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Milena Radenković1, Ana Maksimović2 Faculty of Natural Sciences Niš [email protected] USAGE OF ADDITIVES AND BACTERIOCINS IN FOOD INDUSTRIES Abstract Progress in technology bears with it a lot of changes in lifestyle and also in diet. A great number of additives is used in the industry of food production for the food to have longer lasting period. However, their harmful effect on people's health is being manifested on daily basis so that is why their usage in food is not safe. On the other hand, microorganisms synthesize a great deal of different compounds which can be used in food industries as natural additives. Except for primary metabolites of microorganisms that are used as additives in food, bacteriocins, secondary bacterial metabolites have the biggest potential as natural preservatives. Key words: additives, bacteriocins, fermented sausages INTRODUCTION From early times in food and drinks substances that emphasized or enhanced certain characteristics of this products were being added. Many of this substances, however, had negative effect on people's health. That is why international congress was held in 1909. where positive list of substances allowed for usage in food production was accepted. All of these compounds are called additives. 1 ADDITIVES Additives are substances which, no matter their nutritious value, are not used as foodstuff nor represent characteristic ingredients of foodstuff but are added to food with a purpose, for technological reasons, during production, modification, preparing, processing, packing, transporting or keeping, whereby they themselves, directly or indirectly, become ingredients of food or affect organoleptic characteristics of the product. Bearing in mind not only the way of life but as well the upgrowth of the human population, additive usage is rising. They are used as antioxidants that extend product's validity, as artifical dyes and aromas that ameliorate the smell, taste and appearance of the product that was lost during the processing, as preservatives that keep from deterioration, thickeners, gelatinizing agents, sweeteners, anti-thickener agents, stabilizators, modified starches, emulsifiers and emulsifying salts and many other classes. All additives are marked as En where n is the ordinal number under which one is listed wheather on the list of the approved additives or on the list of the banned ones. Regading that additives are not common ingredients of food, it is necessary to execute a number of researches that will show weather a substance is suitable for use in the food industry. We cannot talk about harmlessness in using additives but only about the harmfulness threshold in their usage. Some additives become toxic when they are used for a long time (cumulative harmful effect). Taking in account that toxicity analysis are very extensive (going from their effect on intestinal microflorae, additive metabolism, cumulative effect to effect on offsprings, their alergic, mutagenicity and cancerous effects), it often happens that substances are taken off the positive list after the reevaluation. JEFCA (Joint Expert Comittee on Food Additives), WHO (World Health Organization) and FAO (Food and Agriculture Organization) in USA decide about additive traits, way of usage, health safety and recommend their daily dose worldwide. In Europe there is EFSA (European Food Safety Authority). An additive has to satisfy three terms in order to be found on the approved additive list: it must not be harmful to human health, it has to be required in the production process and it must not serve for the sake of deceiving buyers. In recent years, EFSA requested complementary research and reevaluation of harmful effects and recommended daily dose for a great number of substances, mostly artifical dyes and preservatives, some of which have been taken off the list of the approved additives after establishing certain effects that have not been detected earlier or have not been considered harmful enough to influence the final grade.For some additives the RDI (Recommended daily intake) dose was changed. RDI is recommended quantity of a substance that is considered not to be harmful for humans if taken daily throughout the whole life. In order to calculate RDI the amount of a certain additive that is not harmful to animals, on which the research is conducted, is reduced a hundred times. In this way, yhe risk which exists because of the difference in human and animal metabolism is minimized. It is expressed in milligrams per kilogram of body mass. Not one scientist and not one helth organization can state with certainty that any of the food additives is completely harmless. Each of us have different bodies and different 2 tolerance limits for different substance concentration. Besides, interactions of chemical compounds in products can not be fully forseen. Some substances which are commonly found in food can interact with an additive and form a nuisance compound. Interactions can not be predicted, and research of each of the possible ones in different conditions (ex. termic process, freezing and so) is economically and physically impossible if you take in account the diversity of substances in the nature. Practical example that confirms this assumption is the fenomena that scientists Lawrence and Gardnet noticed in 1993. In soft drinks benzene is formed in the interaction between benzoic acid (E210) and antioxidant citric acid (E300), under temperature, light and acid reaction of the environment. Concentration of benzene created this way is a hundred times greater than the one permitted. Benzene is a cancerous substance and should not be taken into human body bacause it causes severe damages as leukemia, tumors, nervous system damage and other. That is why benzoic acid should not be used in drinks that already contain vitamine C. Second story is about artifical dyes whose interaction with preservative sodium benzoat has been known at least four decades before detailed research presented by scientific studies has been done. Some of the compounds from this group, which were proven to react with sodium – benzoat (E211) and form cancerous derivate aniline, were finally banned from food industry in 2007. One of the consequences that consuming aniline induces also is ADHD (Attention Deficit Hyperactivity Disorder) common in children who mostly use foodstuff rich in azo dyes like ice cream and sweets. Most products that contain this dyes have to be marked in manifest: “Can induce attention deficit and hyperactivity in children.“ In year 1973. american scientist Ben Fiangold created a hypothessis that hyperactivity problems and attention deficit in children were induced by consuming food rich in artifical dyes and aromas. Also, some additives can be used in small quantities so they do not immediatelly provoke harmful effect, but can be proven harmful further on because they, rather than being dissolved, accumulate in the body until critical quantity is reached. What is not harmful for healthy adult can be for pregnant woman or a child. In children less than three years old, detoxication system is still insufficiently developed, and in older individuals its capacity is considerably reduced. Additives can induce different reactions in people as allergies, headaches, nausea, hyperactivity, nerovus system damage and tumors. Monotony of cursory world of today induces increased intake of cartain additives in relation to recommended daily intake dose, so cases of intolerance are more and more common in children as well as in adults. Aspartam (E951) from artifical sweeteners group is used in thousands of products like chewing gum, sweets, toothpastes and diabetes products. Italian scientists carried out detailed examinations in Centre for cancer research in Bologne and got discomforting results that confirm that aspartam is cause of different types of cancer and tissue changes. Although valid results did get published, they are not fully validated. EFSA published public announcement of harmfulness but did not take this additive from the list of the acceptable ones. Similar effect on the body have other artifical sweeteners (acesulfame, sacharine) aswell. Potassium bromate, which is used in baking industry, can cause diarrhoea, nausea, vomiting and alike. Nitrozamines can trigger stomach cancer. Some additives as tatrazine, butylated hydroxytoluene (BHT) and 3 butylated hydroxyanisole (BHA) can induce allergic rash in people with chronic nettle rash. Additives used in food very easily reach environment. Frightening research first in Switzerland, then in Germany, came to results of existance of great concentration of acesulfame and other artifical sweeteners by the outpour of fecal water in river flows. Many examples can be found, and parellel to establishing more or less harmful effect of additives, food industry invests a great amount of money to find and use new additives. On the other side, so called „green technology“ and continuous progress of contemporary, conservation technologies of XX and XXI century, include exploatation from the field of biological food protection using different bacterium products as bioprotectors in the service of protecting people's health and increasing conservation value and organoleptic attribute of the product. BACTERIOCINS The ability of LAB (Lactic acid bacteria) to produce antimicrobial substances has been exploited for centuries as the means of conserving food. In fermentation occurs an oxydation process of available carbon hydrates to organic compounds with smaller molecular mass , which show significant antimicrobial activity, firstly lactic, acetic and propionic acid. Aside of the production of primary metabolits, LAB produce a variety of other inorganic compounds and secondary metabolits which show antimicrobial effect. A great deal of LAB synthesize protein inhibition substances that are called bacteriocins, which give the producing variety a selective advantage over similar natural competitors. Bacteriocins are substances, originally proteins, with bacteriocidic or bacteriostatic activity which is limited on closly related species and towards which the variety that produces it has specific mechanisms of autoprotection. Relatively narrow inhibitory activity spectrum of bacteriocins is a result of filogenic relateness of producing cells and aimed cells that are usually allied with bacteria producers, in other words, that are in similar ecological niches. In common circumstances, effect on the gram-negative bacteria is not recorded because bacteriocins mostly affect cytoplasmatic membranes of other bacteria cells, so inhibition effect of bacteriocins on the gram-negative bacteria is reduced because of the protective lipoprotein layer in their cell walls. Influence on the gram-positive bacteria is known, and the effect on pathogenic bacteria Lysteria monocitogenes, closly related to genus Lactobaccilus, which represent great problem to the meat production industry because of their common occurence, is of special importance. Although in the last few years their usage in the food industry has been considered a possibility, as well as the usage for the purpose of a complete health safety of the products and for prevention and protection from bacterial infections, in order for bacteriocins, as natural perservatives, to be applied in the food industry, they primarily have to be approved as legal additives, in other words, they must have GRAS (Generally Recognized As Safe) status. That is the case with nizin, a product of 4 Lactococcus sub. sp. lactis. Nizin is used as refined bacteriocin or is sintetised in situ by bacteria that are used as starter cultures. At the moment, in the meat industry, none of the bacteriocins have that kind of status, but utilization of protective cultures which contain varieties that produce bacteriocins is widely spread. Lactococcus sub. sp. lactis with confirmed production of bacteriocins today can be found as a part of functional starter for fermented sausages production, in spite of inadequate adaptation in the sausage production technology alone (shows sensitivity to nitrites). In the last few years interest for controlled in vivo production exists alongside with bacteriocin production on the part of probiotic varieties in digestive duct. Lactobacilluses isolated from different types of sausages represent common bacteriocin producers: Lb. sakei, Lb. curvatus, Lb. plantarum, Lb. brevis and Lb. casei. Simultaneously, on account of demonstrated antilisterial effect, a great deal of them is used as a part of starter cultures when producing fermented sausages: Lb. sakei, Lb. curvatus and Lb. plantarum. A published research of the Institute of Meat Hygiene and Technology in Belgrade, dealt with the production of a variety of Lactobacillus sakei by the means of isolation of ammonia semirefined bacteriocin (sacacin) by a method of precipitation with sulphate using a local, Serbian sausage which is fermented traditionally. Examined sensitivity of bacteria Listeria monocytogenes to bacteriocin revealed that bacteriocin isolated from Lb. sakei I 154 shows distincive antimicrobial effect towards L. monocitogenes NCTC 10527. Antimicrobial activity is evidented on temperatures 12, 18 и 25ºC and pH values 5,0 , 5,5 и 6,0. The intensity of antilisterial activity of tested bacteriocin is proportional to pH values and the increase in temperature. Bacteriocin effect depends upon a number of factors. Presence of proteolitic enzymes, originally from products or LAB, leads to their retrogression and activity decrement. Elongated bacteriocin action can induce resistance of fastidious bacterium towards this bacteriocin. The presence of the gene responsable for immunity probably contributes to different sensitivity of LAB varieties and species to specific bacteriocins. Lactacin B production is induced with a dimeric protein produced by dart cells. Because of that, bacteriocin production can possibly be induced or amplified by the presence of a sensible variety. Except for the influence on unwanted microorganisms, these substances inhibit the activity and LAB, which should be given special attention when constructing defined mixed starter cultures. Also, other technological factors should be well adapted and researched (pH, salt, nitrites) with a functional attributes of bacteriocin producing varieties. During the application of bacteriocin producing varieties LAB as a starter culture it is very important to optimize time of bacteriocin production. It is very important to differ optimal growth conditions LAB from the optimal conditions for bacteriocin production. For example, optimal temperature for growth of Lb. sakei CTC 494, that leads to generating maximal biomass, is around 35ºC, as opposed to maximal cell bacteriocin production, sacacin K, which is set at the temperature ranging from 20º to 25ºC. CONCLUSION: 5 Everything we ingest through our diet directly influences our bodies and therewith our health and our life. Therefore, it is necessary to maximally test and check substances we use in food production in terms of their influence on people. Additives are indicating higher insalubrious levels, so their usage should be reduced to minimum. Bacteriocins, as primary products of LAB and other bacteria, can contribute to food quality and so we should tend to research them more. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 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