Name: the effect of the application of thyme essential oil on microbial load during meat drying
Uploaded: 2018-03-14T13:30:00
Description: Watch this Scientific Journal Video about The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying at JoVE.com

This work was supported by the Internal Grant Agency of the Faculty of Tropical AgriSciences, (project number: 20175013) and the CIGA 20182023&#160;both grants, from the Czech University of Life Sciences.

1. Meat Preparation
<ol>
	<li>Obtain a short loin of beef (fresh beef from biceps femoris) from a local butchery and transfer it to the lab. 
	NOTE: It is recommended to transport the loin of beef at ambient temperature (20 - 25 &#176;C), for a period not longer than 20 min in a hermetic sealed bag.</li>
	<li>To sterilize the outer surface of the beef muscle, in a laminar safety cabinet, wash the muscle by spraying with 70% (v/v) ethanol for 10 s using a squeeze bottle of 500 mL. Apply 0.025 g of ethanol ...

<ol>
	<li>Eklund, M. W., Peterson, M. E., Poysky, F. T., Paranjpye, R. N., Pelroy, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+bacterial+pathogens+during+processing+of+cold-smoked+and+dried+salmon+strips.">Control of bacterial pathogens during processing of cold-smoked and dried salmon strips.</a> J. Food Prot. 67 (2), 347-351 (2004).</li><li>Mahmoud, B. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservative+effect+of+combined+treatment+with+electrolyzed+NaCl+solutions+and+essential+oil+compounds+on+carp+fillets+during+convectional+air-drying.">Preservative effect of combined treatment with electrolyzed NaCl solutions and essential oil compounds on carp fillets during convectional air-drying.</a> Int. J. Food Microbiol. 106 (3), 331-337 (2006).</li><li>Rahman, M. S., Guizani, N., Al-Ruzeiki, M. H., Al Khalasi, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microflora+Changes+in+Tuna+Mince+During+Convection+Air+Drying.">Microflora Changes in Tuna Mince During Convection Air Drying.</a> Dry. Technol. 18 (10), 2369-2379 (2000).</li><li>Faith, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+of+Escherichia+coli+O157:+H7+in+ground+and+formed+beef+jerky+prepared+at+levels+of+5+and+20%+fat+and+dried+at+52,+57,+63,+or+68+C+in+a+home-style+dehydrator.">Viability of Escherichia coli O157: H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63, or 68 C in a home-style dehydrator.</a> Int. J. Food Microbiol. 41 (3), 213-221 (1998).</li><li>Hierro, E., De La Hoz, L., Ord&#243;&#241;ez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headspace+volatile+compounds+from+salted+and+occasionally+smoked+dried+meats+(cecinas)+as+affected+by+animal+species.">Headspace volatile compounds from salted and occasionally smoked dried meats (cecinas) as affected by animal species.</a> Food Chem. 85 (4), 649-657 (2004).</li><li>Nummer, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Preparation+Methods+on+the+Microbiological+Safety+of+Home-Dried+Meat+Jerky.">Effects of Preparation Methods on the Microbiological Safety of Home-Dried Meat Jerky.</a> J. Food Prot. 67 (10), 2337-2341 (2004).</li><li>Greig, J. D., Ravel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+foodborne+outbreak+data+reported+internationally+for+source+attribution.">Analysis of foodborne outbreak data reported internationally for source attribution.</a> Int. J. Food Microbiol. 130 (2), 77-87 (2009).</li><li>Eidson, M., Sewell, C. M., Graves, G., Olson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+jerky+gastroenteritis+outbreaks.">Beef jerky gastroenteritis outbreaks.</a> J. Environ. Health. 62 (6), 9-13 (2000).</li><li>Allen, K., Cornforth, D., Whittier, D., Vasavada, M., Nummer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+high+humidity+and+wet+marinade+methods+for+pasteurization+of+jerky.">Evaluation of high humidity and wet marinade methods for pasteurization of jerky.</a> J. Food Sci. 72 (7), (2007).</li><li>Levine, P., Rose, B., Green, S., Ransom, G., Hill, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+testing+of+ready-to-eat+meat+and+poultry+products+collected+at+federally+inspected+establishments+in+the+United+States,+1990+to+1999.">Pathogen testing of ready-to-eat meat and poultry products collected at federally inspected establishments in the United States, 1990 to 1999.</a> J. Food Prot. 64 (8), 1188-1193 (1990).</li><li>Keene, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+outbreak+of+Escherichia+coli+O157:H7+infections+traced+to+jerky+made+from+deer+meat.">An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat.</a> JAMA. 277 (15), 1229-1231 (1997).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viable+but+nonculturable+state+in+bacteria.">The viable but nonculturable state in bacteria.</a> J. Microbiol. 43, 93-100 (2005).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+findings+on+the+viable+but+nonculturable+state+in+pathogenic+bacteria.">Recent findings on the viable but nonculturable state in pathogenic bacteria.</a> FEMS Microbiol. Rev. 34 (4), 415-425 (2010).</li><li>Khamisse, E., Firmesse, O., Christieans, S., Chassaing, D., Carpentier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+cleaning+and+disinfection+on+the+non-culturable+and+culturable+bacterial+loads+of+food-contact+surfaces+at+a+beef+processing+plant.">Impact of cleaning and disinfection on the non-culturable and culturable bacterial loads of food-contact surfaces at a beef processing plant.</a> Int. J. Food Microbiol. 158 (2), 163-168 (2012).</li><li>Li, L., Mendis, N., Trigui, H., Oliver, J. D., Faucher, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+viable+but+non-culturable+state+in+human+bacterial+pathogens.">The importance of the viable but non-culturable state in human bacterial pathogens.</a> Front. Microbiol. 5, 258 (2014).</li><li>Hern&#225;ndez, H., Claramount, D., Ku&#269;erov&#225;, I., Banout, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+modified+blanching+and+oregano+essential+oil+on+drying+kinetics+and+sensory+attributes+of+dried+meat.">The effects of modified blanching and oregano essential oil on drying kinetics and sensory attributes of dried meat.</a> J. Food Process. Preserv. , (2016).</li><li>Garc&#237;a-D&#237;ez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Essential+Oils+on+Consumer+Acceptance+of+Chouri&#231;o+de+vinho+-+A+Dry-Cured+Sausage+Made+from+Wine-Marinated+Meat+-+Assessed+by+the+Hedonic+Scale,+JAR+Intensity+Scale+and+Consumers'+&amp;#34;Will+to+Consume+and+Purchase.&amp;#34.">The Impact of Essential Oils on Consumer Acceptance of Chouri&#231;o de vinho - A Dry-Cured Sausage Made from Wine-Marinated Meat - Assessed by the Hedonic Scale, JAR Intensity Scale and Consumers' &amp;#34;Will to Consume and Purchase.&amp;#34.</a> J. Food Process. Preserv. 41 (4), (2017).</li><li>Govaris, A., Solomakos, N., Pexara, A., Chatzopoulou, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+effect+of+oregano+essential+oil,+nisin+and+their+combination+against+Salmonella+Enteritidis+in+minced+sheep+meat+during+refrigerated+storage.">The antimicrobial effect of oregano essential oil, nisin and their combination against Salmonella Enteritidis in minced sheep meat during refrigerated storage.</a> Int. J. Food Microbiol. 137 (2-3), 175-180 (2010).</li><li>Holley, R. A., Patel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+shelf-life+and+safety+of+perishable+foods+by+plant+essential+oils+and+smoke+antimicrobials.">Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials.</a> Food Microbiol. 22 (4), 273-292 (2005).</li><li>Petrou, S., Tsiraki, M., Giatrakou, V., Savvaidis, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+dipping+or+oregano+oil+treatments,+singly+or+combined+on+modified+atmosphere+packaged+chicken+breast+meat.">Chitosan dipping or oregano oil treatments, singly or combined on modified atmosphere packaged chicken breast meat.</a> Int. J. Food Microbiol. 156 (3), 264-271 (2012).</li><li>Ballester-costa, C., Sendra, E., Viuda-martos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Antioxidant+and+Antibacterial+Properties+on+Meat+Homogenates+of+Essential+Oils+Obtained+from+Four+Thymus+Species+Achieved+from+Organic+Growth.">Assessment of Antioxidant and Antibacterial Properties on Meat Homogenates of Essential Oils Obtained from Four Thymus Species Achieved from Organic Growth.</a> Foods. 6 (8), 59 (2017).</li><li>Hern&#225;ndez, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+oregano+essential+oil+on+microbial+load+and+sensory+attributes+of+dried+meat.">The effect of oregano essential oil on microbial load and sensory attributes of dried meat.</a> J. Sci. Food Agric. 97 (1), 82-87 (2017).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterization+and+antimicrobial+properties+of+herbs+and+spices+essential+oils+against+pathogens+and+spoilage+bacteria+associated+to+dry-cured+meat+products.">Chemical characterization and antimicrobial properties of herbs and spices essential oils against pathogens and spoilage bacteria associated to dry-cured meat products.</a> J. Essent. Oil Res. 29 (2), 117-125 (2017).</li><li>Cavanagh, H. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+Activity+of+the+Volatile+Phase+of+Essential+Oils:+A+Brief+Review.">Antifungal Activity of the Volatile Phase of Essential Oils: A Brief Review.</a> Nat. Prod. Commun. 2 (12), 1297-1302 (2007).</li><li>Tajkarimi, M. M., Ibrahim, S. A., Cliver, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+herb+and+spice+compounds+in+food.">Antimicrobial herb and spice compounds in food.</a> Food Control. 21 (9), 1199-1218 (2010).</li><li>Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., Pulkrabek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+selected+essential+oils+in+vapour+phase+against+foodborne+bacteria.">Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria.</a> Food Control. 20 (2), 157-160 (2009).</li><li>Burt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+oils:+Their+antibacterial+properties+and+potential+applications+in+foods+-+A+review.">Essential oils: Their antibacterial properties and potential applications in foods - A review.</a> Int. J. Food Microbiol. 94 (3), 223-253 (2004).</li><li>Ramanathan, L., Das, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+control+of+lipid+oxidation+in+ground+fish+by+some+polyphenolic+natural+products.">Studies on the control of lipid oxidation in ground fish by some polyphenolic natural products.</a> J. Agric. Food Chem. 40 (1), 17-21 (1992).</li><li>Yamazaki, K., Yamamoto, T., Kawai, Y., Inoue, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+antilisterial+activity+of+essential+oil+constituents+by+nisin+and+diglycerol+fatty+acid+ester.">Enhancement of antilisterial activity of essential oil constituents by nisin and diglycerol fatty acid ester.</a> Food Microbiol. 21 (3), 283-289 (2004).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+activity+of+essential+oils+from+herbs+and+spices+used+on+meat+products+against+food+borne+pathogens.">Synergistic activity of essential oils from herbs and spices used on meat products against food borne pathogens.</a> Nat. Prod. Commun. 12 (2), 281-286 (2017).</li><li>Hussein Hamdy Roby, M., Atef Sarhan, M., Abdel-Hamed Selim, K., Ibrahim Khalel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antioxidant+activity,+total+phenols+and+phenolic+compounds+in+thyme+(Thymus+vulgaris+L.),+sage+(Salvia+officinalis+L.),+and+marjoram+(Origanum+majorana+L.)+extracts.">Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and marjoram (Origanum majorana L.) extracts.</a> Ind. Crops Prod. 43, 827-831 (2013).</li><li>Gouveia, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Antimicrobial+Effect+of+Essential+Oils+Against+Listeria+monocytogenes+in+Sous+vide+Cook-Chill+Beef+during+Storage.">The Antimicrobial Effect of Essential Oils Against Listeria monocytogenes in Sous vide Cook-Chill Beef during Storage.</a> J. Food Process. Preserv. 41 (4), (2017).</li><li>Chen, C., Nace, G., Irwin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+6+x+6+drop+plate+method+for+simultaneous+colony+counting+and+MPN+enumeration+of+Campylobacter+jejuni,+Listeria+monocytogenes,+and+Escherichia+coli.">A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli.</a> J. Microbiol. Methods. 55 (2), 475-479 (2003).</li><li>Herigstad, B., Hamilton, M., Heersink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+optimize+the+drop+plate+method+for+enumerating+bacteria.">How to optimize the drop plate method for enumerating bacteria.</a> J. Microbiol. Methods. 44 (2), 121-129 (2001).</li><li>. Practical food microbiology Available from: <a target="_blank" href="https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019">https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019</a> (2003)</li><li>Smith-Palmer, A., Stewart, J., Fyfe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+plant+essential+oils+and+essences+against+five+important+food-borne+pathogens.">Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.</a> Lett. Appl. Microbiol. 26 (2), 118-122 (1998).</li><li>Burt, S. a., Reinders, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+selected+plant+essential+oils+against+Escherichia+coli+O157:H7.">Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7.</a> Lett. Appl. Microbiol. 36 (3), 162-167 (2003).</li></ol>

Previous research has shown that microorganisms causing foodborne diseases survive drying10. It is therefore necessary to apply preservatives before drying to assure food safety. In this study, we focus on using TEO. The reason is twofold: First, there is a high demand from consumers to use natural products as alternative additives to improve food quality16; Second, a previous study demonstrated positive results after using OEO during the meat drying process<sup class="xref...

Drying as a traditional method to preserve foods has been used since ancient times. Nowadays, there is a growing interest in drying as an effective method for food preservation1,2,3. It is used to make a variety of specially processed meats. One of the most well-known is jerky.
Jerky, one of the oldest methods for meat preservation, is based on curing and drying to lower water activity and therefore to extend its shelf life4. Nowadays, jerky as a preserved cured meat is still very popular, where food safety, flavor, and texture are essential. Jerky preparation can be used for almost any type of meat, including beef, pork, poultry, or game5, and it requires chopping the meat in lean strips and drying it. Usually, marinating the meat in a curing solution or smoking are used along with drying to give the jerky its characteristic flavor6.
Despite the vast interest of drying to truly preserve food, the risk of foodborne outbreaks by E. coli from poorly dried meat is critical and needs to be controlled. There are some studies reporting foodborne gastroenteritis outbreaks particularly with E. coli O157:H7, attributed to inadequate heat processing during home-drying. Similar cases have occurred even in commercially prepared jerky7,8,9. Levine et al.10 proposed that foodborne microorganisms can survive moderate drying conditions (approximately 60 &#176;C) used by commercial jerky producers. E. coli O157:H7 outbreaks of foodborne diseases in the middle of the 1990s were attributed to ground dried meat products6,11. Interestingly, in all the previous cases, the main risk is caused by bacterial pathogens recognized as viable but non-culturable (VBNC). Under various stresses such as temperature changes or starvation, the E. coli cells could enter a particular state known as the VBNC state12,13. The VBNC cells may then be resuscitated back to culturable cells by exposure to suitable conditions and then present a threat to human health due to foodborne contamination14,15. This means that if the meat is consumed immediately after drying the product it is safe. However, in the case of inadequate storage, such as increased humidity, there is a high risk of reactivation of pathogens and microbial growth.
Besides drying and marinade methods, there is a high demand from consumers to use natural products as an alternative to additives to improve food quality16,17. There has been a particular interest in the application of natural food additives for meat instead of classical synthetic preservatives18,19,20,21. Even though there is a lack of sufficient experimental evidence in the use of essential oils when drying the meat, early research in this field already demonstrates positive results22,23.
Since the Middle Ages, people have recognized Essential Oil Compounds (EOCs) for their antimicrobial, insecticidal, and antiparasitic chracteristics24,25,26. Today, EOCs are part of one of the most important group of bioactive natural compounds. Among the different EOCs, thymol is one of the most well-known. It is composed of more than 85% of TEO23. This phenol prevents microbial and chemical deterioration when added to food. Additionally, its antibacterial properties might be improved in combination with other natural preservatives2,27,28,29,30. Nowadays, thyme (Thymus vulgaris), a herb that belongs to Lamiaceae family, has been recognized as a flavoring agent as well as a very effective meat preservative31. A study by Garc&#237;a-D&#237;ez et al.30 on meat products found that TEO displayed a wider inhibition pattern against foodborne pathogens when compared to other essential oils. Therefore, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process.
In this protocol, we present a novel method of applying TEO during meat drying, specifically using it in vapor form directly in a drying chamber. For evaluation, we use the MIC to determine the absence of pathogenic bacteria in treated samples compared with raw ones. The preliminary results show that this method is a highly effective alternative to synthetic preservatives and that it significantly reduces microbial load in dried meat.

<table>
	<tbody>
		<tr>
			<td>Meat cutter</td>
			<td>Kalorik</td>
			<td>KP 3530</td>
			<td>from Miami Gardens, FL, USA</td>
		</tr>
		<tr>
			<td>Laminar safety cabinet</td>
			<td>Faster s.r.l</td>
			<td></td>
			<td>from Italy</td>
		</tr>
		<tr>
			<td>Squeeze bottle of 500 mL</td>
			<td>Merci</td>
			<td>632 524 325 025</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Standard laboratory drier UFE 400</td>
			<td>Memmert</td>
			<td>DE 66812464</td>
			<td>from Germany</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>BT 120</td>
			<td>N/A</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Refrigerator and Freezer</td>
			<td>Bosch</td>
			<td>KGN34VW20G</td>
			<td>from DE</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td>Biosan</td>
			<td>220 000 050 122</td>
			<td>Latvia; supplier Merci, CZ</td>
		</tr>
		<tr>
			<td>Escherichia coli ATCC 25922</td>
			<td>Oxoid</td>
			<td>CL7050</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Chromservis</td>
			<td>22008013</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Sterilized plastic tubes 15 mL</td>
			<td>Gama</td>
			<td>331 000 020 115</td>
			<td>from CZ, supplier Merci</td>
		</tr>
		<tr>
			<td>20 mL injection vial</td>
			<td>Healthy vial</td>
			<td>hvft169</td>
			<td>from China</td>
		</tr>
		<tr>
			<td>20 mm sterile butyl rubber stopper</td>
			<td>Merci</td>
			<td>22008013</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>20 mm aluminum cap</td>
			<td>Healthy vial</td>
			<td>N/A</td>
			<td>from China</td>
		</tr>
		<tr>
			<td>Thyme essential oil</td>
			<td>Sigma Aldrich</td>
			<td>W306509</td>
			<td>from St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>Mueller Hinton Broth</td>
			<td>Oxoid</td>
			<td>CM0337</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Penta</td>
			<td>16610-31000</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Oxoid</td>
			<td>LP0034</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Polysorbate 80 (Tween 80)</td>
			<td>Roth</td>
			<td>T 13502</td>
			<td>from DE, supplier P-lab</td>
		</tr>
		<tr>
			<td>Shaker SHO-1D</td>
			<td>Verkon</td>
			<td>DH.WSR04020</td>
			<td>from CZ,&#160; 10 - 300 rpm. 350 x 350 mm with a platform for flasks</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Bioferm</td>
			<td>N/A</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>MacConkey Agar</td>
			<td>Oxoid</td>
			<td>CM007</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Plate Count Agar</td>
			<td>Oxoid</td>
			<td>CM0325</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Merci</td>
			<td>480 622 080 040</td>
			<td>from CZ</td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 250 mL</td>
			<td>Simax</td>
			<td>610 002 122 636</td>
			<td>from CZ; supplier Merci CZ</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Socorex</td>
			<td>S852820</td>
			<td>from Switzerland; supplier P lab, CZ</td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Gamma</td>
			<td>V400916</td>
			<td>CZ</td>
		</tr>
		<tr>
			<td>Microlitre pipette 100-1000 &#956;L</td>
			<td>Eppendorf</td>
			<td>333 120 000 062</td>
			<td>from Germany; supplier Merci, CZ</td>
		</tr>
	</tbody>
</table>

the effect of the application of thyme essential oil on microbial load during meat drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

We had first previously developed this method by using oregano essential oil (OEO) to enhance food safety and increase the value of dried meat. In general, the preceding experiments showed that E. coli goes into the VBNC state during drying as a survival strategy. This is demonstrated by the fact that there were no culturable bacteria after the drying finished22. Therefore, the pre-enrichment process for 6 h was necessary to allow the counting of the strai...

Watch this Scientific Journal Video about The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying at JoVE.com

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

The overall goal of this experiment is to evaluate if thyme essential oil, applied in vapor phase, can serve as effective preservative during meat-drying process. This method can help answer questions in the field of food processing and safety, such as how to effectively apply natural preservatives instead of synthetic ones. The main advantages of this technique is that enables fast and equal distribution of thyme essential oil while the meat dries and that all vapor can be used in lower volumes than in case of liquid oil application.The use of this method can provide insight into improving the safety and quality of dried meat and also can be applied for treatment of other food products against other unfavorable microorganisms. Generally, individuals new to this method will struggle because the drop plate methods used to count the number of the bacteria requires properly prepared plates and practice. Obtain a short loin of beef from a local butchery, and transfer it to the lab in a sealed bag at ambient temperatures within 20 minutes of purchase.In a Laminar safety cabinet, sterilize the outer surface of the beef muscle using a 10-second application of 70%ethanol, sprayed on from a squeeze bottle. Then, using sterile technique, cut out the outer surface of the muscle so that no ethanol remains on the preparation. Then pack the muscle into a sealed bag, and freeze it for one day at negative 18 degrees Celsius.The next day, thaw the frozen muscle at four degrees Celsius over six hours. Then, in a Laminar safety cabinet, use a meat cutter to make 0.5 centimeter thick slices. And further reduce the size of the slices into 5x2.5 square centimeter rectangular samples.For storage, pack the samples into plastic bags and keep them in the freezer. In a Laminar safety cabinet, prepare a standardized inoculum of E.coli ATCC 25922 to inoculate the meat samples. For the inoculation, arrange at least four samples on an aluminum foil sheet.Then load just as many samples on a second foil sheet to serve as uninoculated controls. Next, inoculate each sample by applying 400 microliters of inoculum onto one side and gently spreading it over the surface using a sterile cell-spreader. Then, let the samples dry for 10 minutes.Next, flip the samples over with tweezers, and repeat the procedure for a total of 800 microliters of inoculum per sample. After this, check the inoculation efficiency of two of the inoculated raw samples and two uninoculated controls. And the rest of the samples, wrap them in foil.Wash the raw samples in peptone water for 10 minutes at room temperature with 140 rpm of shaking. After the wash, on a Microtiter plate, make five 10-fold serial dilutions of the bathwater which now contains bacteria. Next, apply five microliter drops of each dilution by using a multichannel pipette onto plates with MacConkey agar and plate count agar using the 6x6 drop method.The drops should absorb quickly into the agar. For the cultivation of the dilutions by the 6x6 drop method, ensure that the plate count agar and MacConkey agar plates are appropriately dried so that the drops will absorb quickly into the agar. Then, culture the inoculated plates at 37 degrees Celsius for 24 hours.Prepare a meat drying oven for the application of thyme essential oil by vapor. Soak a 12x20 centimeter filter paper with the desired volume of TEO containing 79%thymol. Now take the samples left in the foil and transfer them together with the filter paper to the prepared drying oven with TEO to dry for six hours.In parallel, treat a control set of samples in a meat drying oven without TEO. After the drying, to assess the survival of E.coli on the bacteria-treated samples, repeat the same procedure of washing the samples in peptone water with one altercation. In this case, after the 10 minutes of shaking, transfer the samples in the bath to an incubator at 37 degrees Celsius for a six-hour pre-enrichment incubation.After the pre-enrichment, make five 10-fold dilutions of the bathwater as before. Then, use the 6x6 drop method to plant the dilutions on the detection plates and incubate the plates for 24 hours. The next day, determine the colony forming units per gram of meat.Examine the plates for the presence of mesophilic aerobic bacteria which appear as white spots on plate count agar, and count the typical E.coli colonies on the MacConkey agar which appear as red to dark pink. To analyze the microbiological data, convert the counts to the log of the CFU per gram and use an analysis of variants to determine the main affects of treatment. During the drying process, not all E.coli is eliminated.Some bacteria enter a viable but nonculturable state. So, when meat is stored improperly under high temperature and/or humidity, these bacteria can become viable again and potentially harm consumers. Treatment of the meat with different doses of TEO during drying could completely or partially eliminate E.coli.The number of mesophilic aerobic bacteria and enteric bacilli on meat samples was determined on PCA and MCA plates. When 1.5 milliliters of TEO was applied, no growth of bacteria was observed even after a six-hour pre-enrichment phase. This dose corresponds to 0.028 milliliter of TEO vapor per liter of air.After watching this video, you should have a good understanding of how to treat meat with vapor phase of essential oils during drying and evaluate the number of viable bacteria in the final product. In addition to this procedure, other methods like sensory evaluation and GC analysis can be performed in order to answer additional questions about consumer acceptability of the final product and amount of thyme essential oil residue. Don't forget that working with a meat cutter and pathogenic bacteria can be hazardous, and precautions such as wearing appropriate protective equipment and use of a Laminar safety cabinet should always be taken.

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