Nisin Preparation

Nisin Preparation occurs as a white, free-flowing powder. It is a mixture of closely related polypeptides produced by strains of the Lactococcus lactis subsp. lactis Lancefield Group N in a sterilized milk-culture medium. Nisin in the fermentation broth can be recovered by various methods, such as injecting sterile, compressed air (froth concentration) acidification salting out and spray-drying. 

Assay Not less than 900 IU of Nisin per milligram of Nisin Preparation. 

Tolerance o/Lactococcus lactis to High Concentrations of Nisin Prepare cultures of Lactococcus lactis (ATCC 11454, NCIMB 8586) in sterile skim milk by incubating for 18 h at 30°. Prepare one or more flasks containing 100 mL of litmus milk, and sterilize at 121° for 15 min. Suspend 0.1 g of sample in the sterilized litmus milk, and allow to stand at room temperature for 2 h. Add 0.1 mL of the L. lactis culture, and incubate at 30° for 24 h. L. lactis will grow in this concentration of sample (about 1000 IU/mL) however, it will not grow in similar concentrations of other antimicrobial substances. This test will not differentiate Nisin from subtilin. 

Food and Drug Administration. Nisin preparation Affirmation of GRAS status as a direct human food ingredient. Fed Reg 1988 53 11247-11251. 

Nisin has been applied successfully to prepare sterile beverage-quality chocolate milk. The antibiotic serves as a sterilization aid because it inhibits outgrowth of heat-damaged spores and so permits use of less drastic heat treatments for sterilization (Heinemann et al. 1964). 

Heinemann, B., Stumbo, C. R. and Scurlock, A. 1964. Use of nisin in preparing beverage-quality sterile chocolate-flavored milk. J. Dairy Sci. 47, 8-12. 

Nisin Standard Solutions Suspend 100 mg of Nisin International Reference Preparation3 (1000 IU of Nisin per milligram), accurately weighed, in 80 mL of 0.02 N hydrochloric acid. Set aside at room temperature for 2 h. Dilute the suspension to a final volume of 100.0 mL with 0.02 N hydrochloric acid. This standard stock solution contains 1000 IU of Nisin per milliliter. From this solution, pipet 0.5, 1.0, 2.5, 5.0, and 10.0 mL into separate 1000-mL volumetric flasks. Dilute each flask to volume with 0.02 N hydrochloric acid to obtain Nisin Standard Solutions with concentrations of 0.5, 1.0, 2.5, 5.0 and 10.0 IU/mL. Store the standard stock solution for up to 7 days at 4°, or prepare a fresh solution each day. 

Stability to Acid Suspend a 100-mg sample in 0.02 N hydrochloric acid as described in the preparation of the Nisin Standard Solutions under the Assay. Boil this solution for 5 min. Using the Assay method described above, determine the Nisin concentration. No significant loss of activity is noted following this heat treatment. The calculated Nisin concentration of the boiled sample is 100% 5% of the Assay value. Adjust the pH of the Nisin solution to 11.0 by adding 5 N sodium hydroxide. Heat the solution at 65° for 30 min, and... 

The Nisin International Reference Preparation is available from the WHO International Laboratory for Biological Standards, Ministry of Agriculture, Fisheries Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, England. 

Colas JC, Shi WL, Rao VSNM, Omri A, Mozafari MR, Singh H (2007) Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting. Micron 38 841-847... 

Antimicrobial Edible films were prepared from natural fiber of pectin and other food hydrocolloids for food packaging or wrapping by extrusion followed by compression or blown film method. Microscopic analysis revealed a well mixed integrated structure of extruded pellets and an even distribution of the synthetic hydrocolloid in the biopolymers. The resultant composite films possess the mechanical properties that are comparable to films cast from most natural hydrocolloids that consumed as foods or components in processed foods. The inclusion of polyethylene oxide) alters the textures of the resultant composite films and therefore, demonstrating a new technique for the modification of film properties. The composite films were produced in mild processing conditions, thus, the films are able to protect the bioactivity of the incorporated nisin, as shown by the inhibition of Listeria monocytogenes bacterial growth by a liquid incubation method. 

Also in Osaka, another former student of Akabori, professor Tetsuo Shiba (Plate 43) and his associates developed a procedure for the preparation of lanthionine-containing peptides and applied it, in 1988, to the synthesis of the antibiotic nisin (p. 223). In Tokyo, at Rikkyo University, professor Ichiro Muramatsu, also from the Akabori school, proposed a rapid method of synthesis and discovered novel side reactions, such as the formation of guanidine derivatives during coupling with carbodiimides. 

Preparation of Glucomannan-Chitosan-Nisin Ternary Film.199... 

Konjac glucomannan was dissolved in distilled water and filtered out leaving a concentration of 1% (w/w). Chitosan dissolved in 0.8% (w/w) aqueous acetic acid to prepare 1% (w/w) solution. The solutions of konjac glucomannan/chitosan with different mixing ratios [9 1,8 2,7 3,6 4,5 5,4 6,3 7,2 8,1 9 konjac glucomannan/chitosan (w/w) were cast onto polystyrene plates and dried at room temperature and then vacuum dried for 48h to obtain the films. Nisin was incorporated into pure konjac glucomannan, chitosan film or the selected blend film forming solution at various levels to obtain antimicrobial films. 

Chitosan film was prepared by dissolving 1 g of shrimp chitosan in lOOmL of 1% acetic acid solution. The solution was filtered through a silk screen. The three antimicrobial agents—garlic oil, potassium sorbate, and nisin— were incorporated into chitosan film forming solution at various levels. The solutions were cast in polyacrylic plates and dried. The dry films obtained were peeled off and stored in a chamber at 50% RH and 25°C until evaluation. 

Li, B., Kennedy, J. F., Peng, J. L., Yie, X., and Xie, B. J. 2006. Preparation and performance evaluation of glucomannan-chitosan-nisin ternary antimicrobial blend film. Carbohydrate Polymers, 65(4) 488-494. 

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