Antimicrobial Activity of Natural Compounds

Antimicrobial Activity of Natural Compounds: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Microbiology

Contributor: Rui Meneses , Paula Teixeira

Using natural compounds as antimicrobial agents in food is not a new idea. For ages, humans have used a variety of compounds, ranging from organic acids, essential oils (EOs), and wines to salts and seasonings, all with the intent of improving the sensorial experience and the product’s lifespan. Essential oils, present in numerous seasonings (e.g., garlic, oregano, thyme, and rosemary), and the organic acids present in various fruits (e.g., grapes, tomatoes, and citrus fruits), have been studied for their natural preservative properties. Heavily complex fermented substances, such as vinegar, wines, and sauces such as soy sauce, that possess various known antimicrobial compounds are also frequently used. Although some of the substances mentioned above mainly have high concentrations of organic acids, others, such as wines, hold highly complex matrices containing several compounds known for their antimicrobial activity.

natural antimicrobial compounds
essential oils
organic acids

1. Essential Oils and Extracts from Aromatic Herbs

Although most studies reporting the antimicrobial activity of aromatic herbs delve into EOs, studies employing plant extracts have also been conducted. However, there is still a lack of knowledge on how the extraction processes affect the bioactivity of most aromatic plant extracts. Nonetheless, the plant extracts from common aromatic herbs such as oregano and thyme have shown antimicrobial activity against foodborne pathogens and spoilage bacteria.

Jovanović et al. (2021) studied the inhibitory and bactericidal activity of two polyphenol-rich wild thyme extracts against seven foodborne pathogens. Of all the tested bacteria, Enterococcus faecalis was the most susceptible to inhibition from the tested extracts, with a minimum inhibitory concentration (MIC) of 0.313 mg/mL. Moreover, L. monocytogenes and Bacillus cereus presented the highest tolerances, with MIC values of 0.625 mg/mL for both organisms. Staphylococcus aureus and Yersinia enterocolitica were considerably less susceptible to the inhibitory effect of the wild thyme extracts (MIC 1.25 mg/mL). Regarding the bactericidal effect, L. monocytogenes proved to be the most susceptible of all the tested microorganisms, with a minimum bactericidal concentration (MBC) of 2.5 mg/mL. On the other hand, S. aureus and Y. enterocolitica showed the highest resistance to the extracts, with an MBC value of 10 mg/mL [67].

Teixeira et al. (2013) evaluated the antimicrobial activity of oregano extracts and the EOs obtained through hot water extraction against seven foodborne pathogens and spoilage bacteria. The tested extracts inhibited the growth of the tested bacteria. Nonetheless, the EOs inhibited the growth (MIC value < 5 mg/mL) and reduced the levels of the tested microorganisms [68].

Essential oils are volatile and aromatic oily liquids extracted from plant components (e.g., leaves or seeds, fruits, stems, roots, and buds) and possess hundreds of low molecular weight secondary metabolites [69].

Although terpenes and terpenoids are the most prevalent class in EOs [70], with phenolic compounds only representing a smaller fraction of the total constituents of EOs, the phenolic contents of an EO have been found to be directly correlated with its antimicrobial activity [56]. Nonetheless, studies have shown that terpenes such as carvacrol and thymol present in oregano and thyme possess promising antimicrobial capacity [71,72,73,74]. For instance, Thanissery et al. (2014) tested, in vitro, the antimicrobial capabilities of rosemary, clove, thyme, and orange EOs against Salmonella spp., C. coli, and C. jejuni. Overall, Campylobacter isolates proved to be more susceptible to the antibacterial activity of the tested EOs, with synergistic effects occurring between the EOs [75].

Additionally, along with terpenes and phenylpropanoids, flavonoids and alkaloids also demonstrated significant antimicrobial activity when administered isolated or as part of an extract [76,77,78].

The mechanisms behind the antimicrobial activity of EOs vary, either leading to a bacteriostatic effect, where the bacterial growth inhibition occurs, or to a bactericidal action, where EOs kill the bacterial cells. Essential oils most commonly target the cytoplasmic membrane, damaging it while disrupting efflux pumps and other proteins embedded into the cell membrane, leading to leakage of intracellular components and subsequent membrane rupture and cell death [2,79]. Carvacrol, thymol, and garlic extracts were shown to exert this mechanism of antimicrobial action [80,81]. Essential oils can also exhibit antibacterial activity through other means. For instance, andrographolide, a terpene, can interfere with protein and DNA synthesis [82], while quercetin inhibits the biosynthesis of unsaturated fatty acids [83]. The United States Food and Drug Administration (FDA) classifies several EOs as generally recognised as safe (GRAS).

However, as with any other compound, using EOs as antimicrobials in meat products has certain limitations. Meat is a complex matrix that encompasses high quantities of saturated fatty acids and proteins, which may decrease the activity of EOs due to their high binding capacity of volatile compounds present in the EO. Consequently, they lead to the need to administer higher concentrations of EOs in meat products, raising another question; adding high concentrations of EOs to meat products creates powerful and unpleasant aromas and off-flavours, making them unacceptable to the consumer.

The use of EOs in edible coatings, active packaging, microencapsulation, and nanoparticles has been proposed [84,85,86,87]. Nonetheless, although many of these approaches are highly effective, they carry intrinsic costs which increase the price of an otherwise relatively inexpensive product. Hence, industries dissent from adopting these methods on a large scale.

A summary of the studies evaluating the antimicrobial activity of the EOs of different plants and the various delivery methods is presented in Table 2.

Table 2. Studies evaluating the antimicrobial activity of essential oils (EOs) of different plants.

EOs Employed	Concentrations Applied	Microorganisms Tested	Major Effects	Matrix	Marination and Storage	References
21 different EOs and several combinations	0.50%	Spoilage bacteria	Only eight of all tested EOs produced antimicrobial activity. The optimal compound spices extract, for reducing spoilage bacteria, consisted of 2.4 µL/mL of cassia bark EO, 1.0 µL/mL of cinnamon EO, 3.5 µL/mL of tea tree EO, and 9.0 µL/mL of angelica EO.	In vitro	The essential oils were directly applied on plates coated with putrefying bacteria liquid.	[88]
Thyme and orange EOs		Salmonella Enteritidis Campylobacter coli	Treatments with thyme, orange oils, and vacuum tumbling significantly reduced the viable counts of S. Enteritidis and C. coli by 2.3–2.6 and 3.1–3.6 log₁₀ CFU/g, respectively.	Chicken breast fillets and wings	Vacuum tumbling for 20′ with a10% (v/w) pre-chilled (4 °C) marinade solution.	[89]
Thyme and orange EOs	1.0% (w/w)	Escherichia coli Staphylococcus aureus	Treatments with EOs and atmospheric cold plasma (APC), along with their combinations, reduced bacterial growth. EOs contributed to the increased sensibility of E. coli to APC treatment.	Chicken breast fillets	Immersion in a marinade solution for 2 min followed by storage at 4 °C and exposure to APC.	[90]
Carvacrol (CA) and thymol (TH)	0.4 and 0.8% (v/w)	Pseudomonas spp. Brochothrix thermosphacta E. coli Yeast and moulds Total coliforms Total viable count (TVC)	Together with vacuum packaging, EOs at 0.8% delayed the growth of spoilage bacteria. The combination of EOs at 0.4% with both packaging methods increased the products’ shelf-life by 6 to >12 days.	Chicken breast fillets	Immersion in a marinade solution with storage at 4 °C under aerobic or vacuum packaging.	[91]
Carvacrol, cinnamaldehyde (CI) and thymol	1.0 and 2.0% (v/v)	Listeria monocytogenes Salmonella spp. E. coli O157:H7	The marination decreased all pathogen counts. EOs did not enhance the antimicrobial action against L. monocytogenes. 1.0% CI decreased Salmonella counts by 1.0 log10 CFU/g. For E. coli O157:H7 EOs lead to a ≤2.4 log₁₀ CFU/g reduction.	Chicken breast	Immersion in a marinade solution with storage at 4 or 10 °C for 1, 4, and 7 days.	[92]
Propolis extract	4.0, 8.0, 12.0% (v/w)	S. aureus E. coli Yeast and moulds TVC	During storage, bacterial growth was decreased by the propolis extracts, with higher concentrations yielding higher antimicrobial activities. Furthermore, propolis reduced the changes in meat texture quality throughout the storage period.	Chicken breast	Immersion in a marinade solution with storage at 4 °C for 3, 6, 9, and 12 days.	[93]

2. Organic Acids

Like EOs, organic acids are granted GRAS status by the EU and the FDA [93,94], allowing their use in various technical purposes for poultry products, from preservatives and antioxidants to pH adjusters and flavouring agents [95]. Recipes such as Ceviche, a Peruvian dish containing raw fish, rely for their safety on the large quantities of organic acids added, citric acid in the case of Ceviche [96]. In addition, vinaigrette, besides improving the flavour of meat, has a high acetic acid content, which presents another hurdle for bacteria to overcome.

There are two main phenomena responsible for the antimicrobial activity of organic acids. The first one, cytoplasm acidification, hinders cellular metabolic reactions. The second process, the intracellular accumulation of toxic dissociated acid forms, prompts the cell to pointlessly spend large amounts of energy to counteract the natural acid influx, leading to its demise [97,98,99,100].

As previously mentioned, some problems arise when using organic acids in meat products. Generally, meat possesses a high buffering capacity, allowing the maintenance of a relatively high pH even when exposed to acidic solutions, with poultry meat having an average pH value of approximately 6. Under these conditions, organic acids dissociate and, in this form, lose their ability to enter the cell, reducing their effectiveness [54]. The addition of organic acids at higher concentrations would overcome this limitation. However, this would impose new obstacles. High concentrations of organic solutions would considerably alter meat properties such as its colour, smell, taste, water holding capacity, and binding capacity, thus creating a darker meat product with a pungent acidic smell and taste, losing significant amounts of water when cooked and being more brittle when cut [101].

The studies evaluating organic acid’s antimicrobial activity are presented in Table 3.

Table 3. Studies evaluating the antimicrobial activity of organic acids.

Organic Acids Employed	Concentrations Applied	Microorganisms Tested	Major Effects	Food Matrix	Exposure Conditions	References
Malic acid (MA) and Acetic acid (AC)	5 mg/mL	Five Salmonella serovars: Typhimurium, Heidelberg, Copenhagen, Enteritidis, and Kentucky.	At 4 °C, the solutions containing both malic and acetic acid were able to ensure a 5-log reduction in Salmonella on the chicken breast while also reducing mesophilic aerobic bacteria and lactic acid bacteria.	Chicken breast	Immersion for 5 min with shaking at 150 rpm/min followed by storage at 4 °C for 10 days.	[102]
Vinegar (Acetic acid-AC) and lemon juice (Citric acid-CA)	4%, 2%, 1.5%, 1% and 0.5% (v/v)	Three Salmonella serovars: Typhimurium, Enteritidis and Infantis.	Higher concentrations of organic acids (2–4% v/v) were the most effective against the tested pathogens. The effect of AC on the pathogen was more pronounced compared to CA. The response to acid stress was strain-dependent.	Chicken breast fillets	Immersion for 1 h at 4 °C. Storage at 4, 8, 12 and 16 °C for 9 days.	[103]
Citric acid (CA), Latic acid (LA)	0.2–10%	Chicken skin microbiota: mesophilic and psychotropic bacteria, coliforms, yeasts, and moulds.	The organic acids improved the shelf life of the tested carcasses while significantly reducing the microbial load of the carcass.	Chicken skin	Immersion for 1 min followed by storage for 3 days at 6 ± 2 °C.	[104]
LA, MA and Fumaric acid (FA)	3%	Salmonella spp.	All tested acids reduced Salmonella counts by more than 1 log₁₀ CFU/g, with FA being the most effective one.	Chicken breast	Immersion for 15 s followed by storage at 4 °C for 10 days.	[105]
LA and CA	0.5%, 1.0%, 1.5%, 2.0% (w/v)	Chicken meat natural microbiota, Salmonella spp. and Staphylococcus aureus.	After the administration of the spray-washing treatment with lactic acids and citric acid, microbial loads on the chicken drumsticks significantly decreased—most effective: 0.5% LA, 1% CA, spray-washing for 30 s.	Chicken drumstick	Spray-washing for 15, 30, 45, 60 s.	[106]

3. Wines

The literature extensively reports wine as a digestion aid and protector against infections from common foodborne pathogens such as C. jejuni, Salmonella spp., E. coli O157:H7, B. cereus, and L. monocytogenes [107,108,109,110].

The antimicrobial mechanism of wines is still not fully understood. Wine is an exceedingly complex matrix possessing low pH, high content of organic acids (such as malic and tartaric acid), and high ethanol concentration [111]. In addition, polyphenols and fatty acids are also present in wines [112], alongside sulphur dioxide, often added to wines as an antioxidation agent [113]. All these compounds are known for possessing antimicrobial action.

The antibacterial activity of wine might not be due to one single constituent but the overall combination of such components. Just and Daeschel (2003) observed that the wine and grape juice from the same grapes had similar pH levels and showed different antimicrobial strengths against E. coli O157:H7 and Salmonella spp. Furthermore, although the notion that ethanol could have been chiefly involved in the wine’s bactericidal effect was perfectly valid, it was disproven by the lack of antimicrobial activity shown by ethanol solutions diluted to concentrations commonly found in wines (10 to 15% ethanol) [109].

The work of Santoro et al. (2020), assessing the antimicrobial activity of various wines tested on a fish matrix, also supported the notion that the antimicrobial capacity of wine may come from a synergistic action of its components rather than a particular constituent. None of the tested wine constituents matched the wines’ antimicrobial action [114].

The antimicrobial activity of wine was evaluated in broth models in which a selected concentration of pathogens was added directly to the wine or to the wine solution. In these environments, variables such as matrix protection are not observed or even considered. As such, any compound will achieve its highest antimicrobial potential. Therefore, when performing the same experiment on food matrices, we may not reach the same result due to the protection offered by the food matrix. The parallelism between broth models and food matrices does not translate well. Isohanni et al. (2010) demonstrated that, in a broth model, wine reduced Campylobacter by 7 log cycles in just 15 min. However, when applied to a food matrix, the same wine only decreased Campylobacter by 1 log cycle after 48 h of exposure [115].

According to Friedman et al. (2007), wine acts as a good solvent for EOs, for instance, thymol and carvacrol. Accordingly, wine may be used not only for its bactericidal effect but also as a solvent for EOs, thus enabling the creation of complex solutions containing organic acids and EOs. The synergistic effect of these compounds would lead to effective antimicrobial action by the marinade without resorting to synthetic food preservatives [116].

In Table 4, summaries of the various studies evaluating the antimicrobial activity of wines are presented.

Table 4. Studies evaluating the antimicrobial activity of wines.

Wine Employed	Microorganisms Tested	Major Effects	Matrix	Exposure Conditions	References
Red wine (Sauvignon Blanc) and White wine (Cabernet Sauvignon)	Campylobacter jejuni Campylobacter coli	For the broth models, white wine reduced up to 7 log10 CFU/mL of Campylobacter spp. in just 15 min. However, in the food matrix, the identical wine only reduced Campylobacter loads by 1.0 log10 CFU/mL over 48 h.	Broth model and Chicken breast fillets	Immersion for 10, 15, and 30 min, and 1, 3 h at room temperature. 24 and 48 h at 4 °C.	[115]
Red wine (Douro)	C. jejuni	In broth, undiluted wine and its components drastically reduced the C. jejuni counts by approximately 7.0 log10 CFU/g. Furthermore, ethanol and the organic acids present in the wine is suggested to work synergistically. Additionally, in the stomach model, the wine enhanced the antimicrobial activity of the gastric fluid against C. jejuni.	Broth and stomach model	The pathogen was directly exposed to the wine solution both in broth and in the stomach model.	[107]
Red wine (Pinot Noir) and white wine (Chardonnay)	Escherichia coli O157:H7 Salmonella Typhimurium	When added directly into wine solutions, both pathogens were rapidly inactivated after 1 h for E. coli and half an hour for Salmonella. However, in the stomach model, the wine showed no antimicrobial action against E. coli O157:H7, whereas Salmonella was reduced to undetectable levels after 2 h of exposure to the wine. For Salmonella, the primary antimicrobial activity of the tested wine showed to be acid related.	Broth and stomach model	The pathogen was directly exposed to the wine solution both in broth and in the stomach model.	[109]
Red wine (Cabernet) and white wine (Chardonnay)	E. coli O157:H7 Listeria monocytogenes S. Typhimurium Staphylococcus aureus	Of all the tested pathogens, Salmonella was the most susceptible pathogen to red wine, with S. aureus presenting itself as the least susceptible to both wines. Ethanol and organic acids appeared to work synergistically with one another.	Broth model	The pathogen was directly exposed to the wine solution.	[117]

This entry is adapted from the peer-reviewed paper 10.3390/app122211774

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