Mechanism of action and safety of food additive lactic acid streptococcin


Mechanism of action and safety of food additive lactic acid streptococcin

Streptococcin lactisin, also known as nisin peptide, is a natural biological antimicrobial peptide produced by the fermentation of Streptococcus lactis naturally occurring in milk and cheese, which has a broad-spectrum antibacterial effect and can effectively inhibit the growth and reproduction of most gram-positive bacteria and their spores. In particular, it has an obvious inhibitory effect on common bacteria such as Staphylococcus aureus, Streptococcus haemolyticus, and Clostridium botulinum, and can play a role in preservative and preservation of many foods. Moreover, lactic acid streptococcin has good stability, heat and acid resistance, and has a good application prospect in the food industry.

Streptococcin lactis a world-recognized, safe and natural biological food preservative and antibacterial agent, mainly used for the preservation and preservation of milk and dairy products, meat and meat products. The discovery of lactic acid streptococcin dates back to the 20s of the last century, and in 1928, American researchers such as LA. Rogers first reported that the metabolites of Streptococcus lactis can inhibit the growth of other lactic acid bacteria. In 1947, A.T.R. Mattick et al. found that some lactic acid streptococci in serological N group can produce protein bacteriostatic substances, and prepared this polypeptide substance from the fermentation broth of lactic acid streptococcus.

White lactic acid streptococcin powder

Nisin is a natural product of Streptococcus lactis and toxicity studies of Streptococcus lactis in excess of food applications have shown that it is non-toxic. Because it is particularly sensitive to proteolytic enzymes (α-trypsin), it is quickly hydrolyzed into amino acids by proteolytic enzymes in the digestive tract after consumption. In 1953, Nisaplin, a batch of commercial products of streptococcin lactis, was launched in the United Kingdom; In 1969, the Joint FAO/WHO Expert Committee on Food Additives approved streptococcin lactis as a food additive; In 1988, the U.S. Food and Drug Administration (FDA) also officially approved the use of lactic acid streptococcin in food; In 1990, the Food Supervision Department of the Ministry of Health of China issued a certificate of conformity for the use of lactic acid streptococcin in China. More than 50 countries have approved the use of lactic acid streptococcin.

Bacteriostatic range of lactic acid streptococcin

Streptococcin lactis can effectively inhibit or kill the gram-positive bacteria that cause food spoilage, and can kill most of the gram-positive bacteria at a concentration of 100ppm, and can inhibit some species of staphylococcus, streptococcus, lactobacillus and micrococcus; It also has a significant inhibitory effect on most of the clostridia and bacillus spp. and their spores. Hitchins, A.D. et al. have shown that the spores of Bacillus thermophilus are the most sensitive to Nisin, and a very small amount of Nisin can kill its spores, and the effect of Nisin on the spores is to inhibit their germination at the initial stage of spore expansion, rather than kill them.

Due to the narrow antibacterial spectrum of Nisin, it can only kill or inhibit Gram-positive bacteria, but has no obvious effect on Gram-negative bacteria, molds and yeasts, so its application is limited, and the combination of Nisin with other preservatives can make up for this shortcoming, and then play a broad-spectrum antibacterial effect. Studies have shown that the combination of Nisin and other preservatives can enhance its own antibacterial effect, and a wide antibacterial spectrum can be obtained. The combination of Nisin and lactic acid can inhibit Salmonella and Staphylococcus aureus in meat. Nisin is used in combination with chelating agents (such as EDTA) to have 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.

Lactic acid streptococcin molecular structure

Antibacterial mechanism of lactic acid streptococcin

Studies have shown that Nisin is a hydrophobic and positively charged small peptide, which 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), and can invade the cell membrane to form permeable holes through the action of the C-terminus, inhibit the synthesis of the cell wall of Gram-positive bacteria, change the permeability of the cell membrane, cause the outflow of small molecule substances in the cell, and at the same time, the inflow of extracellular water molecules, and finally lead to the autolysis and death of the cell.

Nisin has antimicrobial activity against many G+ bacteria, but has no effect on G-bacteria, yeasts, and molds. Comparing the cell wall of G+ bacteria and G-bacteria, it can be found that the peptidoglycan layer of G+ bacteria is much thicker than that of G- bacteria, and the cell wall composition of G-bacteria is more complex, mainly including proteins, phospholipids and lipopolysaccharides, etc., which are very dense and can only allow small molecules with a molecular weight of less than 600Da to pass through, while Nisin has a molecular weight of about 3510Da, so it cannot pass through the dense cell wall, therefore, Nisin cannot reach the cell membrane and play a bactericidal role. To further corroborate this claim, Steven and Kordel et al. reported that after treatment changed the permeability 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 for Nisin molecules to enter. In the process of Nisin sterilization, the rare amino acids contained in its molecule have a great effect on Nisin, and the research on this problem is quite active, mainly through the means of protein engineering, and the composition of one or a section of amino acids in the Nisin molecule is changed in a directional manner to study its function. Research in this area will lead to a better understanding of Nisin's mechanism of action in order to expand the scope of application or enhance the effect of action.

Safety of lactic acid streptococcin

Streptococcin lactis a natural peptide substance, which can be digested and decomposed into amino acids by proteases in the human body (such as trypsin, pancreatic enzymes, sialases, etc.) after consumption, without microbial toxicity or pathogenic effects, so its safety is high.

In 1956, the British Food Preservatives Commission confirmed that different amounts of streptococcin lactis are naturally present in milk and cheese; In 1962, Hara et al. of Japan confirmed that the semi-lethal LD50 of lactic acid streptococcin in mice was about 7g/kg bw, which was similar to the LD50 value of ordinary salt. The commercial sector in the United Kingdom and the former Soviet Union has conducted extensive toxicity and biological studies on the toxicity and biology of the lactic acid streptococcin produced, including carcinogenicity, survival, regeneration, blood chemistry, kidney function, brain function, stress response, and animal organ pathology, and many other studies have proved that Nisin is safe. Toxicological tests proved that the acute oral toxicity of lactate streptococcin mice: the LD50 of female mice was 6.81g/kg bw, and the LD50 of male mice was 9.26g/kg bw, which was actually non-toxic. Acute oral toxicity of lactic acid streptococcin rats: the LD50 of female rats was 6.81g/kg bw, and the LD50 of male rats was 14.70g/kg bw, which was actually non-toxic. The results showed that lactic acid streptococcin was safe. U.S. federal regulations have classified lactic acid streptococcin as "generally recognized as safe" (GRAS). In March 1992, China's Ministry of Health clearly pointed out in the document approved for implementation: "Lactic acid streptococcin can be scientifically considered safe as a food preservation agent". At present, nearly 50 countries around the world have enacted laws to regulate the scope and limit of the use of lactic acid streptococcin as a food preservative, while more than 20 countries such as the United Kingdom, France, and Australia have no regulations on the use of limited amounts.

Uses of lactic acid streptococcin

1. Application in milk and dairy products
1. Domestic Nisin was first successfully applied in high-temperature sterilized milk (UHT). Usually, UHT milk is instantaneously sterilized by UHT and aseptically packaged without the need to add food preservatives. However, in the hot summer, the quality of raw milk is often difficult to guarantee, and as a result, there are more microbial residues, resulting in an increase in the rate of bad bags (acid packets, swollen bags, etc.). The addition of Nisin can greatly reduce the rate of UHT milk spoilage and prolong the retention period.
2. Application in secondary sterilized milk. Most of the secondary sterilized milk is produced by small and medium-sized dairy enterprises, and the products are medium and low-grade. Fresh milk needs to be sterilized by secondary heating for a long time, resulting in non-enzymatic browning and darkening of the product. The addition of 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 qualified rate of pasteurized milk in the Shanghai market was only 73.37%, and the qualified products at the time of delivery were distributed to consumers through the distribution station, due to the lack of cold chain support (the longest time without cold chain was 8 hours), resulting in a serious decline in the quality of processed milk and excessive coliform bacteria. The addition of Nisin can improve the safety of pasteurized milk.
4. Application in fresh milk. According to the new international standard GB5408.2-1999, food preservatives shall not be added to UHT milk and secondary sterilized milk. To achieve this, it is necessary to improve the quality of raw milk (fresh milk) and strictly control the microbiological indicators in raw milk. According to the pollution-free food standard issued by the Ministry of Agriculture, the total number of colonies in each milliliter of fresh milk should be less than 500,000. However, according to a dairy factory in Shandong, in addition to the direct pasture can be controlled, the total number of colonies of fresh milk transported by refrigerated trucks 400 kilometers away and 5~6 hours will greatly exceed the standard. According to a report from a milk station in Inner Mongolia, even if the milk is collected centrally, mechanically cooled, and transported by refrigerated truck, the milk temperature is controlled at 4~6 °C, and does not exceed 10 °C in summer, and the total number of colonies per milliliter of raw milk is still within the range of 100~2 million, which cannot meet the indicators specified in the standard.
In addition, chemical preservatives can not be used in raw milk, and individual farmers add formaldehyde to prevent fresh milk from deteriorating and rotting, which is a serious violation and illegal behavior. If Nisin is added, it can make the fresh milk maintain its quality and meet the standard (because Nisin is naturally present in some fresh milk).
5. Application in yogurt. Yogurt containing active lactic acid bacteria can be fermented slowly during the shelf life, resulting in a further increase in the acidity of the yogurt. As a result, yogurt tastes worse and is unacceptable to consumers. The addition of Nisin can inhibit the further increase of acidity in yogurt fermentation.
The method of adding yogurt can be before or after the main fermentation. What is the appropriate way to add it? This depends on the variety and process of the product. If the addition is made before the main fermentation, the amount of Nisin added must be strictly controlled. This is because Nisin has an inhibitory effect on yogurt-producing cultures, which prolongs the fermentation time. The criterion for judging is that after the addition of Nisin, the main fermentation time of yogurt should end within 3~6 hours. If the main fermentation time is more than 6 hours, the amount of Nisin should be reduced. Due to the wide variety of yogurt, the specific application of various yogurt 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 used effectively in low-temperature meat products. So far, no matter what kind of packaging form (flexible packaging, canning, bottle, casing), no matter what kind of processing method (pickling, waxing, sauce, smoking, marinade, sweet and sour, dried) poultry (chicken, duck, goose), meat (pig, cattle, sheep, rabbit), fish and shrimp and other products have achieved relatively ideal results.
Because meat products are rich in nutrients, high water activity, and neutral pH, all kinds of harmful microorganisms, such as bacteria, molds, and yeasts, can grow and multiply. In order to expand the antibacterial range and enhance the antiseptic effect, Nisin's composite products should be selected, rather than pure Nisin. The so-called compound additives refer to the mixture of two or more single varieties of additives by physical methods. Usually, it is provided directly by the production unit, and customers who are able to do so can also compound it themselves.
At present, composite products are generally developed according to the following three principles:
(1) Each component in the composite product is a variety approved by the state.
(2) For the convenience of customers and transportation, solid varieties are not compounded with liquid varieties, and there are no composite liquid products. It is generally a solid compound additive. If the customer rejoins on his own, he is not subject to this principle.
(3) In 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, basically the same as natural unsaturated fatty acids, it can be assimilated in the body to produce carbon dioxide and water. Therefore, sorbic acid can be regarded as an ingredient in food. It can be considered harmless to the human body.
It has been shown that Nisin works with chelating agents such as EDTA to inhibit Gram-negative bacteria. In this way, the antimicrobial spectrum of Nisin is expanded. EDTA is a disodium salt of ethylenediaminetetraacetic acid, which is prepared by the reaction of ethylenediamine, sodium cyanide and formaldehyde aqueous solution, and then reacts with sodium hydroxide. The national standard stipulates that it is only allowed to be used in some canned foods. For safety reasons, a company has refused to use EDTA-containing compound additives, which is not unreasonable. If the chelating agent gluconate-δ-lactone is selected, it has greater safety and superiority, and this organic acid is an intermediate product of the body's glucose metabolism. Therefore, it can be considered that it is harmless to the human body. Gluconate-δ-lactone also has the effect of lowering the pH of meat products, which can enhance the effect of colorants and preservatives. In addition, it can also reduce the water activity of meat products, which is an excellent food preservative in itself.

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 prolong the shelf life of food. At present, it is mainly used in some canned fruits and vegetables to reduce the sterilization intensity and make canned fruits and vegetables have better tissue brittleness and eating quality.

Fourth, the application
in plant protein food Boxed lactone tofu, which has a shelf life of less than 12 hours on a hot summer day. After 12 hours, the product will become dehydrated, sour, and spoiled. With the addition of Nisin, the shelf life can be extended to 24 hours. The shelf life of 1 day is safe to meet the needs of this ready-to-eat product circulating in the market. If there are higher requirements for the shelf life, according to the research of Wang Shaolin, Beijing Agricultural University, adding Nisin and microwave sterilization, at room temperature 18 °C, the shelf life can reach 3 days.
5. Application in beverages Adding Nisin
to beverages can inhibit the growth and reproduction of acid-resistant and heat-resistant bacteria (such as Bacillus acidus), prevent rancidity of beverages, and prolong the shelf life of products. At present, the beverage varieties that have been used in beverage production include fruit juice drinks, acetic acid drinks, aloe vera drinks, milk-containing drinks, health drinks (such as ginseng drinks, wolfberry chrysanthemum drinks), etc.
6. Application in brewing wine
Using the property that Nisin does not inhibit yeast fermentation, it can be used in the brewing of beer, rice wine, wine and other alcoholic beverages to prevent rancidity and other diseases caused by lactic acid bacteria. In the brewing of beer and rice wine, at present, it is mainly used in the expansion of brewer's yeast and rice wine yeast to prevent the infection of miscellaneous bacteria. For low-alcohol rice wine, it is often caused by incomplete sterilization, unclean packaging containers, or poor sealing, low alcohol content, etc., which cause sour and deterioration during storage. The addition of Nisin can effectively prevent rancidity and prolong the shelf life of rice wine.
7. Application in convenience food
In small packaging of leisure convenience food, such as chicken legs, chicken feet, dried meat and other poultry products, has been summarized in the "application of meat products" section is elaborated, and will not be repeated here. Vegetable convenience foods, such as low-salt mustard and shredded gourd, are also used normally in production.

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