Applications of antimicrobial bactricocin nisin

Nisin is effective against a broad range of Gram-positive bacteria, including spore-forming bacteria, such as C. botulinum and B. cereus, and used to inhibit vegetative growth from spores in processed cheese, meats, beverages,and some canned foods.

Applications

Processed Cheese Products

Nisin has been established as a most effective preservative in pasteurized processed cheese products, including block cheese, slices, spreads, sauces, and dips. This is because typical heat processing (85–105 C for 5–10 min) of the raw cheese during melting does not eliminate spores. Without the addition of nisin, the composition of the pasteurized processed cheese would favor the outgrowth of the spores. Spore formers associated with processed cheese include Clostridium butyricum, Clostridium tyrobutyricum, Clostridium sporogenes, and Clostridium botulinum. Spore outgrowth of the first three species may result in spoilage due to the production of gas, off odors, and liquefaction of the cheese, whereas C. botulinum more seriously produces a potentially fatal toxin. The level of nisin required to inhibit the outgrowth of spores in processed cheese and other products depends on a number of factors:the level of clostridial spores present, the composition of the food, for example, NaCl, disodium phosphate, pH, and moisture content; the shelf life required, and the temperature of storage. Generally, levels of nisin used to control nonbotulinal spoilage in processed cheese vary from 6.25 to 12.5 mg/kg. For anti-botulinum protection, the level required is 12.5 mg /kg or higher.

Other Pasteurized Dairy Products

Other pasteurized dairy products, such as chilled desserts,cannot be subjected to full sterilization without damaging their organoleptic qualities, and are thus sometimes preserved with nisin to extend their shelf life. For example, tests on chocolate dairy dessert demonstrated a 20-day increase in shelf life with 3.75 mg/kg of nisin added at 7 C. Similarly, canned evaporated milk has an extended shelf life with added nisin. The addition of nisin to milk is permitted in some countries because of shelf-life problems associated with the climate (high ambient temperatures), long-distance transport, and inadequate refrigeration. The use of nisin at levels of 0.75–1.25 mg/litres has been demonstrated to more than double the shelf life of the product. However, it is not permitted in the European Union, the United States, and other countries with temperate climates. The addition of nisin to high-heat-treated flavored milk has also been shown to extend shelf life.

Pasteurised liquid egg products

Pasteurised liquid egg products may contain the whole egg, the yolk or the white. The heat treatment applied (62-65 C for 2-3 min) kills salmonella but not all bacterial spores and vegetative cells. Many of the surviving bacteria are psychrotrophic, so pasteurised liquid egg products usually have a limited shelf life. In a trial with pasteurised liquid whole egg, nisin (5 mg/l) significantly extended the refrigerated shelf life (>60 days). Nisin also protected the egg against the growth of psychrotrophic Bacillus cereus.

High moisture hot plate bakery products

Typical hotplate bakery products include crumpets and potato cakes. They are flour-based, high moisture (48-56%), non-acidic (pH 6-8) and lightly cooked on a hotplate during production. They are sold at room temperature and are traditionally toasted before eating. The flour used to make these products always contains low levels of Bacillus spores and conditions are ideal for growth, so it is not surprising that outbreaks of food poisoning associated with B. cereus toxins have been reported.

These bakery products have a short shelf life (3-5 days), but a recent study in the UK found very high levels of Bacillus (108 cfu/g) in potato cakes well within the sell-by date. The popularity of crumpets in Australia and the risk of B. cereus food poisoning, exacerbated by high ambient temperatures, led to factory trials with nisin. The addition of nisin to crumpet dough at concentrations of 3.75 mg kg1 and above effectively inhibited the growth of B. cereus to safe levels. This led to regulations in Australia allowing the use of nisin in such high-moisture baked goods.

Canned Foods

Nisin is used in canned foods principally to control thermophilic spoilage. It is mandatory in most countries that lowacid canned foods (pH > 4.5) should receive a minimum heat process (F0=3) to ensure the destruction of C. botulinum spores. The survival of heat-resistant spores of the thermophiles Geobacillus stearothermophilus and Thermoanaerobacterium thermosaccharolyticum during this process are responsible for spoilage, particularly under warm conditions. The bacterial spoilage of high-acid foods (pH < 4.5) is restricted to nonpathogenic, heat-resistant, aciduric, sporeforming bacterial species, such as Clostridium pasteurianum,Bacillus macerans, and Bacillus coagulans. Spoilage resulting from the growth of all these bacteria can be effectively controlled by nisin. Addition levels are generally between 2.5 and 5.0 mg kg1 and product examples include canned vegetables, soups, coconut milk, and cereal puddings (such as rice,semolina, and tapioca).

Meat and Fish Products

In relation to processed meat products, nisin has been considered as an alternative preservative system to that of nitrite, due to concerns about toxicological safety. However,various studies have shown that nisin does not perform at its full potential in meat systems. Results generally indicate that nisin is only effective at high levels, that is, 12.5 mg/kg and above. Proposed reasons for the inadequacy of a nisin preservative system in meats include: poor solubility in meat systems;binding of nisin onto meat particles and surfaces; uneven distribution; and possible interference with nisin’s mode of action by phospholipids. However, both modified atmosphere and vacuum packaging in combination with nisin have shown more promising results.

Relatively few studies have been carried out on the use of nisin as a preservative of fish and shellfish. However, the potential hazard of botulism from chilled fish packed under vacuum or modified gas atmospheres prompted a trial application of nisin by spray to fillets of cod, herring, and smoked mackerel inoculated with C. botulinum type-E spores. Toxin production was delayed by 5 days compared with the control at 10 C, but only by half a day at 26 C. Recent research into the application of nisin to canned lobster meat, to control Listeria monocytogenes, has been very positive. Heat processing of canned lobster, which is retailed frozen, can only be achieved by heating at 60 C for 5 min without undesirable product shrinkage occurring. Such heat processing results in a 2 log reduction of L. monocytogenes, whereas the addition of nisin to the brine at 25 mg/l increases the reduction by 5–6 logs.

Natural Cheese

Nisin can be used to prevent blowing in some hard and semihard ripened cheeses, such as Emmental and Gouda.This blowing is caused by contamination with the anaerobic spore-formers C. butyricum and C. tyrobutyricum, usually from a milk source when the cow has been fed with silage. The bacteria convert lactic acid into butyric acid, which causes the off-flavor and aroma of the cheese. The formation of H2 and CO2 gas during ripening also results in the development of too many large holes in the cheese. Cheeses can also be contaminated with Lactobacillus spp., causing off-flavors and gas production, and with the food-poisoning pathogens L. monocytogenes and Staphylococcus aureus, all of which are susceptible to nisin.

The use of nisin is an attractive alternative to other agents, including sodium nitrate, which has become increasingly unpopular and usually only works against specific microorganisms (e.g., Clostridium). However, nisin resistant starter cultures must be used in conjunction with nisin to ensure successful development of the cheese. The use of nisin producing starter cultures to manufacture cheese with significant levels of nisin is also being investigated. At present, no existing nisin-producing starters have the flavor-generating, eye-forming, and acidifying activities and the bacteriophage resistance that are suitable for the manufacture of most cheese types. However, a nisin producing starter culture for Gouda production has been developed using the food-grade genetic transfer technique of conjugation. During production, clostridial blowing and S. aureus growth were both inhibited over the whole period of ripening.

Soft white fresh cheeses (e.g., ricotta, paneer) do not require starter cultures, being alternatively coagulated by direct acidification, calcium chloride, or rennet. In these cheeses, nisin very effectively controls the growth of L. monocytogenes. Shelf-life analysis of ricotta in an inoculated trial demonstrated that the addition of 2.5 mg/l nisin to the milk preproduction could effectively inhibit the growth ofL.monocytogenes at 6–8/C for at least 8 weeks. Ricotta made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation.

Yogurt

The addition of nisin to stirred yogurt postproduction has an inhibitory effect on the starter culture (a mixture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains), thereby preventing subsequent overacidification of the yogurt. Thus, an increase in shelf life is attained by maintaining the flavor of the yogurt (less sour)

Salad Dressings

The development of salad dressings with reduced acidity gives improved flavor and protects the added ingredients. However,raising the pH (from 3.8 to 4.2) can make salad dressings prone to lactic acid bacterial spoilage during ambient storage. Such growth has been successfully controlled by the addition of nisin at levels of 2.5–5 mg/ l.

Alcoholic Beverages

Nisin has a potential role in the production of alcoholic beverages. It has been demonstrated that nisin is effective in controlling spoilage by lactic acid bacteria, such as Lactobacillus, Pediococcus, Leuconostoc, and Oenococcus at a level of 0.25–2.5 mg/ l in both beer and wine. Yeasts are completely unaffected by nisin, which allows its addition during the fermentation. Identified applications of nisin in the brewing and wine industry include: its addition to fermenters to prevent or control contamination, increasing the shelf life of unpasteurized beers, reducing pasteurization regimes, and washing pitching yeast to eliminate contaminating bacteria (as an alternative method to acid washing,which affects yeast viability). Formerly, nisin could not be used during wine fermentations that depend on malolactic acid fermentation. However, this problem has been overcome by developing nisin-resistant strains of Oenococcusoenos, which can grow and maintain malolactic fermentation in the presence of nisin. In the production of fruit brandies, the addition of nisin reduces the growth of competitive lactic acid bacteria and directly favors the growth of the fermenting yeast, to increase alcohol content by at least 10%.

Reference: ENCYCLOPEDIA OF FOOD MICROBIOLOGY