What is Nisin?
Nisin is a natural biological antimicrobial peptide produced by the fermentation of nisin naturally occurring in milk and cheese. It has a broad-spectrum antibacterial effect and can effectively inhibit most gram-positive bacteria. and the growth and reproduction of its spores. In particular, it has a significant inhibitory effect on common bacteria such as Staphylococcus aureus, hemolytic Streptococcus, and Clostridium botulinum, and can play a preservative and preservation role in many foods. Moreover, nisin has good stability, heat and acid resistance, and has good application prospects in the food industry.
Nisin is a world-recognized and safe natural biological food preservative and antibacterial agent. It is mainly used for the preservation and preservation of milk and dairy products, meat and meat products. The discovery of nisin dates back to the 1920s. In 1928, American researchers such as LA. Rogers first reported that nisin metabolites can inhibit the growth of other lactic acid bacteria. In 1947, ATR Mattick et al. discovered that some lactic acid streptococci in the serological N group could produce protein-based bacteriostatic substances, and prepared this polypeptide substance from the lactic acid streptococcus fermentation broth, because it was produced by the lactic acid bacteria in the N group. It is an antibacterial substance, so it is named N-inhibitory Substance, that is, N group antibacterial substance, abbreviated as Nisin.
White nisin powder
Nisin is a natural product of nisin. Studies on the toxicity of nisin in amounts far exceeding food applications have shown that it is non-toxic. Because it is particularly sensitive to proteolytic enzymes (α-trypsin), it can be quickly hydrolyzed into amino acids by proteolytic enzymes in the digestive tract after consumption. In 1953, Nisaplin, the first commercial product of nisin, was launched in the UK; in 1969, the FAO/WHO Joint Expert Committee on Food Additives approved nisin as a food additive; in 1988, the U.S. Food and Drug Administration (FDA) ) also officially approved the use of nisin in food; in 1990, the Food Supervision Department of the Ministry of Health of my country issued a certificate of conformity for the use of nisin in China. Currently, more than 50 countries have approved the use of nisin.
Antibacterial scope of nisin
Nisin can effectively inhibit or kill Gram-positive bacteria that cause food spoilage. At a concentration of 100 ppm, it can kill most Gram-positive bacteria and can inhibit Staphylococcus, Streptococcus, and Lactobacillus. It also has a significant inhibitory effect on most Clostridium and Bacillus genus and their spores. Hitchins, AD and other studies have shown that the spores of Bacillus stearothermophilus in the genus Bacillus are the most sensitive to Nisin, and a very small amount of Nisin can kill the spores. The effect of Nisin on the spores is to inhibit their germination at the initial stage of spore expansion. Instead of killing.
Because Nisin has a narrow antibacterial spectrum and can only kill or inhibit Gram-positive bacteria, it has no obvious effect on Gram-negative bacteria, molds and yeasts, so its application is limited and Nisin is used in conjunction with other preservatives. It can make up for this shortcoming and then exert a broad-spectrum antibacterial effect. Studies have shown that Nisin can enhance its antibacterial effect when used in combination with other preservatives, and can obtain a wider antibacterial spectrum. The combination of Nisin and lactic acid can inhibit Salmonella and Staphylococcus aureus in meat. Nisin, used in combination with chelating agents (such as EDTA), has a certain inhibitory effect on Salmonella and can effectively reduce the number of other G-bacteria. In addition, the combination of Nisin with phosphate and citrate can also improve its inhibitory effect on G-bacteria.
Nisin molecular structure
Antibacterial mechanism of nisin
Research shows that Nisin is a hydrophobic, positively charged small peptide that can be adsorbed on the cell membrane of Gram-positive sensitive bacteria and interact with negatively charged substances in the cell wall (such as teichoic acid, uronic acid, acidic polysaccharides or Phospholipids) can invade into the cell membrane through the C-terminal function to form permeable pores, inhibit the synthesis of the cell wall of Gram-positive bacteria, change the permeability of the cell membrane, and cause small molecule substances in the cell to flow out. At the same time, extracellular water Molecules flow in, eventually leading to cell autolysis and death.
Nisin has antibacterial activity against many G+ bacteria, but has no effect on G- bacteria, yeasts and molds. Comparing the cell walls of G+ bacteria and G- bacteria, it can be found that the peptidoglycan layer of G+ bacteria is significantly thicker than that of G- bacteria, while the cell wall composition of G- bacteria is more complex, mainly including proteins, phospholipids, lipopolysaccharides, etc., and is very dense. , can only allow small molecules with a molecular weight of less than 600 Da to pass, and Nisin has a molecular weight of about 3510 Da, so it cannot pass through the dense cell wall. Therefore, Nisin cannot reach the cell membrane and exert its bactericidal effect. In order to further confirm this statement, Steven and Kordel et al. reported that after treatment to change the permeability properties of the outer wall of G-bacteria, G-bacteria also became sensitive to Nisin and could also be inhibited or killed. This strongly proves that G-bacteria are not sensitive to Nisin because the cell wall is too thick and Nisin molecules cannot enter. During the sterilization process of Nisin, the rare amino acids contained in the molecule have a great effect on Nisin. Research on this issue is quite active, mainly through protein engineering methods to directionally change one or a section of amino acids in the Nisin molecule. composition to study its functions. Research in this area will allow people to further understand the mechanism of action of Nisin in order to expand its application scope or enhance its effect.
Safety of nisin
Nisin is a natural polypeptide substance that can be digested and decomposed into amino acids by proteases (such as trypsin, trypsin, salivary enzymes, etc.) in the human body after consumption. It has no microbial toxicity or pathogenic effects, so it is relatively safe. high.
In 1956, the British Food Preservatives Committee confirmed that different amounts of nisin naturally exist in milk and cheese; in 1962, Japan's Hara et al. confirmed that the half-lethal dose LD50 of nisin in mice was about 7g/kg bw, has an LD50 value similar to that of ordinary salt; the commercial sector in the UK and the former Soviet Union conducted extensive toxicity and biological studies on nisin produced by it, including carcinogenicity, viability, regeneration, blood chemistry, kidney function , brain function, stress response and animal organ pathology and many other aspects of research have proven that Nisin is safe. Toxicology tests have proven that the oral acute toxicity of nisin in mice: the LD50 of female mice is 6.81g/kg bw and the LD50 of male mice is 9.26g/kg bw, which is actually non-toxic. Oral acute toxicity of nisin in rats: the LD50 of female rats is 6.81g/kg bw, and the LD50 of male rats is 14.70g/kg bw, which is practically non-toxic. The results show that nisin is safe. U.S. federal regulations have classified nisin as "generally recognized as safe (GRAS)". In March 1992, my country's Ministry of Health clearly stated in the document approving the implementation: "Nisin can be scientifically considered safe as a food preservative." At present, nearly 50 countries around the world have formulated laws stipulating the scope and limit of use of nisin as a food preservative, while more than 20 countries such as the United Kingdom, France, and Australia have no regulations on limited use.
Uses of nisin
1. Application in milk and dairy products
1. Domestic Nisin was first successfully applied in high-temperature sterilized milk (UHT). Usually, UHT milk is instant sterilized by ultra-high temperature and packaged aseptically without adding food preservatives. However, in the hot summer, the quality of raw milk is often difficult to guarantee. As a result, more microorganisms remain, resulting in an increase in the rate of bad bags (sour bags, bloated bags, etc.). Adding Nisin can significantly reduce the spoilage rate of UHT milk and extend the shelf life.
2. Application in twice sterilized milk. Most of the secondary sterilized milk is produced by small and medium-sized dairy enterprises, and the products are medium and low-end. Fresh milk needs to be heated and sterilized for a long time, which will lead to non-enzymatic browning and the color of the product will become darker. Adding Nisin can reduce the sterilization intensity, reduce the process of non-enzymatic browning, and extend the shelf life of the product.
3. Application in pasteurized milk. According to reports, in August last year, the pass rate of pasteurized milk on the Shanghai market was only 73.37%. Qualified products when leaving the factory were then distributed to consumers through distribution stations. Due to the lack of cold chain support (no cold chain status) The longest time is 8 hours), resulting in a serious decline in the quality of processed milk and excessive coliform bacteria. Adding Nisin can improve the safety of pasteurized milk.
4．Application in fresh milk. According to the new international standard GB5408.2-1999, food preservatives are not allowed to be added to ultra-high temperature sterilized milk and secondary sterilized milk. To this end, the quality of raw milk (fresh milk) must be improved and the microbial indicators in raw milk must be strictly controlled. According to the pollution-free food standards promulgated by the Ministry of Agriculture, the total number of bacterial colonies per milliliter of fresh milk should be less than 500,000. However, according to a dairy factory in Shandong, in addition to the control of directly owned farms, the total number of bacterial colonies in fresh milk transported by refrigerated trucks 400 kilometers away and taking 5 to 6 hours will greatly exceed the standard. According to a report from a milk station in Inner Mongolia, even if milk is collected intensively, mechanically cooled, and transported by refrigerated trucks, and the milk temperature is controlled at 4 to 6°C and does not exceed 10°C in summer, the total number of bacterial colonies per milliliter of raw milk is still in the range of 1 to 2 million. Within, the indicators specified in the standards cannot be met.
In addition, chemical preservatives cannot be used in raw milk. In order to prevent fresh milk from deteriorating and spoiling, some farmers add formaldehyde for preservatives, which is a serious violation and illegal behavior. If Nisin is added, the quality of fresh milk can be maintained and meet the standards (because Nisin naturally exists in some fresh milk).
5. Application in yogurt. During the shelf life of yogurt containing active lactic acid bacteria, the lactic acid bacteria can also slowly ferment, causing the acidity of the yogurt to continue to increase. As a result, the taste of yogurt deteriorates and is unacceptable to consumers. Adding Nisin can inhibit the continued increase in acidity of yogurt fermentation.
The addition method to yogurt can be before the main fermentation or after the main fermentation. What is the appropriate way to add it? This depends on the product variety and process. If added before the main fermentation, the amount of Nisin must be strictly controlled. Because Nisin has an inhibitory effect on the yogurt-producing strains, which prolongs the fermentation time. The criterion for judgment is that after adding Nisin, the main fermentation time of yogurt should end within 3 to 6 hours, which is normal. If the main fermentation time exceeds 6 hours, the amount of Nisin added should be reduced. Since there are many varieties of yoghurt, the specific applications of each type of yoghurt will not be introduced one by one.
2. Application in meat products
Domestic Nisin was the first to be successfully applied in high-temperature meat products. Then, it is also effectively used in low-temperature meat products. So far, no matter what kind of packaging form (flexible packaging, canned, bottled, casing filling), no matter what kind of processing method (pickled, cured, sauced, smoked, brine, sweet and sour, dried) poultry (chicken, duck, Goose), meat (pig, beef, sheep, rabbit), fish and shrimp and other products have achieved ideal results.
Because meat products are rich in nutrients, have high water activity, and have a neutral pH, various harmful microorganisms, such as bacteria, molds, and yeasts, can grow and reproduce. In order to expand the antibacterial range and enhance the antiseptic effect, Nisin compound products should be used instead of pure Nisin. The so-called composite additives refer to two or more single types of additives mixed by physical methods. Usually, it is provided directly by the production unit, and qualified customers can also compound it by themselves.
Nowadays, composite products are generally developed according to the following three principles:
① Each component in the composite product is a species approved for use by the state.
②In order to facilitate customer use and transportation, solid varieties are not compounded with liquid varieties, and there are no compounded liquid products. They are generally solid composite additives. If the customer makes the compound on their own, they are not subject to this principle.
③In order to improve the safety and quality of food, natural food preservatives are not compounded with chemical preservatives, except for sorbic acid. Because sorbic acid is an unsaturated fatty acid, it is basically the same as natural unsaturated fatty acids and can be assimilated in the body to produce carbon dioxide and water. Therefore, sorbic acid can be regarded as a component of food. It can be considered harmless to the human body.
Some data show that Nisin works together with chelating agents such as EDTA to inhibit Gram-negative bacteria. In this way, the antibacterial spectrum of Nisin is expanded. EDTA is the disodium salt of ethylenediaminetetraacetic acid, which is produced by reacting ethylenediamine, sodium cyanide and formaldehyde aqueous solution, and then reacting with sodium hydroxide. National standards stipulate that it is only allowed to be used in some canned foods. For safety reasons, a certain company once refused to use compound additives containing EDTA, which is not unreasonable. If you choose the chelating agent glucono-delta-lactone, it will have greater safety and superiority. This organic acid is an intermediate product of the body's sugar metabolism. Therefore, it can be considered that it is harmless to the human body. Glucono-delta-lactone also has the effect of lowering the pH of meat products and can enhance the effects of color-forming agents and preservatives. In addition, it can also reduce the water activity of meat products and is itself an excellent food preservative.
3. Application in canned food
Adding Nisin to canned food has many advantages:
① It can reduce the intensity of heat treatment, reduce the loss of food nutrients, and improve the eating quality of the product.
② Inhibit the growth and reproduction of heat-resistant bacterial spores and extend the storage time of food. At present, it is mainly used in some canned fruits and vegetables to reduce the sterilization intensity and make the canned fruits and vegetables have better tissue crispness and eating quality.
4. Application in plant protein foods
Boxed lactone tofu has a shelf life of less than 12 hours in the hot summer. Over 12 hours, the product will become dehydrated, sour, and deteriorate. With the addition of Nisin, the shelf life can be extended to 24 hours. The shelf life of 1 day is safe and can meet the needs of this kind of ready-to-eat product for circulation in the market. If there are higher requirements for the shelf life, according to research by Professor Wang Shaolin of Beijing Agricultural University, adding Nisin and combining it with microwave sterilization can achieve a shelf life of 3 days at room temperature of 18°C.
5. Application in beverages
Adding Nisin to beverages can inhibit the growth and reproduction of acid-resistant and heat-resistant bacteria (such as Geobacillus acidophilum), prevent rancidity of beverages, and extend the shelf life of products. At present, the types of beverages that have been used in beverage production include fruit juice beverages, acetic acid beverages, aloe vera beverages, milk-containing beverages, health beverages (such as ginseng beverages, wolfberry and chrysanthemum beverages), etc.
6. Application in brewing wine Taking
advantage of the fact that Nisin does not inhibit yeast fermentation, it can be used in the brewing of beer, rice wine, wine and other alcohol-containing beverages to prevent lactic acid bacteria from causing rancidity of wine and other diseases. In the brewing of beer and rice wine, it is currently mainly used for the expanded culture of beer yeast and rice wine yeast to prevent the infection of miscellaneous bacteria. For low-alcohol rice wine, rice wine often becomes sour and deteriorates during storage due to incomplete sterilization, unclean packaging containers, poor sealing, low alcohol content, etc. Adding Nisin can effectively prevent the rancidity of rice wine and extend its shelf life.
7. Application in convenience foods
Small packaged snack convenience foods, such as chicken legs, chicken feet, jerky and other poultry products, have been summarized in the "Application in Meat Products" section and will not be repeated here. Vegetable convenience foods, such as low-salt pickled mustard, pumpkin shreds, etc., are also commonly used in production.