Preservatives: Classifications and Analysis E Mani-Lipez, € Palou, nd A Lépez-Malo, Universidad de las Américas Puebla, Cholula, Mexico (© 20%6 Elsevier Lia lights resend Chemical reservatives Chemical preservatives are defined in the Code of Federal Regulations (CFR) by the US Food and Drug Administration (FDA; 21. CER 101.22 (a) (5)) as any chemical that, when added to food, tends to prevent or retard deterioration thereof, but does not include common salt, sugars, vinegars, spices, or oils exacted from spices, substances added to food by direct exposure thereof to wood smoke, or chemicals applied fr their insecticidal or herbicidal properties.” Preservatives are used to prevent or retard both chemical and biological deteriorations ff foods. The FDA includes in ther lit of food preservatives (21. CFR 172, subpart B) compounds with different preventive functions, such as antioxidant, color, flavor, and texture retainers, a well as antimicrobial agents. Only two functional classes of preservatives are clearly defined by the agency, ant ‘oxidant that is defined as “substances used to preserve food by retatding deterioration, rancidity, or discoloration due to oxi dation” (21 CFR 170.3 (0) (3)) and antimicrobial agents as “substances used to preserve food by preventing growth of microorganisms and subsequent spoilage, including fungistas, ‘mold and rope inhibitors” (21 CFR 170.3 (0) (2). ‘On the other hand, the Food and Agricultural Organization (FAO) and the World Health Organization (WHO) of the United Nations through the Codex Alimentarius international food standards define preservative as “a food additive, which prolongs the shelf life ofa food by protecting against deterio ration caused by microorganisms’ and include the following categorization: antimicrobial preservative, antimicrobial syne fist antimold and antirope agent, antimycotic agent, bac fophage control agent, fungistatic agent, and preservative, A more specific definition is defined and established by the FAO/WHO for preservative, which only considers the biologi cal deterioration, neglecting chemical deterioration. ‘The approved preservatives for use in foodstufs by two main reg tulatory agencies FAO/WHO and FDA are presented in Tables 1 and 2, respectively. Therefore, according © these 1wo major regulatory agencies and their consistency in preservatives def nition, preservatives inthis article will refer only to chemical compounds that prevent biological food spoilage Classification of Preservatives Antimicrobial agents can be classified with regard to different aspects for example, based on their spectrum of action as bacte- Fostatic bacercdes,fungistatic, and fungicide, based on their origin as synthetic oF natural and based on chemical structure “Mos preservatives ae uilized to delay or inhibit the growth ofa _04p of microorganisms, but thee are some that are assigned to 8 particular microorganism, asi the case of nitrite against Clo ‘ridwn tomlin in cured meat products, nisin against CC toulinum in pasteurized milkcheeses, and lactate and diacetate to inactivate Listeria monoctogenes in processed meats, There are not established criteria fr classifying preservatives in some cases, more than one classification is utilized according to ther func: tion, itended used, chemical structure, and origin. For example, the FAO/WHO established a elasification based on thei action spectrum and finction, Furthermore, the FDA clases preserva tives based on their function, Some scientific reports utilize a 13) as the solvent for standard preparation and sample extraction. Five different fruit matrices (apricot, golden ‘grape, white peach ig. and mango) were sucessfully tested and analyzed with this method. Nowadays, the most common tech nique proposed for determining sulfites is HPLC (reversed-phase502__ Proservatives: Classifications and Analysis ion-pairing liquid chromatography with postcolumn detection of HPLC-fluorescencespectromety), Nitrates and Nitrites Nitrates and nitrites are used as curing and antimicrobial agents and in cured products. Their main purpose as an ant ricrobial agent isto inhibit the growth of Clostridium boul: ‘num and the production of botulinum toxin in these cured products. ts use is restricted to these products. There are four permissible salts of these compounds for use in foods: sodium nitrate, potasium nitrate, sodium nitrite, and. potassium nitrite. The use of nitrite and nitrate in the processing of fish products is specified in ttle 21 of the CFR: potassium nitrate can be used as a curing agent in the processing of cod roe, without exceeding 200 ppm in finished roe (21CFRI72.160). Furthermore, sodium nitrate can be used as a preservative and «olor fixative, with or without sodium nite, in smoked, cured sablefsh; smoked, cured salmon; and smoked, cured shad and in meat-curing preparations for the home curing of meat and ‘meat products (including poultry and wild game) so that the level of sodium nitrate does not excced 150 ppm and the level ‘of sodium nitrite does not exceed 150 ppm in the finished product. Sodium nititehas the same specifications as sodium nitrate In addition, sodium nitrite could be use as color fa tive in smoked, cured tuna fish products without exceeding 10 ppm in the finished product. Sodium nitrite can also be used in processing smoked chub; the maximum content in the edible portion ofthe finished smoked product is 150 ppm, as measured in the loin muscle, Procedures for calculating the amount of nitrite and nitrate that can be added to curing solutions and meat products ae specified by the USDA. ‘ince control and regulation of nitrates and nitrite use in meat products is crucial to ensure consumer sae methodologies have been utilized to determine nitrites, including ion chromatography, CE, differential pulse voltammetry, enzymatic reduction, and electrochemistry. The ‘extraction of nitrate and nitrite from meat products is critical in terms of obtaining adequate recoveries and reducing possible imerferences. When nitvite is added to a biological material, such as meat product, it reacts with various components pre sent naturally inthe matrix, which means that the levels of nitrite analytically detectable (known a5 residual) necessarily vary. Some compounds in meat products (ascorbic acid, fat, protein, et) that can interfere in the determination of nitrates land nitrites may be present in the extract, which is why puri «ation or clarification steps are necessary. Several clearing tech: niques have been applied forthe analysis of nitrite and nitrate in food, especially in meat products these are based on de- protinization and clarifying agents, using reagents, such as Potassium aluminum sulfate, borax (anhydrous sodium tet borate), mercuric chloride, zine slate, or potassium hexacya- noferrate plus zine acetate. Other clearing techniques include active carbon or ge filtration on Sephadex G-25, the latter used for beverages only AOAC official methods established one method to deter mine nitates and nites in meat (935.48 xylenol method), fone method to analyze nitrites in curing preparations that includes dry cure mix and curing pickle (964.13 tirimetic method), and one method for nitrites in cured meat (973.31 colorimetric method). These methods ae simple. FAO/WHO describes nisin A as an antimicrobial preservative th chemical formula C1ssHaseOu7Nea8? and molar mass of 3354.12 gmol- it physically appears white to light brown ‘micronized powder, is soluble in water and insoluble in non- polar solvents, and is not less than 900 IU of nisin per mili tram, The assay method consists in a bioassay that compares the activity ofa standard solution against unknown sample by ‘uorescent light. FDA permits its use to inhibit the outgrowth fof C. hotlinum spores and toxin formation in pasteurized cheeses, its maximum concentration is determined by GMP (<250 ppm in the finished product). The Codex General Standard for Food Additives permits its use in cheeses (125 ppm max. evel), cloted cream (10 ppm max. level), and cereal and starch desserts (3 ppm max. level). Many ‘more applications have been suggested in foods like fresh pizza, salad dressings, soy and vegetarian products, pasteurized liquid eg, feuit juices, fish and shellfish, and meat products. Nisin antimicrobial spectrum is against Gram-positive bacte- Fla. FDA established the quantitative method for nisin, the one designated by British Standards Insttation ‘Methods for the sstimation and differentiation of nisin in processed cheese’ Alternatively, there ae several suggested methods for quanti cation in foods (milk, cheese, liquid egg, canned tomatoes, and salad dressings), which include horizontal agar diffusion, immunochemisty, nisin-induced reporter gene expression, and turbidimetric bioassay. The widely used agar diffusion assay is not able to distinguish nisin from other interfering substances present in the food, causing false-positive results Although immunochemical methods are more sensitive than the agar diflsion assay, they are not totaly reliable, due 10 cross-reactions with compounds structurally elated to nisin that can be present in the testing material also known as pimaricin is defined a a fungiidal antimycotic ofthe polyene macrolide group. tis produced by several species of Sepiomyas. The commercial product may Contain up to nee mole of water Hs chemical formula (CyligNOy ts molar mase i 66.74 g mal tis a white 10 creamy-white, almost odores,entlline powder practi tally insoluble in wate, guid, and mineral ls andi slighty soluble in methanol and soluble in glacial acetic acid and dlimethylformamide. The assay method is by HPLC with a Column packed with octadecyilanized silica and utaviolet detector measuring at 303 am. FDA permitted suse in cheese in amounts not exceeding 20 ppm in the finished product FAO/WHO permits ts use in nipened, pened, procesed, analogue, and whey protein cheeses at maximum level of 410 ppm and in cared and dried nom heat tented procesed ‘meat, pouluy, and game products in whole pieces or cuts ta ‘maximum level of 20 ppm. Other applications are suggested fornatamycin in fiuitjuces, bakery products olives, and winePreservatives: Classifications and Analysis 503 FDA established as quantitative the one method designed by International Dairy Federation (IDF) Standard 1404:1992, ‘Cheese and cheese rind ~ determination of natamycin content method by molecular absorption spectrometry and by HPLC: (Other suggested methods are ultraviolet spectrometry, liquid chromatography, and HPLC for natamycin in cheeses. Parabens Methyl, ethyl, propyl, butyl, and heptyl esters of phydrospbenzoate are denominated parabens, They are odor less, almost colorless crystals or white crystalline powders. Water solubility is inversely related to alkyl chain length. The pH range of antimicrobial activity is 3-8; their antimicrobial activity is ‘mainly against bacteria and fungi. Combinations of two or more parabens ae often used since they have synergistic effects, FDA seis the maximum level of application of methyl (2ICERISA.1490) and propyl (21CFRI84.1670) esters of Ihydroxybenzoic acid at 0.19, Both methylparaben and propyl parahen are permitted as antimycotic agents in food packaging material (2CFR1S1.23). Heptyparaben (21CFRI72.145) is also approved for use in fermented malt beverages at a maxi ‘mum of 12 ppm and in noncasbonated soft drinks and fruit based beverages at a maximum level of 20 ppm, FAO/WHO Permits the use of methylparaben, ethylparaben, and propyl paraben in foods. The Joint FAO/WHO Expert Committee on Food Additives (FECEA) established the assay method of methyl paraben and ethylparaben asa tvimetrie method. Regulatory agencies have not established an oficial method forts dete nation in foods. Nevertheless a great number of methods are proposed to quantify parabens in foods, such as spectrophotometry injection gas chromatography, HPLC-mass spectrometry, and CE with amperometric detection, Dimethyl Dicarbonate Dimethyl dicarbonate (CHO) is described by the FAO/ WHO asa colorless liquid with a molar mass of 139.09 gmol~ and soluble in water and miscible with tol ‘ene. The method of assay is tsimettc; dimethyl dicarbonate (DMC) is mixed with an excess ofesoburylamine, which it reacts quantitatively. The excess of amine is backtitrated with acid. DMDC has antimicrobial activity against yeast and bacte Fa. FDA approves the use of DMC in certain beverages in ‘which the mirobial population has been reduced to 500 micro- bonganisms per ml oF less by curent GMP in wine, dealcoho lized wine, and low-alcohol wine at maximum of 200 ppm: and in ready-o-drink teas at a maximum of 250 ppm. For carbonated oF noncatbonated, non-juice-containing. (1% juice), flavored and unflavored beverages containing added ‘lectrolytes (5-20 mequiv.I~! Na+ and 3-7 mequv.I™! K+), 4 maximum of 250 ppm is permitted, while for carbonated, diluted beverages containing juice, fruit favor, or both, in ‘which the juice content does not exceed 50%, a maximum of 250 ppm. FAO/WHO permits its use in water-based flavored drinks including ‘spor. “energy electrolyte drinks and pa ticulate drinks, coffee, coffee substitutes, tea, herbal infusions, grain beverages, cider, perry, and wines at a maximum of 250 ppm and in grape wines and mead at a maximum of 200 ppm. AOAC official methods are not available for the determination of DMDC in food. The FDA designates gs chromatography to determine DMDC. Lysozyme Lysozyme i defined by FAO/WHO as a polypeptide obisined from heats eg whites consisting of 129 amino acids, having a molar mass of about 14000 gmol-! and an iroslectric point of 10.7 and possessing enzymatic activity to hyroyze the (1-1) linkages beeen N-aceyimuramicacd and N-aceyglucosiine inthe outer membranes of bacterial species in particular Gra positive organiams usually obtained in the hydrochloride form for food use. 1s describe asa white, adovtess powder, a 3 preservative to prevent the late blowing of cheese caused by Clostitium tyroburricum, and a8 an assay =950 ygmg* and is soluble in water and insoluble inorganic solvents and in con centzated saline soltions. The assay method is based om the changes of turbidity of «suspension of Micosceus leu ATCC 44698. Codex General Standard for Food Additives permits Iso _zyme use in pened cheese, and maximum levels according to GMI, hile for cider, ery, and gape wines, a maximum of S00 ppm i permitted. Several food applications have been sg ested fr lysozyme in meat products, dary products, seafood, tofu vegetables imei sae, and wine Offical methods arenot sailale for the determination of lysozyme in foods. Recent studies show that online eapillay zone electrophoresis and mass spectrometry can be used at 4 method for quantiying Iysoayme in chose. [See alsa: Acids Properties and Datnination, Baciocns, Bosc} Peptides in Foods; Chromatography: Focus on Multidimensional GC Chromatography: High-Pesormance Liquid Chromatography Consumer Protection Legislation, Food and Agriculture Organization of te United Nations; Inared Spectroscopy. Aplications; Nalamycin, Nites and Niles; Preservation of Foods; Presenatves: Food Use Word Healt Organization, Further Reading ‘Sanam AL Davison FM, Slnnn San Tonga ds) (00) Fd aes. aC Pas (huss DF an ane FW (1972 Aiionbal nd ase Fr TE (et) Haibaot oad aes. al 1, p 15-184 Cel CRC Press. ‘ison Pan Pash ME (180 Matos asin ety ol ood _puizobals Food Tasty): 148-15 ‘isco PH an Tal T2007) Chemises and nua elimi exmpouns. Dale MP apd Boal Lt (is) Food Icrebilogefdansits and ots. 2 9p. 73-74. Wein, Dc: ASM Pace ura TE (97) CAC haa dae, a 1. CRC Pass insin.ara sandr WE (1098) Nsin as 2 malo pes, Ci Ravens In Fod Seance & Aon 241 59-8 RS (16 ira ies ood ad a, v7. CRE Ps, ‘ot FADNHD Ext Cons nFod Av, (2000, Eraation latin ood sand cra: Sa st oat el jt FAOMED ot504 Proservatives: Classifications and Analysis ‘anit ono assis 61}. Wats Hath Onaniztion a). Wak ath Onanition Lipo A Pac E, Pash ME, ane Dison FM (25) Mad oat ys of sls i Dan PM, Soe Nand rn Ln) ‘Anica in fats, 3 ep, 69-EE0. Nov Yak CRC Pes. Lu a Liisi (18D Fo 3. Fad ates. Wiy-V0H, Nisin SS 2010) United Stes Gon Rapin avd iran Stas ‘la bod aa. Spring pp. 1-2. Not LM ae Toe es )(212) Fad aris by HPLC (ra 10). CRC Pes. Panacea Mion CE (0) Fad aa aya pact. Sp rar VA, Curing FE, el Fang DY (156) Techni ol sary is eased gesaveand paraxial Cal vin nFood Scns & tion 254): 358-298 ul Wand Go GW (as) (208) Fo sates. Sgt Sos an Basa FF (105) Sri and sores. Fd sec and ety, 9p 49-94. New Yar: Maral. Relevant Websites pew codeine Cote Alena ap fosarepacufoaifeoaAeaiveiss ashore enn, Euopan Carmi, ap:focareacufaeoa Aan. — Eopan Carmen, pew ot tnd ty iti igen — Th SOF ‘WHO Exper Connie ca ocd Alois ECFA). p:tewu ta oftndingeeisaagiielnghesvesngese) vend hn = FOR. ap faa go uodlginlPadaginLabelng Foodies on 5225 hn ~ Ey Adela Fad in he Unt Sts (AUS). up fawn ovfedingabesaagigabelngya da in ~ Canal Fecal Sale (GS) pen ensayo — WHOL