL coating effectively suppressed the numbers of TPC, coliforms, yeasts, and molds on peeled eggs during the 6-wk storage (P < 0.05). The results suggest that CL coating on hard-boiled eggs can control the growth of S. Enteritidis and reduce undesirable changes in the interior quality of eggs.

Applications of Bacteriocins in Food Systems

Renewed interest in the use of bactericidal proteins (bacteriocins) as food protection and preservation agents is due in part to new food safety challenges, the affirmation of the bacteriocin Nisin as a GRAS additive in certain foods, the potential of using molecular biology to manipulate bacteriocin production, and a growing interest in so-called “natural” food preservatives. Bacteriocins may be introduced into foods by different mechanisms: 1) In lactic fermented foods the bacteriocins may be present as a result of the growth of bacteriocinogenic lactic acid bacteria. 2) Bacteriocins may be introduced as a constitutent of lactic food fermentation by-products (such as whey) into other foods. 3) Bacteriocins may be purified, concentrated and added directly as an additive. The activity of bacteriocins in foods will be highly dependent on physical and chemical factors such as pH, ionic strength, solubility and temperature. Bacteriocins as proteins may also be susceptible to enzymatic degradation and non-specific binding. The narrow spectrum of activity of certain bacteriocins is also a limiting factor in food applications. An effective bacteriocin for use as a food preservative should meet minimum requirements such as: 1) Non-toxic. 2) Stable and highly active. 3) Possessing a broad spectrum of activity. 4) No effect on sensory attributes of food. 5) Inexpensive and simple to use. The bacteriocin Nisin has shown the most promise as an effective food preservative, however, other bacteriocins produced by lactic acid bacteria are now being investigated for potential application in foods.

Reduction of Salmonella enterica on Alfalfa Seeds with Acidic Electrolyzed Oxidizing Water and Enhanced Uptake of Acidic Electrolyzed Oxidizing Water into Seeds by Gas Exchange

Alfalfa sprouts have been implicated in several salmonellosis outbreaks in recent years. The disinfectant effects of acidic electrolyzed oxidizing (EO) water against Salmonella enterica both in an aqueous system and on artificially contaminated alfalfa seeds were determined. The optimum ratio of seeds to EO water was determined in order to maximize the antimicrobial effect of EO water. Seeds were combined with EO water at ratios (wt/vol) of 1:4, 1:10, 1:20, 1:40, and 1:100, and the characteristics of EO water (pH, oxidation reduction potential [ORP], and free chlorine concentration) were determined. When the ratio of seeds to EO water was increased from 1:4 to 1:100, the pH decreased from 3.82 to 2.63, while the ORP increased from +455 to +1,073 mV. EO water (with a pH of 2.54 to 2.38 and an ORP of +1,083 to +1,092 mV) exhibited strong potential for the inactivation of S. enterica in an aqueous system (producing a reduction of at least 6.6 log CFU/ml). Treatment of artificially contaminated alfalfa seeds with EO water at a seed-to-EO water ratio of 1:100 for 15 and 60 min significantly reduced Salmonella populations by 2.04 and 1.96 log CFU/g, respectively (P < 0.05), while a Butterfields buffer wash decreased Salmonella populations by 0.18 and 0.23 log CFU/g, respectively. After treatment, EO water was Salmonella negative by enrichment with or without neutralization. Germination of seeds was not significantly affected (P > 0.05) by treatment for up to 60 min in electrolyzed water. The uptake of liquid into the seeds was influenced by the internal gas composition (air, N2, or O2) of seeds before the liquid was added.

Effects of Water, Sodium Hypochlorite, Peroxyacetic Acid, and Acidified Sodium Chlorite on In-Shell Hazelnuts Inoculated with Salmonella Enterica Serovar Panama

Recent foodborne disease outbreaks involving minimally processed tree nuts have generated a need for improved sanitation procedures. Chemical sprays and dips have shown promise for reducing pathogens on fresh produce, but little research has been conducted for in-shell hazelnuts. This study analyzed the effectiveness of 3 chemical sanitizers for reducing Salmonella on in-shell hazelnuts. Treatments of water, sodium hypochlorite (NaOCl; 25 and 50 ppm), peroxyacetic acid (PAA; 80 and 120 ppm), and acidified sodium chlorite (ASC; 450, 830, and 1013 ppm) were sprayed onto hazelnut samples inoculated with Salmonella enterica serovar Panama. Hazelnut samples were immersed in liquid cultures of S. Panama for 24 h, air-dried, and then sprayed with water and chemical treatments. Inoculation achieved S. Panama populations of approximately 8.04 log CFU/hazelnut. Surviving S. panama populations were evaluated using a nonselective medium (tryptic soy agar), incubated 3 h, and then overlaid with selective media (xylose lysine deoxycholate agar). All of the chemical treatments significantly reduced S. Panama populations (P ≤ 0.0001). The most effective concentrations of ASC, PAA, and NaOCl treatments reduced populations by 2.65, 1.46, and 0.66 log units, respectively. ASC showed the greatest potential for use as a postharvest sanitation treatment.