Prof. Naresh Magan and Dr. Ángel Medina
Applied Mycology Group, Environment and Agrifood Theme, Cranfield University, Cranfield, MK43 0AL, U.K.
However, when we examine the ingredients which are used, especially in Europe, a significant number of these are rejected at the borders because of mycotoxins, especially those originating from sub-tropical and tropical regions (RASFF, 2019).
We will address the use of such preservatives and their potential drawbacks, especially when using sub-optimal concentrations which may exacerbate mycotoxin contamination.
There has also been a lot of interest in finding alternative food/feed grade preservatives based on antioxidants and plant extracts, especially essential oils. However, many of these are not classed as GRAS compounds because the necessary toxicology has not been completed and in addition the cost-benefit analyses needs to be included to evaluate their potential for commercial use. These aspects will also be addressed.
ALIPHATIC ACIDS AND THEIR SALTS FOR CONTROLLING SPOILAGE AND MYCOTOXIGENIC MOULDS
Animal feed consists of mixtures of ingredients including cereals, cereal by-products, cotton seed cake, groundnut cake, soya extracts, palm pulp, pulses and other additives depending on whether the product is destined for cattle, swine, or poultry.
The production of pelleted feed results in a relatively dry feed product that can be stored effectively in the short and medium term, provided they do not absorb moisture during the storage phase, due to poor management post-harvest.
This can occur because of ineffective partitioning of different types of commodities during transport or storage, since feed ingredients can re-absorb moisture. This is especially so if some of the ingredients are hygroscopic, which can allow the initiation of growth by spoilage and indeed mycotoxigenic moulds resulting in contamination with mycotoxins (Perreira et al., 2019).
Economically, spoilage moulds and mycotoxin contamination are very important.
In a 10-year study in the USA, it was shown that animal feed ingredients and forage samples contained significant amounts of mycotoxins. For example:
Forages including concentrates, maize grain, soybean meal, cottonseed cake, corn silage, grass hay, small grain and grass silage have been surveyed for the presence of mycotoxins.
These have all shown a significant increase over recent years in the percentage of samples of feed which are contaminated with different mycotoxins including those by Biomin, Alltech, Randox and Patent Co, entre otras (Biomin survey, 2019; Alltech survey, 2019; Randox survey, 2019, Patent Co 2019).
Non-extruded feedstuffs, including hay and mixed feeds, are often treated with anti-mould preservatives, usually based on individual or mixtures of aliphatic acids or their salts. This can reduce the potential for initiation of mould spoilage and mycotoxin contamination.
Most of the preservative mixtures used commercially are, however, fungistatic not fungicidal, and thus are not lethal to contaminating mould spores.
Under-treated or non-treated pockets of feed, even very small ones, of a few grams, may provide initial foci for the initiation of germination of spoilage/mycotoxigenic mould spores if the feed moisture content is conducive to growth (Lord et al., 1981, 1983; Lacey et al., 1982).
It is thus critical that, when preservatives are added, effective mixing of the preservative with the feed is achieved to ensure coverage of the feed with the right concentration to minimize opportunities for spoilage moulds/ mycotoxigenic mould initiation and toxin contamination from occurring during short- and medium-term storage.
Indeed, elegant studies by Lord et al. (1981) showed the importance of effective mixing of aliphatic acids or their salts with the feed to prevent undertreated or untreated pockets of the feed.
They suggested that mould spoilage may be initiated as well as contamination with aflatoxins (AFs; class 1a carcinogens), other mycotoxins such as ochratoxin A (OTA) or type B trichothecenes, including DON, ZEN, and FUMs.
Studies of different poultry mix feeds used as starter and finisher blends were shown to have different mycobiota diversity depending on the season and the way the feed raw ingredients are processed.
Often the predominant spoilage mould species were from the Aspergillus, Penicillium and Fusarium, responsible for mycotoxin contamination, especially in warmer regions of the world, e.g., South-East Asia (Alam et al., 2010 a,b).
In Asia, poultry feed is produced by commercial feed mills as well as by local cooperatives. The feed mill owners purchase these ingredients in bulk in the production season and store them for processing into feed throughout the year.
However, often in tropical and sub-tropical climatic regions, harvested raw commodities from the fields often have a relatively high moisture content, which makes them unsafe for even short-term storage without mould spoilage. Thus, without effective drying to <0.70 water activity, then intermediate moisture conditions will allow the development of dry loving (xerophilic) moulds mentioned previously, many of which are mycotoxigenic (Ilangantileke, 1987; Sanchis, 2000; Figure 1).
A number of commercial companies sell preservative formulations based on aliphatic acids including formic, propionic, or sorbic acids or mixtures thereof.
Sometimes their salts are preferred because they have a reduced volatility and are more user friendly.
Figure 1. Diagrammatic profiles of growth/no growth limits for the three key mycotoxigenic genera in relation to the two key interaction abiotic factors.
CONCENTRATION IS CRITICAL
Of course, the formulation methods are confidential to each industrial producer. However, using the right concentration is critical to achieve effective control of spoilage and mycotoxigenic moulds.
Table 1 shows difference in concentrations that can occur when mixing different ratios of aliphatic acids or their salts and their actual concentrations.
Table 1. Preservative concentrations of stock solutions (g in 100ml water) (from Marin et al., 1999).
Table 2 compares the effect of two commercial products and propionic acid on the Minimum Inhibitory Concentration (MIC) required for inhibiting growth of three important mycotoxigenic fungi on a maize-based medium (A. flavus, AFs; A. ochraceus/P. verrucosum, Ocratoxina A, OTA).
On moist maize, 0.25% was necessary for similar control to be achieved of A. flavus and A. parasiticus.
Table 2. Comparison of minimum inhibitory concentration (%) required for propionic acid and two commercial formulations of aliphatic acids in vitro on maize-based media (Marin et al., 2000).
Table 3 shows the effect of sorbic acid on different spoilage moulds, the pH and concentration range.
Table 3. Minimum Inhibitory concentration (MIC) of sorbic acid against different spoilage mould genera or specific species (adaptado de Lück y Jager, 1997).
Figure 2 compares the effect of using mixtures of propionic acid and its sodium salt on the growth of Fusarium section Liseola species (F. verticillioides, F. proliferatum) responsible for contamination of maize with FUMs (Marin et al., 2000).
This clearly shows that the ratio of the mixture and, more importantly, the water availability of the maize significantly affected the colonization of maize by these species. Under wetter conditions, there was practically no control of F. verticillioides, although there is some control of F. proliferatum, but only at 0.93-0.95 aw.
Figure 2. Effect of different ratios of propionic acid/sodium propionate on maize grain at 25ºC on growth rates (Marin et al., 2000).
Table 4 shows the effect of different concentrations of propionic acid and aw levels on fumonisin B1 contamination of naturally contaminated maize or that with an additional inoculum of F. verticillioides after 4 weeks storage at 25ºC. This shows that regardless of conditions, only at the highest concentration tested was there a significant decrease of contamination with this mycotoxin.
Table 4. Fumonisin B1 (µg/ml) in natural maize treated with propionic acid concentrations and stored for 4 weeks at 25oC. (from Marin et al., 2000). Statistically, Fusarium, Preservative dose, and aw interactions: only Fusarium x Preservative was statistically significant.
Some studies have examined the changes in populations of different starter and finisher poultry feeds in Asian countries (Alam et al., 2012). This showed the dominance of the different mycobiota present in different feeds and the relative contamination with mycotoxins, especially aflatoxins in different seasons in different feeds (Table 5).
The impact of calcium propionate to both starter and finisher feed were shown to have significant benefits, decreasing the fungal populations, especially of A. flavus and A. parasiticus, and contamination with aflatoxins (AFB1, AFB2, AFG1, AFG2; Alam et al., 2014a,b).
It is important to reduce aflatoxin contamination in poultry feeds because of their significant impact in causing aflatoxicosis characterized by listlessness, anorexia with lowered growth rate, poor feed utilization, decreased weight gain, decreased egg weight and production, increased susceptibility to environmental and microbial stresses and increased mortality (Leeson, 1995; Wild, 2000).
Thus, more effective mixtures of preservatives may provide benefits of either additive or synergistic effect on relative control, especially of these important mycotoxins, during short- and medium-term storage.
Table 5. Seasonal occurrence of AFB1 (ng g-1) in poultry feeds and their ingredients collected from Peshawar (from Alam et al., 2012).
There have been reports of the use of other acids such as adipic, ferulic, fumaric, tartaric and trans-cinnamic acid (plant extract) against mycotoxigenic moulds for feed applications. The effects have been variable with in some cases good efficacy in controlling growth by >50-75%.
Table 6 summarizes the effect of different acids on growth and mycotoxin production by different mycotoxigenic moulds.
Table 6. Summary of the effects of different acids in the aqueous or ethanolic form on the growth and mycotoxin production by different fungal species (adapted from Mylona, 2013).
Previous studies have also examined the different chain lengths of a range of aliphatic acids for the control of spoilage moulds.
⇰ Previous studies with moist hay have shown that the chain length of the aliphatic acid may influence the relative control of spoilage moulds in feed such as Paecilomyces variotii and Aspergillus glaucus group (= Eurotium species). Thus, fatty acids up to a chain length of C9-C10 were very effective for controlling spoilage moulds.
However, anti-mould activity subsequently decreased with increasing chain length with solutions of dodecanoic acid (C12) unable to control growth at all.
⇰ Studies of moist barley (22, 28% m.c.) with recommended commercial doses of propionic acid showed that over a 6 months period A. flavus and AFs contamination was controlled. However, atter 4-6 months the treatment was less effective against P. verrucosum resulting in an increase in OTA contamination (Skudamore et al., 2004).
Thus, while one mycotoxigenic species was controlled, another was more resilient and increased the risk of OTA contamination during medium term storage, especially in the 22% moist barley.
Studies have also suggested that intermediate concentrations of such preservatives may result in a stimulation of the production of mycotoxins.
- ⇰ Arroyo et al. (2000) showed that the production of OTA by P. verrucosum was stimulated by the decimal reduction of the recommended doses of calcium propionate or potassium sorbate.
- ⇰ Intermediate concentrations of propionic acid have also been shown to stimulate the production of AFs by A. flavus (Al Hilli and Smith, 1979).
USE OF ANTIOXIDANTS, PLANT EXTRACTS AND BIOPRESERVATIVES
There has been significant interest in the use of alternative compounds, especially antioxidants, plant extracts (both essential oils and their volatile components) and biopreservative inoculants to control mould spoilage of individual ingredients or mixed feeds for poultry, cattle, and swine.
Other phenolic-derived antioxidants have been screened for their possible antimicrobial efficacy. Butylated hydroxyoluene (BHT), butylated hydroxyanisole (BHA), propylgallate (PG), 2-tert-butylhydroxybenzoic (TBHQ) and propyl parabens are among them.
In contrast, while many crude essential oils (ESOs) or their components (e.g., eugenol, thymol, cinnamaldehyde, vanillin, carvacrol, linalool) have been screened for controlling spoilage and mycotoxigenic moulds for feed applications many of these have not had the necessary toxicology tests for approved for use as antimicrobials.
Biopreservatives have become more attractive in recent years as many countries, especially the EU, have banned a range of crop protection chemical groups because of safety and environmental concerns.
This has been a driver for the development of biocontrol strategies as part of an IPM approach.
Originally, propyl parabens, esters of 4-hydroxybenzoic acid (PHB), were synthesized as a possible replacement of existing preservatives like salicylic and benzoic acids, effective only in the highly acid pH range.
Although Lück and Jager (1997) maintained that one of the most important characteristics of parabens, together with a much higher antimicrobial action to that of phenols and organic acids, is their pH independent activity, some studies have reported a slight influence of pH on their activity (Thomson et al., 1993).
Due to their high pKa value (8.5), parabens are chemical preservatives effective over a wider range of pH (3-8). Antimicrobial activity of parabens is related to the length of the ester group of the molecule.
As additives, parabens have been extensively applied as alkali solutions or as ethanol or propyl glycol solutions in a range of food products, including pickled vegetables (Belitz and Grosch, 1999). However, few studies have examined their potential applications for use in feed.
Kubo y otros (2001) compared the antifungal activity of three gallates, propyl (C3), octyl (C8) and dodecyl (C12) and found that only octyl gallate (OG) was the active compound against four different fungal genera with a MIC of 25ppm.
These results did not however, take account of environmental conditions, especially aw and temperature, which have been shown to have an impact of efficacy.
Table 7 shows more recent studies on the comparison of the efficacy of OG and other antioxidants as well as salts of aliphatic acids and the ED50 concentrations necessary for inhibiting AFB1 production by A. flavus.
This clearly showed that OG was better than many of the other compounds screened.
Table 7. Effective dose for 50% control (ED50 values, ppm) of antioxidants and comparison with aliphatic acids for inhibition of AFB1 production by A. flavus when grown at different water availability conditions at 25°C for 10 days (Sultan, 2011).
Subsequent studies were carried out by treating stored peanuts with different concentrations of OG and examining the effect on temporal populations changes of A. flavus (CFUs/g peanuts) over storage periods of 14 days at25ºC (Figura 3).
Figure 3. Temporal effect of different octyl gallate concentrations (OG, ppm) on populations of A. flavus isolated over 14 days in peanuts stored at 25°C. Bars indicate standard error of the means (from Sultan, 2011).
The populations significantly increased, especially between 7 and 14 days of storage, suggesting that in situ application was not as effective as in vitro studies on peanut-based media.
Statistically, single treatments, OG x Time, aw x Time Interactions were all significant. However, there was no effect of the interactions of OG x aw y OG x Time x aw.
Storage time was the main significant effect followed by the storage aw conditions.
The peanuts were subsequently analyzed for AFB1 contamination levels.
- ⇰ All the factors (OG, time, aw and their interactions) significantly influenced toxin production, except OG x aw (P< 0.05).
- ⇰ Storage time was the major significant effect followed by the initial aw of the peanuts entering storage (Sultan, 2011).
- ⇰ The final A. flavus populations and the AFB1 contamination levels were significantly higher after 14 days of storage, indicating that even with these concentrations of this antioxidant, efficacy was not good enough relative to the legislative limits.
Mixtures of compounds may be one way forward provided that they can provide a synergistic effect rather than just an additive one in terms of mould spoilage control or toxin production in feeds, especially in mixtures of cereals, cotton seed meal, pulses and other additives.
A significant body of work exists on the efficacy of plant essential oils (ESOs) or compounds extracted from mixtures of plant ESOs.
The efficacy of ESOs was recently reviewed in relation to control of crude extracts as well as purified components of ESOs by Prakash et al. (2015a,b).
However, while this examined their role for control of spoilage moulds and mycotoxin production in different agrifood chains the potential for use in feed was not prioritized.
Figure 4 shows an example where 25 ESOs were screened for efficacy and control of P. verrucosum, responsible for OTA contamination. This clearly showed that only a few had significant efficacy in controlling growth of this mycotoxigenic mould.
Figure 4. The effect of 25 different ESOs diluted 1:10 after 48 hours at 25ºC on the inhibition zones against P. verrucosum (OTA11; ochratoxin A producer) on a wheat-based medium at 25ºC. Bars indicate SEM. The circle indicates the best treatments (from Cairns et al., 2003).
Figure 5 shows the example of cinnamon oil concentrations on the relative control of growth of a range of spoilage and mycotoxigenic fungi. This shows that only higher concentrations will have efficacy for complete control (MIC) values for a range of these fungi.
Figure 5. Effect of Cinnamon oil at different concentrations on growth of six different spoilage and mycotoxigenic species at 0.97 water activity and pH 4.5 at 25ºC on a cereal-based medium. CH, Cladosporium herbarum; 453; P. verrucosum strain 453); PC, Penicillium corylophilum; PR, Penicillium roqueforti; ERE, Eurotium repens (=Aspergillus glaucus); AO, Aspergillus ochraceus (=A. westerdijkiae) (from Arroyo, 2000).
Subsequent more detailed studies have examined the comparison of ESOs and, in some cases, antioxidants for control of specific mycotoxins for which legislative limits exist. Figure 6 shows the effect of 200 ppm of different ESOs and antioxidants on the control of DON in stored naturally contaminated moist wheat grain inoculated with F. culmorum. This shows that there was good efficacy at 0.93 and 0.95 aw. However, at 0.97 aw DON contamination could not be controlled.
Figure 6. Examples of the effect of different 200 ppm essential oils and comparison with two antioxidants on control of dexoynivalenol production by Fusarium culmorum in naturally contaminated stored wheat at different water activity levels for 30 days at 25ºC.
PP, Propyl paraben; Clove, Clove essential oil; Cin, Cinnamon essential oil; BHA, Butylhydroxy anisole; Bay, Bay leaf essential oil. (Hope, Aldred and Magan, unpublished data).
⇰ In vitro studies showed that 200 ppm of either PTS or PTSO reduced fungal growth by 50-100% and mycotoxin production by >90% depending on species, mycotoxin and aw conditions on milled wheat, oats and maize respectively. PTS was generally more effective than PTSO.
⇰ However, in situ studies showed that:
- ⇰ DON and ZEN were decreased by 50% with 80 ppm PTSO.
- ⇰ 100 ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 awfor 20 days, although contamination was still above the legislative limits.
⇰ Contrasting effects on T-2/HT-2 toxin contamination of oats were found, depending on aw, with PTS stimulating production under marginal conditions (0.93 aw), but at 0.95 aw effective control was achieved with 100 ppm.
⇰ Treatment of stored maize inoculated with F. verticillioides resulted in a stimulation of total fumonisins in most treatments.
The approach here is to add the inoculants to moist cereals or mixed feed and then seal these to allow colonization and preservation by the production of naturally produced antimicrobial compounds, including bacteriocins (LABs), and colonization of the feed by inoculants (e.g., Pichia (Olstorpe et al., 2011).
A significant amount of work has been carried out on moist cereals as animal feed using yeasts.
For example, P. anomala was developed as an inoculant for the preservation of moist wheat and barley for feed used for cattle. The yeast was formulated and the effect of different formulations examined for both spoilage and mycotoxin control (Druverfors et al., 2002; Mokiou y Magan, 2008).
Figure 7 shows the effect of different formulations of P. anomala on the control of ochratoxin A production by P. verrucosum when stored for up to 30 days (Mokiou y Magan, 2008).
Figure 7. Effect of biopreservative formulations of P. anomala on Ochratoxin A contamination of stored moist wheat grain (0.93 aw) co-inoculated with Penicillium verrucosum for 30 days and 25ºC.
A: unmodified yeast biopreservative cells; B: proline-modified; C: proline+cottonseed flour+ skimmed milk in water; D: same as C plus isotonic solutions (de Mokiou y Magan, 2008).
Subsequently, longer term studies were carried out in pilot scale storage silos with inoculation with P. anomala fresh cells, the best formulation identified.
Figure 8 shows the long-term changes in fresh cells, formulated cells and that of P. roqueforti which can grow under microaerophilic conditions. The key period for control was achieved for 8-9 months but that subsequently the inoculant was less effective.
Figure 8. Pilot scale study of formulated and fresh Pichia anomala cells over a 15 month stored period in moist wheat to control spoilage moulds, especially Penicillium roqueforti (from Druverfors, Mokiou, Magan y Schnurer, unplublished data).
⇰ Regardless of the preservatives used it is critical that nutritional value of the final feed, whether cereal-based, mixed feed or extruded pelleted feed, are conserved for ensuring that the nutritional value and necessary animal growth rates can be maintained.
⇰ The use of existing mixtures or these with alternative natural preservatives may be the main areas for development in the future.
However, this must be with the proviso that effective coverage of the treated feed type can be achieved with the necessary recommended rates.
⇰ In addition, some of the preservative mixtures are not proving to be resilient enough, especially in cereal-based mixed feeds, when controlling spoilage and mycotoxigenic moulds.
⇰ With the impact of interacting climate-related abiotic factors becoming more important, the effect on the resident microbiome of commodities used for making different feeds may change significantly.
⇰ Preservation systems for animal feeds may also have to be modified to take into account of this as the dominant fungal populations may change and the mixtures of mycotoxins produced may also be modified (Medina et al., 2017).
The relative cost-benefit analyses of the use of ESOs, antioxidants alone or with aliphatic acids or their salts as mixtures needs to be compared for successful application in the feed industry.
For example, studies by Aldred y otros (2008) compared a range of ESOs and antioxidants for controlling P. verrucosum and OTA in wheat stored under different conditions. Resveratrol was found to be superior to the others.
However, the costs were prohibitive for the use of such compounds when compared to the aliphatic acids which are economically more cost effective, even if application rates and effective mixing and coverage is necessary for efficacy.
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