Use of Plant Extracts and Essential Oils to Inhibit Listeria Monocytogenes

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8th Feb 2020 Biology Reference this

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Use of Plant Extracts and Essential Oils to Inhibit Listeria monocytogenesand Possible Applications to Foods and Food Production Surfaces

  Listeria monocytogenes is a major concern to the food production industry. It is characterized as a gram positive, facultative anaerobic, non-spore forming rod (Seaberg et. al, 2003). Why this foodborne pathogen is of high importance is mostly due to its high mortality rate, as high as 34%, making it the third leading cause of foodborne death in the United States as of 2011 (Hill et. al, 2013, Olaimat & Holley, 2014). Due to its resistance to temperatures ranging from 2.5 to 44.0°C, pH range of 6.0 to 8.0, aw <0.93, and biofilm production, it is extremely difficult to deal with in a production setting (Seaberg et. al, 2003). Listeria monocytogenes is found mostly in meats, dairy products, and ready-to-eat foods (Wu et. al, 2010). Proper cooking and pasteurization can eliminate most L. monocytogenes cells, but cross-contaminated fruits and vegetables are left vulnerable.

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  Today’s consumers are now migrating from “chemical” and synthetic forms of food preservation to more natural methods (Seaberg et. al, 2003). A natural solution lies in plant extracts and essential oils, which are generally recognized as safe (GRAS). Prior research has determined that many herb and spice extracts inhibit the growth of bacteria, yeasts, and molds. Besides being inhibitory towards potential pathogens, extracts also contain antioxidants and have anti-inflammatory properties that make them even more attractive to consumers.

  Several compounds found in oregano extracts exhibit antimicrobial properties. The major components of oregano oil are carvacrol and thymol, both inhibitory to an array of organisms, including Listeria monocytogenes (Seaberg et. al, 2003). In the Seaberg et. al (2003) study it was found that at a pH of 5.5-6.0 thymol at 100-150 ppm inhibited growth of L. monocytogenes. At 50 ppm, thymol reduced L. monocytogenes counts by 91.8% and completely inhibited them at 100 ppm. Similar amounts of carvacrol were needed at pH of 5.5, but increased levels at 150-200 ppm were necessary for inhibition at pH 6.0. This difference in amounts due to pH is attributed to thymol’s slightly different structure that allows for more penetration of the cell membrane at higher pHs.

  Mustards are also a source of compounds that have antimicrobial properties (Olaimat & Holley, 2014). They contain glucosinolates, like sinalbin and sinigrin, that, when degraded by myrosinases, form antimicrobial isothiocynates that are autotoxic. Bacteria, including Listeria monocytogenes, produce myrosinase-like compounds that can degrade glucosinolates, leading to inhibition. The oriental mustard extract in the Olaimat & Holley (2014) study contained 16% sinigrin. At 10°C, L. monocytogenes growth was inhibited for seven days. Inoculated bologna reached undetectable levels of L. monocytogenes after 52 days at 4°C. There was no significant degradation of sinigrin at any of the temperatures. Myrosinase-like activity in L. monocytogenes was highest at 21°C. The 0.5% extract could inhibit growth for 21 days at 4°C, seven days at 10°C, and could not inhibit growth for 21 days at 21°C. This means that mustard compounds could be good for long-term inhibition of bacteria that have myrosinase-like activity at refrigeration temperatures.

  Other plant extracts and essential oils derived from unique plant sources are being studied. A main course of interest is found in medicinal Chinese plants. Three medicinal plants–Juniperus phoenicea, Pistacia atlantica, and Oudneya adricana–were used to create extracts that exhibited inhibitory effects on Listeria monocytogenes (Hammami et. al, 2009). Extracts made from J. phoenicea and P. atlantica completely inhibited growth. All three of the extracts had high heat resistance, remaining 96% active after being heated at 100°C for 30 minutes. Juniperus and Oudneya remained 70% active after 121°C for 20 minutes. The heat stability of these extracts could be useful in combination with pasteurized and canned foods.

Medicinal pomegranate (Punica grahtum L.) peel and Chinese gallnut (Galla chinesis) combinations have been shown to inhibit L. monocytogenes in agar and broth studies (Wu et. al, 2016). Both were tested at refrigeration temperatures (4°C) and abuse temperatures (12°C) to determine if these extracts would remain inhibitory in food samples. Tuna patties and cooked shrimp were macerated and inoculated with 103 CFU/g. Inhibition was observed with Chinese gallnut extract after seven days and had decreased two logs after 10 days at refrigeration temperatures. Pomegranate peel had less of an inhibitory effect than Chinese gallnut. At 12°C there was increased growth when compared with 4°C. Chinese gallnut was able to inhibit L. monocytogenes for

4-10 days and decrease bacterial counts two logs, but pomegranate peel showed no inhibitory effect at this temperature. While Chinese gallnut could not completely inhibit growth, though it did postpone the exponential growth of L. monocytogenes. One issue with using the Chinese gallnut extract is that it whitened the tuna used in the study. Under the circumstances of food production, it is important to keep foods physically appealing to the customer. Further research will need to be done to determine the appearance changes of food with natural preservation methods.

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The synergistic effects of various plant extracts and essential oils cannot be ignored. Carvacrol found in oregano oil in combination with nisin reduces the amount of nisin needed to observe inhibitory effects sixteen-fold (Seaberg et. al, 2003, Klancnik et. al, 2014). Combinations of thymol and carvacrol, in addition to other phenolic compounds found in oregano, functioned more efficiently as inhibitors than pure thymol or pure carvacrol, possibly because they could better work at water and lipid soluble portions of the meat samples. Application of Alpinia katsumadai seed extract (AlpE) has exhibited inhibitory effects in Campylobacter, antioxidant activity, and antiviral activity (Klancnik et. al, 2014). The tea flavonoid epigallocatehin (EGCG) has also shown strong antibacterial properties. Both 1.03 mg/mL AlpE and 0.62 mg/mL EGCG had inhibitory effects on Listeria monocytogenes, but together the oil combination had a greater effect at 8°C. Different combinations of effective extracts and oils could be used to have compounding effects on L. monocytogenes and other bacteria. Compounds that are not plant extracts or essential oils can also improve the effectiveness of a plant extract’s/essential oil’s antimicrobial properties. Malic acid and ethylenediaminetetraacetic acid (EDTA) in combination with mustard extracts improved antimicrobial activity (Olaimat & Holley, 2014). At 21°C malic acid/EDTA/mustard extracts were bactericidal and bacteriostatic at 10°C. Cranberry-oregano extracts with sodium lactate prove to be very inhibitory according to Apostolidis et. al (2008). The combination at 2% sodium lactate with 750 ppm cranberry-oregano (50:50) extract have inhibitory effects on Listeria monocytogenes, reducing CFU/mL by one log. Extracts/oils and other inhibitory methods that target specific organisms could be used to create a larger synergistic effect when applied to food production.

It is thought that the essential oils function by several mechanisms that lead to growth inhibition and microbial death. Many plant extracts’ and essential oils’ mechanisms of action involve disruption of the cell membrane (Gill & Holley, 2006a). Gill & Holley (2006a) studied the effects of eugenol, carvacrol, and cinnamaldehyde on Listeria monocytogenes. They used L. monocytogenes cells energized with glucose and observed propidium iodine uptake when eugenol, carvacrol, and cinnamaldehyde were applied. Cinnamaldehyde cause a rapid depletion of cellular ATP, but its permeabilization into the cell is less definitive. Lower number of cells were recovered when treated with 10 mM eugenol for four minutes and 10 mM carvacrol for two minutes. Eugenol and carvacrol were determined to both be cell membrane disruptors. Extracellular ATP increased as intracellular ATP decreased when treated with 10 mM eugenol and 10 mM carvacrol. This could also be what caused reduced motility in eugenol/carvacrol treated cells because the flagella gain energy from the proton gradient, which is dissipated at 10 mM eugenol and 5 mM carvacrol. Their ability to permeabilize the membrane could be due to increased solubility from the hydroxyl group on the cyclic hydrocarbons. The inhibitory effects eugenol and carvacrol via cytoplasmic membrane disruption may induce ATPase inhibitory activity in cells. A later study by Gill & Holley (2006b) studied the inhibition of ATPase activity in cells. Inhibition was seen using 5 mM and 10 mM eugenol, 10 mM carvacrol, and 10 mM cinnamaldehyde. The inhibited membrane-bound ATPase activity resulted in impaired cell survival. It is suggested that ATPase inhibition is a secondary cause of death because inhibition occurs at concentrations within the essential oils’ bactericidal ranges. At sublethal concentrations though, growth is reduced, likely from enzyme inhibition.

  Issues involving essential oil use in foodborne pathogen inhibition are mostly due to the concentrations needed to have bactericidal and inhibitory effects (Hill et. al, 2013). At these concentrations the extracts and oils could be smelled and/or tasted in the foods they are being applied to. There are also issues pertaining to solubility with many concentrations being at the limit of the oil/extract to remain in solution. Hill et. al (2013) conducted a study that nanoencapsulates compounds in poly-(D,L-lactide-co-glycolide) (PLGA), a technique commonly used in the pharmaceutical industry that is undetectable by the naked eye or mouth. This method can successfully mask the undesired attributes from using higher essential oil/plant extracts concentrations and improve delivery of compounds. The study used PLGA50 and PLGA65 to encapsulate cinnamon bark extract (CBE). Results showed that encapsulated CBE in PLGA inhibited Listeria monocytogenes at 24 hours and 72 hours, but none were bactericidal. Other benefits of this technique would be increased pH from PLGA degradation that would allow CBE to easily move into the cell due to higher affinity for the membrane. The use of PLGA with other plant extracts and essential oils could allow increased concentrations of more antimicrobial compounds to be used.

  Essential oils are also applicable beyond direct use on foods, especially in the elimination of pathogens from production surfaces. Listeria monocytogenes is an especially difficult organism in food production since it can create a biofilm on surfaces (Oliveira et. al, 2010). These biofilms must normally be removed via mechanical force, through use of chemical compounds, and/or heat. Oliveira et. al (2010) used a disinfectant (ethanol + saline) with added Cymbopogan nardus and/or Cymbopogan citratus essential oils. The combinations were applied to stainless steel surfaces with biofilm adherence. Results showed that longer contact with essential oil combinations with biofilm lead to increased reduction of adhered cells. C. nardus oil applied to a biofilm (3 hours) for 60 minutes showed an 88.13% reduction of adhered cells. Combination of C. nardus and C. citratus oils when applied to a biofilm (240 hours) for 60 minutes showed a 100% reduction of adhered cells. This information can be used to reduce L. monocytogenes biofilms on stainless steel production surfaces and reduce contamination of other foods.

  As customers begin to search for more naturally-preserved foods, it will be necessary to begin experimenting with the application of plant extracts and essential oils. The synergistic effect of essential oils and plant extracts could be used to inhibit the growth of bacterial cocktails because many of the mechanisms of action are applicable to general categories of gram-positive and gram-negative bacteria. Their applications in combination with other hurdle technologies could reduce the incidences of Listeria monocytogenes food contamination by eliminating cells/biofilms, decreasing cross-contamination, and by inhibiting growth within foods.

References

Use of Plant Extracts and Essential Oils to Inhibit Listeria monocytogenesand Possible Applications to Foods and Food Production Surfaces

  Listeria monocytogenes is a major concern to the food production industry. It is characterized as a gram positive, facultative anaerobic, non-spore forming rod (Seaberg et. al, 2003). Why this foodborne pathogen is of high importance is mostly due to its high mortality rate, as high as 34%, making it the third leading cause of foodborne death in the United States as of 2011 (Hill et. al, 2013, Olaimat & Holley, 2014). Due to its resistance to temperatures ranging from 2.5 to 44.0°C, pH range of 6.0 to 8.0, aw <0.93, and biofilm production, it is extremely difficult to deal with in a production setting (Seaberg et. al, 2003). Listeria monocytogenes is found mostly in meats, dairy products, and ready-to-eat foods (Wu et. al, 2010). Proper cooking and pasteurization can eliminate most L. monocytogenes cells, but cross-contaminated fruits and vegetables are left vulnerable.

  Today’s consumers are now migrating from “chemical” and synthetic forms of food preservation to more natural methods (Seaberg et. al, 2003). A natural solution lies in plant extracts and essential oils, which are generally recognized as safe (GRAS). Prior research has determined that many herb and spice extracts inhibit the growth of bacteria, yeasts, and molds. Besides being inhibitory towards potential pathogens, extracts also contain antioxidants and have anti-inflammatory properties that make them even more attractive to consumers.

  Several compounds found in oregano extracts exhibit antimicrobial properties. The major components of oregano oil are carvacrol and thymol, both inhibitory to an array of organisms, including Listeria monocytogenes (Seaberg et. al, 2003). In the Seaberg et. al (2003) study it was found that at a pH of 5.5-6.0 thymol at 100-150 ppm inhibited growth of L. monocytogenes. At 50 ppm, thymol reduced L. monocytogenes counts by 91.8% and completely inhibited them at 100 ppm. Similar amounts of carvacrol were needed at pH of 5.5, but increased levels at 150-200 ppm were necessary for inhibition at pH 6.0. This difference in amounts due to pH is attributed to thymol’s slightly different structure that allows for more penetration of the cell membrane at higher pHs.

  Mustards are also a source of compounds that have antimicrobial properties (Olaimat & Holley, 2014). They contain glucosinolates, like sinalbin and sinigrin, that, when degraded by myrosinases, form antimicrobial isothiocynates that are autotoxic. Bacteria, including Listeria monocytogenes, produce myrosinase-like compounds that can degrade glucosinolates, leading to inhibition. The oriental mustard extract in the Olaimat & Holley (2014) study contained 16% sinigrin. At 10°C, L. monocytogenes growth was inhibited for seven days. Inoculated bologna reached undetectable levels of L. monocytogenes after 52 days at 4°C. There was no significant degradation of sinigrin at any of the temperatures. Myrosinase-like activity in L. monocytogenes was highest at 21°C. The 0.5% extract could inhibit growth for 21 days at 4°C, seven days at 10°C, and could not inhibit growth for 21 days at 21°C. This means that mustard compounds could be good for long-term inhibition of bacteria that have myrosinase-like activity at refrigeration temperatures.

  Other plant extracts and essential oils derived from unique plant sources are being studied. A main course of interest is found in medicinal Chinese plants. Three medicinal plants–Juniperus phoenicea, Pistacia atlantica, and Oudneya adricana–were used to create extracts that exhibited inhibitory effects on Listeria monocytogenes (Hammami et. al, 2009). Extracts made from J. phoenicea and P. atlantica completely inhibited growth. All three of the extracts had high heat resistance, remaining 96% active after being heated at 100°C for 30 minutes. Juniperus and Oudneya remained 70% active after 121°C for 20 minutes. The heat stability of these extracts could be useful in combination with pasteurized and canned foods.

Medicinal pomegranate (Punica grahtum L.) peel and Chinese gallnut (Galla chinesis) combinations have been shown to inhibit L. monocytogenes in agar and broth studies (Wu et. al, 2016). Both were tested at refrigeration temperatures (4°C) and abuse temperatures (12°C) to determine if these extracts would remain inhibitory in food samples. Tuna patties and cooked shrimp were macerated and inoculated with 103 CFU/g. Inhibition was observed with Chinese gallnut extract after seven days and had decreased two logs after 10 days at refrigeration temperatures. Pomegranate peel had less of an inhibitory effect than Chinese gallnut. At 12°C there was increased growth when compared with 4°C. Chinese gallnut was able to inhibit L. monocytogenes for

4-10 days and decrease bacterial counts two logs, but pomegranate peel showed no inhibitory effect at this temperature. While Chinese gallnut could not completely inhibit growth, though it did postpone the exponential growth of L. monocytogenes. One issue with using the Chinese gallnut extract is that it whitened the tuna used in the study. Under the circumstances of food production, it is important to keep foods physically appealing to the customer. Further research will need to be done to determine the appearance changes of food with natural preservation methods.

The synergistic effects of various plant extracts and essential oils cannot be ignored. Carvacrol found in oregano oil in combination with nisin reduces the amount of nisin needed to observe inhibitory effects sixteen-fold (Seaberg et. al, 2003, Klancnik et. al, 2014). Combinations of thymol and carvacrol, in addition to other phenolic compounds found in oregano, functioned more efficiently as inhibitors than pure thymol or pure carvacrol, possibly because they could better work at water and lipid soluble portions of the meat samples. Application of Alpinia katsumadai seed extract (AlpE) has exhibited inhibitory effects in Campylobacter, antioxidant activity, and antiviral activity (Klancnik et. al, 2014). The tea flavonoid epigallocatehin (EGCG) has also shown strong antibacterial properties. Both 1.03 mg/mL AlpE and 0.62 mg/mL EGCG had inhibitory effects on Listeria monocytogenes, but together the oil combination had a greater effect at 8°C. Different combinations of effective extracts and oils could be used to have compounding effects on L. monocytogenes and other bacteria. Compounds that are not plant extracts or essential oils can also improve the effectiveness of a plant extract’s/essential oil’s antimicrobial properties. Malic acid and ethylenediaminetetraacetic acid (EDTA) in combination with mustard extracts improved antimicrobial activity (Olaimat & Holley, 2014). At 21°C malic acid/EDTA/mustard extracts were bactericidal and bacteriostatic at 10°C. Cranberry-oregano extracts with sodium lactate prove to be very inhibitory according to Apostolidis et. al (2008). The combination at 2% sodium lactate with 750 ppm cranberry-oregano (50:50) extract have inhibitory effects on Listeria monocytogenes, reducing CFU/mL by one log. Extracts/oils and other inhibitory methods that target specific organisms could be used to create a larger synergistic effect when applied to food production.

It is thought that the essential oils function by several mechanisms that lead to growth inhibition and microbial death. Many plant extracts’ and essential oils’ mechanisms of action involve disruption of the cell membrane (Gill & Holley, 2006a). Gill & Holley (2006a) studied the effects of eugenol, carvacrol, and cinnamaldehyde on Listeria monocytogenes. They used L. monocytogenes cells energized with glucose and observed propidium iodine uptake when eugenol, carvacrol, and cinnamaldehyde were applied. Cinnamaldehyde cause a rapid depletion of cellular ATP, but its permeabilization into the cell is less definitive. Lower number of cells were recovered when treated with 10 mM eugenol for four minutes and 10 mM carvacrol for two minutes. Eugenol and carvacrol were determined to both be cell membrane disruptors. Extracellular ATP increased as intracellular ATP decreased when treated with 10 mM eugenol and 10 mM carvacrol. This could also be what caused reduced motility in eugenol/carvacrol treated cells because the flagella gain energy from the proton gradient, which is dissipated at 10 mM eugenol and 5 mM carvacrol. Their ability to permeabilize the membrane could be due to increased solubility from the hydroxyl group on the cyclic hydrocarbons. The inhibitory effects eugenol and carvacrol via cytoplasmic membrane disruption may induce ATPase inhibitory activity in cells. A later study by Gill & Holley (2006b) studied the inhibition of ATPase activity in cells. Inhibition was seen using 5 mM and 10 mM eugenol, 10 mM carvacrol, and 10 mM cinnamaldehyde. The inhibited membrane-bound ATPase activity resulted in impaired cell survival. It is suggested that ATPase inhibition is a secondary cause of death because inhibition occurs at concentrations within the essential oils’ bactericidal ranges. At sublethal concentrations though, growth is reduced, likely from enzyme inhibition.

  Issues involving essential oil use in foodborne pathogen inhibition are mostly due to the concentrations needed to have bactericidal and inhibitory effects (Hill et. al, 2013). At these concentrations the extracts and oils could be smelled and/or tasted in the foods they are being applied to. There are also issues pertaining to solubility with many concentrations being at the limit of the oil/extract to remain in solution. Hill et. al (2013) conducted a study that nanoencapsulates compounds in poly-(D,L-lactide-co-glycolide) (PLGA), a technique commonly used in the pharmaceutical industry that is undetectable by the naked eye or mouth. This method can successfully mask the undesired attributes from using higher essential oil/plant extracts concentrations and improve delivery of compounds. The study used PLGA50 and PLGA65 to encapsulate cinnamon bark extract (CBE). Results showed that encapsulated CBE in PLGA inhibited Listeria monocytogenes at 24 hours and 72 hours, but none were bactericidal. Other benefits of this technique would be increased pH from PLGA degradation that would allow CBE to easily move into the cell due to higher affinity for the membrane. The use of PLGA with other plant extracts and essential oils could allow increased concentrations of more antimicrobial compounds to be used.

  Essential oils are also applicable beyond direct use on foods, especially in the elimination of pathogens from production surfaces. Listeria monocytogenes is an especially difficult organism in food production since it can create a biofilm on surfaces (Oliveira et. al, 2010). These biofilms must normally be removed via mechanical force, through use of chemical compounds, and/or heat. Oliveira et. al (2010) used a disinfectant (ethanol + saline) with added Cymbopogan nardus and/or Cymbopogan citratus essential oils. The combinations were applied to stainless steel surfaces with biofilm adherence. Results showed that longer contact with essential oil combinations with biofilm lead to increased reduction of adhered cells. C. nardus oil applied to a biofilm (3 hours) for 60 minutes showed an 88.13% reduction of adhered cells. Combination of C. nardus and C. citratus oils when applied to a biofilm (240 hours) for 60 minutes showed a 100% reduction of adhered cells. This information can be used to reduce L. monocytogenes biofilms on stainless steel production surfaces and reduce contamination of other foods.

  As customers begin to search for more naturally-preserved foods, it will be necessary to begin experimenting with the application of plant extracts and essential oils. The synergistic effect of essential oils and plant extracts could be used to inhibit the growth of bacterial cocktails because many of the mechanisms of action are applicable to general categories of gram-positive and gram-negative bacteria. Their applications in combination with other hurdle technologies could reduce the incidences of Listeria monocytogenes food contamination by eliminating cells/biofilms, decreasing cross-contamination, and by inhibiting growth within foods.

References

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