Ascopyrone P, a novel antibacterial derived from fungi.

AIMS: To assess the antimicrobial efficacy of ascopyrone P (APP), a secondary metabolite formed by the fungi Anthracobia melaloma, Plicaria anthracina, Plic. leiocarpa and Peziza petersi belonging to the order Pezizales. METHODS AND RESULTS: In vitro testing using a well diffusion procedure showed that APP at a high concentration (approximately 5%) inhibited the growth of Gram-positive and Gram-negative bacteria. Using an automated microbiology reader, growth curve analysis showed that 2000-4000 mg l(-1) APP caused total or significant bacterial inhibition after incubation for 24 h at 30 degrees C. Against certain yeast strains, 1000- 2000 mg l(-1) APP enhanced growth, although at higher concentrations inhibition of some yeasts was observed. Clostridium and fungal strains were not sensitive to 2000 mg l(-1) APP. No significant cidal effect was observed after 2 h against Listeria monocytogenes or Escherichia coli. Results were identical whether the APP samples tested had been produced enzymatically or chemically. CONCLUSIONS: At a level of 2000 mg l(-1), APP demonstrated growth inhibitory activity against a broad range of bacteria, but not yeasts or moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible application for this novel natural antimicrobial is in food preservation, to control the growth of Gram-negative and Gram-positive bacteria in raw and cooked foods. Effective dosage levels would be 500-4000 mg kg(-1), depending on food type. The efficacy, organoleptic and safety aspects of this compound in food still need to be assessed.

The effect of nisin on the keeping quality of reduced heat-treated milks.

Milk was subjected to a combination process involving reduced heat treatment (RHT) of 117 degrees C for 2 s and nisin (75 and 150 IU ml(-1)). The microbial activity and other quality aspects were compared with a RHT control (without nisin) and with a ultrahigh temperature (UHT) milk processed at 142 degrees C for 2 s. Nisin was found to inhibit microbial growth for products stored without refrigeration, and RHT-nisin samples stored at 30 degrees C showed very low spoilage rates during 150 days, although not low enough to satisfy requirements for commercial sterility. RHT-nisin samples could be distinguished from and were preferred to the UHT control. Significant browning occurred during storage at 30 degrees C and above but was less in the RHT-nisin milk samples compared with the UHT milk. In RHT-nisin milk samples stored at 20 and 10 degrees C, no microbial activity could be detected in most samples after storage for 1 year. The effectiveness of this combination of RHT, nisin, and low storage temperatures against gram-positive spore-forming bacteria suggests potential for use of nisin in extended shelf life products.

Effective use of nisin to control lactic acid bacterial spoilage in vacuum-packed bologna-type sausage.

Lactic acid bacteria (LAB) commonly cause spoilage in minimal heat-treated vacuum-packed cured delicatessen meats. Predominant species are Lactobacillus sake and L. curvatus. LAB strains isolated from spoiled products of this type (liver sausage, ham and bologna sausage) were found to be sensitive to low nisin concentrations (maximum of 1.25 microg g(-1)). Addition of 25 microg g(-1) nisin (as Nisaplin) inhibited the growth of LAB spoilage organisms inoculated into vacuum-packed pasteurized bologna-type sausages stored at 8 degrees C. Control sausages became spoiled (>10(8) LAB CFU g(-1)) by day 7, whereas sausages containing nisin remained unspoiled for >50 days. The effect of three types of phosphates (used as emulsifiers) on nisin activity in the sausages was compared. LAB growth rate was fastest in samples containing orthophosphate, and slowest in sausages containing diphosphate. The shelf life was also greatly extended in the latter. Fat content also affected nisin activity. Nisin activity (as indicated by LAB inhibition) was greatest in samples containing 15% > 25% > 37% (wt/wt) fat. In a sausage formulation containing 37% fat and incorporating diphosphate as emulsifier, levels of nisin as low as 2.5 microg g(-1) showed antibacterial effects. A nisin level of 6.25 microg g(-1) totally inhibited LAB growth for over 4 weeks and 25 microg g(-1) for 5 weeks. Spoilage control was achieved in the same sausage formulation but with 25% (wt/wt) fat; 12.5 microg g(-1) nisin prevented LAB growth for 5 weeks.

Combined effect of nisin and moderate heat on destruction of Listeria monocytogenes in cold-pack lobster meat.

The combined effect of nisin and moderate heat to increase the killing of Listeria monocytogenes in cans of "cold-pack" lobster was investigated. Adding nisin at a level of 25 mg/kg of can contents to the brine surrounding the lobster, in combination with a heat process giving internal can temperatures of 60 degrees C for 5 min and 65 degrees C for 2 min, resulted in decimal reductions of inoculated L. monocytogenes of 3 to 5 logs, whereas heat or nisin alone resulted in decimal reductions of 1 to 3 logs. Such a reduced heat process to that currently commercially used (65.5 degrees C for 13 to 18 min, depending on the can size) results in significant reduction in drained weight loss, thus allowing considerable cost savings to the lobster-processing industry.

Synergist effect of sucrose fatty acid esters on nisin inhibition of gram-positive bacteria.

Nisin in combination with the sucrose fatty acid esters, sucrose palmitate (P-1570 and P-1670) or sucrose stearate (S-1570 and S-1670) was tested against a range of Gram-negative and Gram-positive bacteria. Initial liquid culture investigation showed that the sugar ester P-1670 resulted in a synergist enhancement of the bacteriostatic activity of nisin against Gram-positive bacteria and not Gram-negative bacteria. Some enhancement of the bactericidal activity of nisin against Listeria monocytogenes was also observed. This increased nisin inhibitory effect was confirmed on solid media using plates with gradients of pH and NaCl. Synergism was observed with all four sucrose fatty acid esters, which enhanced the antimicrobial activity of nisin against several strains of L. monocytogenes, Bacillus cereus (both cells and spores), Lactobacillus plantarum and Staphylococcus aureus. The combination of nisin and the sucrose fatty acid esters showed no inhibition of Gram-negative bacteria (Salmonella enteritidis, Salm. typhimurium and Pseudomonas aeruginosa).

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes.

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6-8 degrees C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5.9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin pre-production to the milk. Each batch of cheese was inoculated with 10(2)-10(3) cfu g-1 of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2.5 mg l-1 could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1-2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6-8 degrees C, with only 10-32% nisin loss.

Applications of the bacteriocin, nisin.

Nisin was first introduced commercially as a food preservative in the UK approximately 30 years ago. First established use was as a preservative in processed cheese products and since then numerous other applications in foods and beverages have been identified. It is currently recognised as a safe food preservative in approximately 50 countries. The established uses of nisin as a preservative in processed cheese, various pasteurised dairy products, and canned vegetables will be briefly reviewed. More recent applications of nisin include its use as a preservative in high moisture, hot baked flour products (crumpets) and pasteurised liquid egg. Renewed interest is evident in the use of nisin in natural cheese production. Considerable research has been carried out on the antilisterial properties of nisin in foods and a number of applications have been proposed. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production and low pH foods such as salad dressings. Further developments of nisin are likely to include synergistic action of nisin with chelators and other bacteriocins, and its use as an adjunct in novel food processing technology such as higher pressure sterilisation and electroporation. Production of highly purified nisin preparations and enhancement by chelators has led to interest in the use of nisin for human ulcer therapy, and mastitis control in cattle.

The Use of Nisin as a Preservative in Crumpets.

Crumpets, a high moisture flour based product, have been implicated in food poisoning due to growth and toxin production by naturally occurring Bacillus cereus during 5-day storage at ambient temperature. Bacillus cereus isolates from untreated crumpets at the end of their shelf-life were shown to be sensitive to nisin. Addition of nisin to the batter at levels of 3.75 µg/g and above effectively prevented the growth to levels capable of causing food poisoning. The fate of nisin during the production and shelf-life of the crumpet was determined.

The use of the bacteriocin, nisin, as a preservative in pasteurized liquid whole egg.

Nisin used at a level of 5 mg/1 resulted in a significant increase in refrigerated shelf life of pasteurized liquid whole egg from between 6-11 d to 17-20 d. In the first of two trials, nisin also protected the liquid egg from growth of Bacillus cereus. Bacillus cereus was not presented in the egg in the second trial. Effective residual levels of nisin were detected in the liquid egg post-pasteurization.