Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.
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References
1.
AbbasiMA, GhazanfariS, SharifiSD, et al.Effect of rosemary essential oil as nitrite substitute on quality of sausage produced using chicken fed by thymus essential oil and rapeseed oil. J Food Sci Technol, 2020; 60:856–867; doi: 10.1007/s13197-020-04786-8
2.
AleixoGC, SilvaMS, MartinsHHA, et al.Effect of essential oils and major compound on Clostridium botulinum endospores inoculated in meat product. Res Soc Dev, 2022; 11(7):e38811729854; doi: 10.3344/8/rsd-v11i7.29854
3.
Álvarez-MartínezFJ, Barrajón-CatalánE, Herranz-LópezM, et al.Antibacterial plant compounds, extracts and essential oils: An updated review on their effects and putative mechanisms of action. Phytomedicine, 2021; 90:153626; doi: 10.1016/j.phymed.2021.153626
4.
AndradePL, BressanMC, GamaLTD, et al.Qualidade da carne maturada de bovinos Red Norte e Nelore. R Bras Zootec, 2010; 39:1791–1800; doi: 10.1590/S1516-35982010000800023
5.
BarradasTN, de Holanda e SilvaKG. Nanoemulsions of essential oils to improve solubility, stability and permeability: A review. Environ Chem Lett, 2021; 19(2):1153–1171; doi: 10.1007/s10311-020-01142-2
6.
BakhtiaryF, SayevandHR, KhaneghahA, et al.Antibacterial efficacy of essential oils and sodium nitrite in vacuum processed beef fillet. Appl Food Biotechnol, 2018; 5:1–10; doi: 10.2203/7/afb.v5i1.17118
7.
BedaleW, SindelarJJ, MilkowskiAL. Dietary nitrate and nitrite: Benefits, risks, and evolving perceptions. Meat Sci, 2016; 120:85–92; doi: 10.1016/j.meatsci.2016.03.009
8.
BenjemaaM, NevesMA, FallehH, et al.Nanoencapsulation of Thymus capitatus essential oil: Formulation process, physical stability characterization and antibacterial efficiency monitoring. Ind Crops Prod, 2018; 113:414–421; doi: 10.1016/j.indcrop.2018.01.062
9.
BozzolaJJ, RussellLD. Electron microscopy. Jones and Bartlett: Boston; 1999.
10.
BrewerMS, ZhuLG, BidnerB, et al.Measuring pork color: Effects of bloom time, muscle, pH and relationship to instrumental parameters. Meat Sci, 2001; 57(2):169–176; doi: 10.1016/S0309-1740(00)00089-9
11.
BrownJL, Tran-DinhN, ChapmanB. Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum. J Food Prot, 2012; 75(4):779–792; doi: 10.4315/0362-028X.JFP-11-391
12.
BruntJ, PlowmanJ, GaskinDJ, et al.Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems. PLoS Pathog, 2014; 10(9):e1004382; doi: 10.1371/journal.ppat.1004382
13.
BruntJ, van VlietAH, CarterAT, et al.Diversity of the genomes and neurotoxins of strains of Clostridium botulinum group I and Clostridium sporogenes associated with foodborne, infant and wound botulism. Toxins, 2020; 12(9):586; doi: 10.3390/toxins12090586
14.
CakirA, KordaliS, ZenginH, et al.Composition and antifungal activity of essential oils isolated from Hypericum hyssopifolium and Hypericum heterophyllum. Flavour Fragr J, 2004; 19(1):62–68; doi: 10.1002/ffj.1279
15.
CecchiniME, PaoloniC, CampraN, et al.Nanoemulsion of Minthostachys verticillata essential oil. In-vitro evaluation of its antibacterial activity. Heliyon, 2021; 7(1):e05896; doi: 10.1016/j.heliyon.2021.e05896
16.
ChaibiA, AbabouchLH, BelasriK, et al.Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores by essential oils. Food Microbiol, 1997; 14(2):161–174; doi: 10.1006/fmic.1996.0075
17.
ChoWI, ChungMS. Bacillus spores: A review of their properties and inactivation processing technologies. Food Sci Biotechnol, 2020; 29:1447–1461; doi: 10.1007/s10068-020-00809-4
18.
ChouhanS, SharmaK, GuleriaS. Antimicrobial activity of some essential oils—present status and future perspectives. Medicines, 2017; 4(3):58; doi: 10.3390/medicines4030058
19.
CofradesS, SerranoA, AyoJ, et al.Restructured beef with different proportions of walnut as affected by meat particle size. Eur Food Res Technol, 2004; 218:230–236; doi: 10.1007/s00217-003-0808-y
20.
de Abreu MartinsHH, Turmo-IbarzA, PiccoliRH, et al.Influence of lipid nanoparticle physical state on β-carotene stability kinetics under different environmental conditions. Food Funct, 2021; 12:840–851; doi: 10.1039/D0FO01980A
21.
de Oca-ÁvalosJMM, CandalRJ, HerreraML. Nanoemulsions: Stability and physical properties. Curr Opin Food Sci, 2017; 16:1–6; doi: 10.1016/j.cofs.2017.06.003
22.
de OliveiraTLC, de Araújo SoaresR, RamosEM, et al.Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite. Int J Food Microbiol, 2011; 144(3):546–555; doi: 10.1016/j.ijfoodmicro.2010.11.022
23.
DeviKP, NishaSA, SakthivelR, et al.Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol, 2010; 130(1):107–115; doi: 10.1016/j.jep.2010.04.025
24.
DiasNAA, PinelliJJ, SilvaMS, et al.Viability of Clostridium difficile in mortadella with added essential oils and reduced sodium nitrite content. Res Soc Dev, 2022; 11(9):e22111931106; doi: 10.3344/8/rsdv11i9.31106
25.
DjihaneB, WafaN, ElkhamssaS, et al.Chemical constituents of Helichrysum italicum (Roth) G. Don essential oil and their antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi and Candida albicans. Saudi Pharm J, 2017; 25(5):780–787; doi: 10.1016/j.jsps.2016.11.001
26.
do AmaralMT, VianaCE, AranhaBC, et al.CIELAB system for evaluating the color of meat products. In: Annals of the International Teaching, Research and Extension Salon [in Portuguese]. 2013; 4(1).
27.
DobritsaAP, KutumbakaKK, WernerK, et al.Clostridium tepidum sp. nov., a close relative of Clostridium sporogenes and Clostridium botulinum Group I. Int J Syst Evol Microbiol, 2017; 67(7):2317–2322; doi: 10.1099/ijsem.0.001948
28.
DonsìF, FerrariG. Essential oil nanoemulsions as antimicrobial agents in food. J Biotechnol, 2016; 233:106–120; doi: 10.1016/j.jbiotec.2016.07.005
29.
DutraMP, de Cássia AleixoG, RamosADLS, et al.Use of gamma radiation on control of Clostridium botulinum in mortadella formulated with different nitrite levels. Radiat Phys Chem, 2016; 119:125–129; doi: 10.1016/j.radphyschem.2015.10.008
30.
DutraMP, RamosEM, RamosADLS, et al.Gamma radiation and storage time on lipidic oxidation, color, heme pigments and residual nitrite of bologna-type sausages formulated with different nitrite additions. Cienc Rural, 2011; 41(12):2203–2210; doi: 10.1590/S0103-84782011005000143
31.
Fernández-LópezJ, Viuda-MartosM. Introduction to the special issue: Application of essential oils in food systems. Foods, 2018; 7(4):56; doi: 10.3390/foods7040056
32.
FiordaFA, de SiqueiraMID. Assessment of pH and water activity in meat products [in Portuguese]. Environ Sci Health, 2009; 36(4):817–826; doi: 10.1822/4/est.v36i4.1132
33.
GillAO, DelaquisP, RussoP, et al.Evaluation of antilisterial action of cilantro oil on vacuum packed ham. Int J Food Microbiol, 2002; 73(1):83–92; doi: 10.1016/S0168-1605(01)00712-7
34.
GutierrezJ, Barry-RyanC, BourkeP. Antimicrobial activity of plant essential oils using food model media: Efficacy, synergistic potential and interactions with food components. Food Microbiol, 2009; 26(2):142–150; doi: 10.1016/j.fm.2008.10.008
35.
HastaoğluE, VuralH, Can ÖP. Effects of thymol and rosemary essential oils and red beet extract on low-nitrite and carmine-free beef mortadella. J Food Process Preserv, 2021; 45:e15855; doi.org/10.1111/jfpp.15855
36.
HerrmannSS, GranbyK, Duedahl-OlesenL. Formation and mitigation of N-nitrosamines in nitrite preserved cooked sausages. Food Chem, 2015; 174:516–526; doi: 10.1016/j.foodchem.2014.11.101
37.
IsmaielAA, PiersonMD. Inhibition of germination, outgrowth, and vegetative growth of Clostridium botulinum 67B by spice oils. J Food Prot, 1990; 53(9):755–758; doi: 10.4315/0362-028X-53.9.755
38.
JafariM, Emam-DjomehZ. Reducing nitrite content in hot dogs by hurdle technology. Food Control, 2007; 18(12):1488–1493; doi: 10.1016/j.foodcont.2006.11.007
39.
JiangY, WuN, FuYJ, et al.Chemical composition and antimicrobial activity of the essential oil of Rosemary. Environ Toxicol Pharmacol, 2011; 32(1):63–68; doi: 10.1016/j.etap.2011.03.011
40.
JoK, LeeS, YongHI, et al.Nitrite sources for cured meat products. LWT, 2020; 129:109583; doi: 10.1016/j.lwt.2020.109583
41.
KumarN, MandalA. Surfactant stabilized oil-in-water nanoemulsion: Stability, interfacial tension, and rheology study for enhanced oil recovery application. Energy Fuels, 2018; 32(6):6452–6466; doi: 10.1021/acs.energyfuels.8b00043
42.
LeeWH, RiemannH. The genetic relatedness of proteolytic Clostridium botulinum strains. Microbiology, 1970; 64(1):85–90; doi: 10.1099/00221287-64-1-85
43.
LiPH, LuWC. Effects of storage conditions on the physical stability of D-limonene nanoemulsion. Food Hydrocoll, 2016; 53:218–224; doi: 10.1016/j.foodhyd.2015.01.031
44.
LiuQ, ZhangM, BhandariB, et al.Effects of nanoemulsion-based active coatings with composite mixture of star anise essential oil, polylysine, and nisin on the quality and shelf life of ready-to-eat Yao meat products. Food Control, 2020; 107:106771; doi: 10.1016/j.foodcont.2019.106771
45.
LonatiD, SchicchiA, CrevaniM, et al.Foodborne botulism: Clinical diagnosis and medical treatment. Toxins, 2020; 12(8):509; doi: 10.3390/toxins12080509
46.
LvF, LiangH, YuanQ, et al.In vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms. Food Res Int, 2011; 44(9):3057–3064; doi: 10.1016/j.foodres.2011.07.030
47.
ManciniS, PreziusoG, Dal BoscoA, et al.Effect of turmeric powder (Curcuma longa L.) and ascorbic acid on physical characteristics and oxidative status of fresh and stored rabbit burgers. Meat Sci, 2015; 110:93–100; doi: 10.1016/j.meatsci.2015.07.005
48.
MartinsHHDA, SimõesLA, IsidoroSR, et al.Preservative of essential oil blends: Control of Clostridium perfringens type a in mortadella. Braz Arch Biol Technol, 2021; 64:e21200106; doi: 10.1590/1678-4324-2021200106
49.
MendesJF, MartinsHHA, OtoniCG, et al.Chemical composition and antibacterial activity of Eugenia brejoensis essential oil nanoemulsions against Pseudomonas fluorescens. LWT, 2018; 93:659–664; doi: 10.1016/j.lwt.2018.04.015
50.
MoazeniM, DavariA, ShabanzadehS, et al.In vitro antifungal activity of Thymus vulgaris essential oil nanoemulsion. J Herb Med, 2021; 28:100452; doi: 10.1016/j.hermed.2021.100452
51.
MoghimiR, GhaderiL, RafatiH, et al.Superior antibacterial activity of nanoemulsion of Thymus daenensis essential oil against E. coli. Food Chem, 2016; 194:410–415; doi: 10.1016/j.foodchem.2015.07.139
52.
MoleyarV, NarasimhamP. Antibacterial activity of essential oil components. Int J Food Microbiol, 1992; 16(4):337–342; doi: 10.1016/0168-1605(92)90035-2
53.
MonteschioJDO, de Vargas JuniorFM, Alves da SilvaAL, et al.Effect of copaíba essential oil (Copaifera officinalis L.) as a natural preservative on the oxidation and shelf life of sheep burgers. PLoS One, 2021; 16(3):e0248499; doi: 10.1371/journal.pone.0248499
54.
Moraes-LovisonM, MarosteganLF, PeresMS, et al.Nanoemulsions encapsulating oregano essential oil: Production, stability, antibacterial activity and incorporation in chicken pâté. LWT, 2017; 77:233–240; doi: 10.1016/j.lwt.2016.11.061
55.
MukurumbiraAR, ShellieRA, KeastR, et al.Encapsulation of essential oils and their application in antimicrobial active packaging. Food Control, 2022; 136:108883; doi: 10.1016/j.foodcont.2022.108883
56.
NCCLS (National Committee for Clinical Laboratory Standards). Performance standards for antimicrobial susceptibility testing; 29th ed.. Clinical and Laboratory Standards Institute, Wayne, PA; 2019.
57.
Odriozola-SerranoI, Oms-OliuG, Martín-BellosoO. Nanoemulsion-based delivery systems to improve functionality of lipophilic components. Front Nutr, 2014; 1:24; doi: 10.3389/fnut.2014.00024
58.
OulkheirS, AghrouchM, El MourabitF, et al.Antibacterial activity of essential oils extracts from cinnamon, thyme, clove and geranium against a gram negative and gram positive pathogenic bacteria. J Dis Med Plants, 2017; 3:1–5; doi: 10.1164/8/j.jdmp.s.2017030201.11
59.
ÖzogulY, ÖzogulF, KulawikP. The antimicrobial effect of grapefruit peel essential oil and its nanoemulsion on fish spoilage bacteria and food-borne pathogens. LWT, 2021; 136:110362; doi: 10.1016/j.lwt.2020.110362
60.
PandeyAK, SinghP, TripathiNN. Chemistry and bioactivities of essential oils of some Ocimum species: An overview. Asian Pac J Trop Biomed, 2014; 4(9):682–694; doi: 10.1298/0/APJTB.4.2014C77
61.
PilevarZ, HosseiniH, HajimehdipoorH, et al.The anti-Staphylococcus aureus effect of combined Echinophora platyloba essential oil and liquid smoke in beef. Food Technol Biotechnol, 2017; 55(1):117; doi: 10.1711/3/ftb.55.01.17.4633
62.
PinelliJJ, de Abreu MartinsHH, GuimarãesAS, et al.Essential oil nanoemulsions for the control of Clostridium sporogenes in cooked meat product: An alternative?. LWT, 2021; 143:111123; doi: 10.1016/j.lwt.2021.111123
63.
QianC, DeckerEA, XiaoH, et al.Physical and chemical stability of β-carotene-enriched nanoemulsions: Influence of pH, ionic strength, temperature, and emulsifier type. Food Chem, 2012; 132(3):1221–1229; doi: 10.1016/j.foodchem.2011.11.091
RamosEM, RamosALS, ScarpaABO, et al.Optimization of the objective evaluation of the color of hams and ham products [in Portuguese]. In: V Brazilian Congress of Meat Science and Technology; São Paulo; 2009.
66.
RaoJ, ChenB, McClementsDJ. Improving the efficacy of essential oils as antimicrobials in foods: Mechanisms of action. Annu Rev Food Sci Technol, 2019; 10:365–387; doi: 10.1146/annurev-food-032818-121727
67.
RödelW, ScheuerR, WagnerH. New method for determining water activity in meat products [in Spanish]. Fleischwirtschaft, 1990; 2:36–41.
68.
RoyA, GuhaP. Formulation and characterization of betel leaf (Piper betle L.) essential oil based nanoemulsion and its in vitro antibacterial efficacy against selected food pathogens. J Food Process Preserv, 2018; 42(6):e13617; doi: 10.3390/molecules23092164
69.
SAS Institute Inc. SAS Statistical Software (Version 9.4). SAS Institute Inc.: Cary, NC; 2018.
70.
SebranekJG, BacusJN. Cured meat products without direct addition of nitrate or nitrite: What are the issues?. Meat Sci, 2007; 77(1):136–147; doi: 10.1016/j.meatsci.2007.03.025
71.
ShaabanHA.Essential oil as antimicrobial agents: Efficacy, stability, and safety issues for food application. In: Essential Oils—Bioactive Compounds, New Perspectives and Applications. IntechOpen: London; 2020.
72.
ShahbaziY. Antioxidant, antibacterial, and antifungal properties of nanoemulsion of clove essential oil. Nanomed Res J, 2019; 4(4):204–208; doi: 10.2203/4/nmrj.2019.04.001
73.
ShanB, CaiYZ, BrooksJD, et al.The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int J Food Microbiol, 2007; 117(1):112–119; doi: 10.1016/j.ijfoodmicro.2007.03.003
74.
SikkemaJ, de BontJA, PoolmanB. Interactions of cyclic hydrocarbons with biological membranes. J Biol Chem, 1994; 269(11):8022–8028; doi: 10.3109/10242429409065221
75.
SindelarJJ, MilkowskiAL. Sodium nitrite in processed meat and poultry meats: A review of curing and examining the risk/benefit of its use. AMSA White Paper Series, 2011; 3:1–14.
76.
SperanzaB, CorboMR. Essential oils for preserving perishable foods: Possibilities and limitations. In: Application of Alternative Food Preservation Technologies to Enhance Food Safety and Stability. (BevilacquaA, CorboMR, SinigagliaM. eds.) Bentham Publisher: Sharjah; 2010; pp. 35–57.
77.
SzymańskiP, ŁaszkiewiczB, SiekierkoU, et al.Effects of the use of Staphylococcus carnosus in the curing process of meat with a reduced amount of sodium nitrite on colour, residue nitrite and nitrate, content of nitrosyl pigments, and microbiological and the sensory quality of cooked meat product. J Food Qual, 2020; 2020:1–10; doi: 10.1155/2020/6141728
78.
TangY, LiF, GuD, et al.Antimicrobial effect and the mechanism of diallyl trisulfide against Campylobacter jejuni. Antibiotics, 2021; 10(3):246; doi: 10.3390/antibiotics10030246
79.
TomovićV, ŠojićB, SavanovićJ, et al.New formulation towards healthier meat products: Juniperus communis L. essential oil as alternative for sodium nitrite in dry fermented sausages. Foods, 2020; 9(8):1066; doi: 10.3390/foods9081066
80.
WangX, ShenY, ThakurK, et al.Antibacterial activity and mechanism of ginger essential oil against Escherichia coli and Staphylococcus aureus. Molecules, 2020; 25(17):3955; doi: 10.3390/molecules25173955
81.
WangY, WeiK, HanX, et al.The antifungal effect of garlic essential oil on Phytophthora nicotianae and the inhibitory component involved. Biomolecules, 2019; 9(10):632; doi: 10.3390/biom9100632
82.
WeigandMR, Pena-GonzalezA, ShireyTB, et al.Implications of genome-based discrimination between Clostridium botulinum group I and Clostridium sporogenes strains for bacterial taxonomy. Appl Environ Microbiol, 2015; 81(16):5420–5429; doi: 10.1128/AEM.01159-15
83.
WentzTG, TremblayBJ, BradshawM, et al.Endogenous CRISPR-Cas systems in group I Clostridium botulinum and Clostridium sporogenes do not directly target the botulinum neurotoxin gene cluster. Front Microbiol, 2022; 12:787726; doi: 10.3389/fmicb.2021.787726
84.
WuJI, RiemannH, LeeWH. Thermal stability of the deoxyribonucleic acid hybrids between the proteolytic strains of Clostridium botulinum and Clostridium sporogenes. Can J Microbiol, 1972; 18(1):97–99.
85.
YazganH. Investigation of antimicrobial properties of sage essential oil and its nanoemulsion as antimicrobial agent. LWT, 2020; 130:109669; doi: 10.1016/j.lwt.2020.109669
86.
YongHI, KimTK, ChoiHD, et al.Clean label meat technology: Pre-converted nitrite as a natural curing. Food Sci Anim Resour, 2021; 41(2):173; doi: 10.5851/kosfa.2020.e96
87.
ZiaratiP, ZahediMT, ShirkhanF, et al.Potential health risks and concerns of high levels of nitrite and nitrate in food sources. SF Pharma J, 2018; 1(3):1–13.
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