MCFA and Monoglycerides – a terminology

MCFAs (defined as saturated fatty acids with C6 to C12 hydrocarbon chains) and their corresponding monoglyceride derivatives are single-chain lipid amphiphiles.

MCFAs have a lower melting point, smaller molecule size, high water solubility and oxidative stability and are less energy-dense (8.3 vs. 9.2 kcal/g) compared to long chain saturated fatty acids (LCFAs; Shah and Limketkai, 2017). These substantial distinctions in chemical and physical properties make MCFA absorption and metabolism different from other fatty acids (Marten et al., 2006).

Medium-chain saturated fatty acids and their corresponding monoglyceride derivatives are of particular interest because of their reported antimicrobial activity.

Alpha-monoglycerides are a class of glycerides that are composed of a fatty acid linked to the sn1-position of glycerol via an ester bond and are known for their strong antimicrobial effects. Figure 1 gives a schematic overview of the esterification reaction to obtain such an α-monoglyceride.

Figure 1. Esterification reaction of a fatty acids and glycerol to form an α-monoglyceride

It is important to understand that only the α-monoglycerides have antimicrobial effects. A triglyceride is a fat, which is broken down during normal digestion processes and is a source of energy.

The strong covalent bond of α-monoglycerides results in multiple benefits compared to free fatty acids. Alpha-monoglycerides are pH independent and consequently do not dissociate. They are non-volatile, non-corrosive and heat stable during feed processing. Furthermore, α-monoglycerides have a neutral taste and odour, but more important, research has shown that α-monoglycerides have a much stronger antimicrobial effect compared to their corresponding free fatty acids (Table 1).

Table 1. The Minimum Inhibitory Concentrations MIC (μg/ml) of lauric acid and monolaurin towards selected bacteria, after Batovska 2009

Moreover, α-monoglycerides are amphiphilic compounds possessing both lipophilic (fat-loving) properties, due to their fatty acid tail and hydrophilic (water-loving) properties, due to their hydrophilic backbone (glycerol). This makes α-monoglycerides self-emulsifying in water. Consequently, they are active in four different environments: water, feed, stomach, and the intestinal tract.

Mode of action

Impact against bacteria

The antibacterial activities of lipids have been known for more than a century. With the advent of antibiotics in the 1940s and 50s the interest in fatty acids and other lipids as antibacterial agents diminished, but in the 1970s there was a renewed interest in antimicrobial lipids, particularly milk lipids.

MCFAs and monoglycerides are antimicrobial agents that can disrupt the phospholipid membrane surrounding membrane-enclosed pathogens such as bacteria and lipid bilayer-enveloped viruses.

Bactericidal activity of MCFAs has been explained by either direct or indirect mechanisms. Researchers have reported that MCFAs target bacterial cell membranes and disrupt their electron transport chain, consequently reducing ATP production and starving them of energy. In this case, after penetrating the membranes in an undissociated form, ionisation of fatty acids take place, which results in increased fluidity, solubility, permeability, and instability of the bacterial membrane (Bergsson et al., 1998; Bergsson et al., 1999). In these processes, the detergent properties of the fatty acids play a substantial role in preventing biofilm formation (Schlievert and Peterson, 2012). As a result of this activity, the internal contents of bacteria leak into the outside of the cell, inhibiting growth and causing bacterial death (Desbois and Smith, 2010). It has been reported that diffusing fatty acids in an undissociated form which then dissociate within the protoplasm, results in intracellular acidification, which adversely affects enzymes and amino acids in the microorganism (Freese et al., 1973; Viegas and Sa-Correia, 1991; Sun, et al. 1998). Aside from these mechanisms, free MCFAs and their corresponding monoglycerides can inhibit synthesis of bacterial toxins and expression of other virulence factors by interfering with signal transduction (Projan et al., 1994; Ruzin and Novick, 2000).

In terms of antibacterial activity, the compounds can inhibit bacterial growth ("bacteriostatic") or induce bacterial cell lysis and cell killing ("bactericidal") (Yoon et al., 2018). In general, MCFAs and monoglycerides exhibit more potent inhibitory activity against Gram-positive bacteria than Gram-negative bacteria (Thormar and Hilmarsson, 2007), because Gram-positive bacteria have simpler, single lipid bilayer cell membrane structures, while Gram-negative ones typically have more complex inner and outer membrane structures.

Spectrum of antimicrobial activity

The extent of the antimicrobial potency of the fatty acids depends on several factors, such as fatty acid structure and concentration, target bacteria, cell membrane properties and the regulatory capacity of the intracellular pH of the bacteria. This has been well documented by in vitro studies.

Pioneering work was completed in the 1970's and the authors conducted detailed structure-function studies investigating how hydrocarbon chain length affects antimicrobial activity (Kabara et al., 1972). In general, capric (C10) and lauric (C12) acids exhibited the highest potencies among fatty acids while corresponding monoglycerides with equivalent chain lengths were typically even more potent.

Research has proven that α-monoglycerides have a much stronger antibacterial effect compared to their corresponding free fatty acids. Depending on the chain length of the fatty acid, they have also antiviral properties. One method to show the antibacterial properties of a substance is to conduct an in vitro Minimal Inhibitory Concentration (MIC) test. MIC values of several fatty acids and their corresponding α-monoglycerides are given in Table 2.

Table 2. MIC-test of capric acid and lauric acid and their corresponding α-monoglycerides on Gram-positive bacteria. Results are given in micromoles per millilitre, Modified after Anonymous, 2017

Lauric acid (C-12) has the greatest antibacterial activity of all medium chain aliphatic fatty acids. This effect is magnified when acid is esterified to glycerol resulting in monolaurin, the best antibacterial monoglyceride with medium chain. Monolaurin is active primarily against Gram-positive pathogenic and spoilage bacteria. However, its spectrum of activity can be broadened when combined with other substances, such as for an example organic acids.

In recent years, there has been interest in exploring the inhibitory activity of MCFAs and monoglycerides against pathogens relevant to the swine industry. It has been reported that GML (C12 monoglyceride), lauric acid (C12) and a mixture of caprylic (C8) and capric (C10) acids exhibit potent antibacterial activity against numerous bacterial pathogens such as Streptococcus suis, and Clostridium perfringens. It has been furthermore demonstrated that MCFAs and monoglycerides often exhibit greater antibacterial potency than commonly studied organic acids - e.g. lactic and citric acids (Skřivanová et al., 2006). It has also been determined that compounds within this class, such as GML, can exhibit nearly complete killing of vegetative cells and spores of aerobic B. anthracis, B. subtilis, and B. cereus and anaerobic Clostridium perfringens and Clostridium (Clostridioides) difficile (Schlievert et al., 2018). Another study has reported that GML and lauric acid also inhibit Listeria monocytogenes, which is a foodborne pathogen that can contaminate swine farms (Wang and Johnson, 1992).

From the need to control pathogenic bacteria (Gram-positive and Gram-negative), ADDCON developed a 3rd generation acidifier (Formi 3G). The product represents an agglomerate of the patented molecule diformate (sodium diformate) and monolaurate.

The double sodium salt of formic acid, sodium diformate, has a strong anti-microbial effect (bacteriostatic and bactericidal effect mainly against Gram-negative pathogenic bacteria), but is proven safe for humans and has no adverse smell. In contrast to formic acid, sodium diformate is a solid product (composition: 40% formic acid, 40% formate and 20% sodium), which is non-corrosive.

Because of its unique chemical structure, sodium diformate combines the chemical properties of both an acid and a salt. The structure of the molecule provides the highest concentration of acid delivered into the small intestine (85%) of the animal, which makes sodium diformate the best alternative for antibiotics in order to control pathogenic bacteria, such as E. coli, Salmonella spp., which are located in the small intestine (duodenum). The diformate is furthermore able to optimize the pH of the proximal parts of the digestive tract and can therefore improve the digestion of proteins and minerals.

Figure 2. Chemical structural formula of formic acid and sodium diformate

On the other hand, formulating 3rd generation acidifier with diformates and monolaurates improves the antimicrobial effect by controlling Gram-positive pathogenic bacteria, and partly virus, as well.

Combining the efficacy of these two known additives, the decontamination effect on Gram-negative and Gram-positive bacteria in sow faeces and the subsequent impact on their suckling piglets is very important and has been tested. The use of dietary diformate and monolaurate in sow diets was shown to be an effective and sustainable tool in improving gut health in sows and performance of their piglets.

Several trials were carried out to test this approach. Multiparous sows on commercial farms in Germany were fed either a commercial lactation diet as control – or a test diet, which contained additionally 1% Formi 3G. On the 21st day of lactation, freshly excreted faecal matter was collected from all sows and analysed for E. coli, Enterococci, Streptococci and the total aerobic bacteria count. Data were analysed using the t-test and a significance level of 0.05 was used in all tests.

Results of the microbial analysis revealed a strong significant impact of the product on the bacterial population in the faecal matter of sows. This holds true for E. coli and Streptococci / Enterococci counts, as well as the total aerobic bacteria count (Table 3). The significant reduction rates in the E. coli counts in the faeces were well above 90% and varied in the trials between 90% and 98%. Furthermore, the reduction in the Streptococci / Enterococci counts within the various trial periods were significant and varied from 75% to 99%. Finally, the count of total aerobic bacteria, among them the group of spoilage indicating bacteria, tended to be reduced (-94%).

Table 3. Bacterial count reduction rates (%) in sow faeces after feeding with 1.0% diformate-monolaurate (Formi 3G) in the lactation diet

The anti-bacterial effect against Clostridia spp.and Streptococcispp.has been shown as well in commercial farms in Australia, were multiparous sows fed either a lactation diet, which contained a regular acidifier at 0.8% as control – or a test diet, which contained 1% of Formi 3G instead (Table 4). The diets were fed from the 90th day of gestation until weaning. Faecal samples (n=24) were collected on the 5thday of lactation. With an analysis of the faecal microbiome significant improvements were noted. The main effects of the test diet were a significant (p<0.018) reduction of the relative abundance of Clostridia spp.by 42.2% and of Streptococci spp.by 42.5% (p<0.013).

Table 4. Relative abundance (%) of Clostridia spp. and Streptococci spp. in the sow faecal microbiome

The addition of the 3rd generation acidifier provides a significant improvement of the health status of sows, based on their faecal microbiome. However, the acidifier not only provides a healthy gut in sows but will furthermore support the piglets indirectly during the suckling period by providing "clean" conditions and thus greatly reducing the spread of E. coli, Streptococci and Clostridia towards the new-born piglets, which would lead to improved performance.

Conclusion

The antimicrobial activities of lipids, particularly of free fatty acids and monoglycerides, have been studied for decades. The activity profiles for many bacteria have been determined, showing that medium-chain saturated fatty acids and monoglycerides thereof are generally most active. With their bactericidal effect against Gram-positive bacteria, monoglycerides are a valuable tool for sustainable healthcare in the post-AGP era. Their combination with an organic acid is preferred because of the effect of organic acids against Gram-negative bacteria.

Formi 3G is therefore the product of choice when it comes to a holistic anti-bacterial approach in swine nutrition.

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Article made possible through the contribution of Dr. S. Petrović, Dr. C. Lückstädt and ADDCON, Bitterfeld – Germany

Impact of medium chain fatty acids, their monoglycerides - and agglomerates of them with diformates against bacteria and viruses - Part I: Anti-bacterial effect