Mould prevention in bread

24 Mould prevention in bread N. Magan, D. Aldred and M. Arroyo, Cranfield University, UK

Abstract: Bread is considered an intermediate-moisture food product that is prone to mould spoilage. Normally bread is eaten fresh or preserved using additives or modified atmosphere packaging. Demands for preservative-free food affects bread production and associated mould spoilage. This chapter considers the important mould species that can cause spoilage depending on the bread. We review the current systems and new methods for preserving bread. Using mixtures of physical methods and natural compounds and the use of nanotechnology in the formulation of products are also discussed in terms of their future use. Key words: moulds, yeasts, bakery products, water activity, pH, mycotoxins, preservatives, essential oils, anti-oxidants, modified atmospheres, packaging, intermediate moisture foods.

24.1

Introduction: the problem of moulds in bread

Bread is a staple food world-wide. UK flour consumption per capita reached 74 kg in 2008/2009 (NABIM, 2009). Of this a substantial proportion is consumed as bread of various kinds and this represents per capita annual bread consumption in the region of 45–50 kg. Bread in the UK is usually very light in texture, almost exclusively made from wheat flour and leavened by yeast fermentation. In the wider European regions including Germany, Denmark, Sweden, Poland and Hungary and other eastern European countries, rye breads made from sourdough fermentation processes are the norm. These are usually of a much lower pH than UK breads. In other parts of the world unleavened breads are much more common. Because of its water activity and storage conditions bread is a highly perishable product. The most common forms of bread deterioration are staling, moisture loss and microbial spoilage (Seiler, 1984; Legan, 1993). Losses due to mould spoilage are not easy to quantify. However, conservative estimates of 1% would result in

© Woodhead Publishing Limited, 2012

598

Breadmaking

losses of £20 million in the UK alone, every year. Across Europe, the economic costs would be expected to be in the region of up to 10 times greater. The reason why moulds are important spoilage organisms in bread is that this food matrix has a relatively high moisture content and thus water activity (aw = 0.94–0.97) and a pH of approximately 6. These properties are conducive to the germination and growth of a wide range of moulds contaminating the product during or post-production. Bread that is sliced, prepacked and wrapped in polyethylene plastic packaging is at the highest risk because the cut surfaces can become contaminated and the wrapping allows moisture condensation where temperature gradients might occur during transport and storage. More than 90% of contamination of bread with spoilage moulds occurs during the cooling, slicing or wrapping operations. Prior to this stage the heating regimes employed in baking mean that most contaminants are eliminated (Legan, 1993; Roessler and Ballenguer, 1996). Most fungal contamination thus occurs from fungal spores in bioaerosols which can become deposited in the factory line from the bakery environment, from flour dust or debris or from the outside atmosphere. Many different filamentous mould species have been implicated in spoilage of bread including Aspergillus and Eurotium, Penicillium, Cladosporium, Mucor and Neurospora (Legan, 1993). Tolerance to a wide range of environmental conditions, and their predominantly mycelia growth habit enable them to colonise food products rapidly by producing a wide range of hydrolytic enzymes to utilise the food matrix. The growth of some spoilage moulds is influenced by changes in water activity and pH values on bread. They are able to grow faster at pH 6 than pH 4.5, and the relative growth rate is decreased as the conditions become drier, regardless of pH. Eurotium repens, a xerophilic species, grows best under this range of conditions, followed by xerotolerant species such as Aspergillus westerdijkae (= A. ochraceus) and Penicillium verrucosum strains. The ecology of the Aspergillus section Circumdati group (previously A. ochraceus group) has recently been determined (Abdel-Hadi and Magan, 2009). Other spoilage Penicillium species grew more slowly. However, some of these are microaerophilic and they can thus grow well in packaged bread systems. Table 24.1 Different allowable preservatives that can be used in bread and bakery products under UK and EU regulations Preservative type

E number

Potassium sorbate Sodium benzoate Calcium propionate Propyl paraben Butylhydroxyanisole (BHA) Butylhydroxytolunene (BHT) Octyl gallate Essential oils

(E202) (E221) (E282) (E216) (E320) (E321) (E311) (None)

} }

Products Regulated for bread (1000>–3000 ppm max dose) Food additives and cosmetics Regulated for foods (100–200 ppm max. levels) No specific regulations

Note: Alcohol, IR and UV are also used on some products.

© Woodhead Publishing Limited, 2012

Mould prevention in bread

599

The predominance of Penicillium species may be partially due to their ability to grow over a wide range of temperatures and water availabilities, and the profuse production of spores which can become airborne and suspended in the atmosphere (Magan, 2007). They often predominate at cooler temperatures, and at 22–4°C there is a 50% reduction in Penicillium contamination, while in warmer climates Aspergillus and Eurotium species prevail. It is important to note that some of the moulds contaminating bread are also able to produce toxic secondary metabolites: mycotoxins. Thus for prolonged shelf life of bread, the control of contamination with spoilage moulds and their toxins is critical. This chapter will examine the current methods for mould control and their limitations, consider the development of new methods of mould control and examine possible future trends.

24.2

Current techniques for mould control and their limitations

Control of mould spoilage in bakery products can be achieved by (1) restricting the access of spoilage moulds to the product, (2) inactivating the fungal material and (3) inhibiting growth of the fungus. However, once the mould gains access to the product, the objective turns into controlling its activity and growth in the product itself. For inactivating or delaying fungal growth in foods several physical, chemical and biological approaches can be used. The most common way to prevent or control mould growth in foodstuffs is by the use of anti-fungal agents. These are chemical compounds that can be added to food which prevent or retard food spoilage moulds from growing. In practice, most of these are fungistatic and not fungicidal. Thus they are effective at stopping germination and subsequent growth where the chemical is present, but good mixing is required to ensure that no under-treated or untreated pockets are present. This can lead to growth of any surface contaminant moulds. Fungicidal compounds are more effective as they destroy the spoilage moulds directly. However, they are often not approved for use in such food products as bread. The concentrations of the added anti-fungal compounds are important. Smid and Gorris (1999) suggested that ideally any anti-microbial substance should inhibit microorganisms during their initial growth phases, and not during subsequent exponential log phase when much higher concentrations may be required, and which may have an impact on the palatability/quality of the food product. To prevent mould spoilage of bread and bakery products and to extend shelf life food grade preservatives based on propionic, sorbic and acetic acids and their salts are used. These are generally recognised as safe (GRAS) compounds (Liewen and Marth, 1985; Binstok et al., 1998). Sorbic acid has a half-life of about 40–110 minutes in the body, and is completely oxidised to CO2 and H2O (Liewen and Marth, 1985). Because these aliphatic acids are quite volatile and corrosive, their sodium, potassium or calcium salts are the forms most commonly used because of their better solubility in water, stability and ease of handling.

© Woodhead Publishing Limited, 2012

600

Breadmaking

There are limits to the type and maximum concentrations that are allowable in bread and bakery products in most countries, including the EU. These are listed in Table 24.1. This may be further influenced by shelf-life consideration, type of bread and wrapping material. In England and Wales for example, sorbates are not permitted in all breads but they are permitted for flour confectionery goods at levels of up to 1000 ppm (Seiler, 1984). In other countries such as Germany, Italy and the Netherlands, sorbic acid and its salts are approved in certain types of breads only. In the USA high-volume loaves of white bread similar to those produced in the UK have successfully been preserved using spray applications of potassium sorbate which is applied immediately after baking (Killian and Krueger, 1983). In the UK, as in many other countries, propionates are the chemical antimicrobial generally used to control moulds in bread, English muffins and crumpets as well as bacterial (Bacillus species) spoilage (Legan, 1983). Propionates are used mainly as potassium or calcium salts because, although more expensive, they are less corrosive and easier to handle than the liquid acid. Their use is permissible at levels of not more than 0.3% (w/w) of propionic acid equivalent (Anon, 1984). The impact of different preservatives and pH of bread on mouldfree shelf life is shown in Table 24.2. It should be noted that propionates have little or no efficacy against yeasts (Sauer, 1977) which make them highly suitable to control mould spoilage in yeast-raised bread products. There are however some disadvantages in the use of weak acids to control mould spoilage in bread/bakery products. Because they are fungistatic and the

Table 24.2 Effect of different concentrations of existing preservatives on mould-free shelf life (days) of bread at 0.95 water activity and different pH values at 25°C Number of mould-free days pH No preservative Potassium sorbate (ppm; w/w) 3000 300 30 Calcium propionate 3000 300 30 Sodium benzoate 3000 300 30

4.5 1.5

6 1.4

7.5 2.5

30 9 1.9

17.2 2.2 1.4

3.2 25 1.8

30 8.9 1.9

3 2.2 1.4

1.3 2.5 1.8

30 3.3 1.0

1.7 0.7 1.8

1.4 1.3 0.6

Source: Arroyo, 2003. Note: Means of effect on 6 spoilage fungi

© Woodhead Publishing Limited, 2012

Mould prevention in bread

601

low absolute efficacy of the propionates means that relatively high concentrations are needed in order to keep bread and baked products free of moulds for more than a few days (Luck and Jager, 1997). They thus only delay germination and subsequent growth of contaminants during production or subsequent packing. Furthermore, at such concentrations serious losses in volume and adverse effects on odour and flavour can occur. Using 0.2% calcium propionate, a reduction of 5–10% of loaf volume occurs in commercial-scale baking because it reduces the yeast activity and also alters the dough rheology. Sorbates have an even greater adverse effect (Legan, 1993). The incorporation of calcium propionate into bread at up to 0.3% concentration at pH 4.5 and 0.93–0.97 aw can effectively control a range of spoilage moulds in bread. However, at sub-optimal concentrations some stimulation was observed. Where pH was 6.0 there was practically no control achieved against E. repens, P. verrucosum, A. westerdijkiae, P. corylophilum and P. roqueforti (Arroyo, 2003). Indeed more recent studies also suggest that intermediate concentrations may influence the interactions between species and may allow mycotoxigenic species to outcompete other spoilage moulds (Arroyo et al., 2008). Sorbic acid and its salts are among the most thoroughly investigated of all preservatives. Use of 0.3% (w/w) potassium sorbate in bread has shown very effective control of a range of spoilage moulds at pH 4.5 and 0.93–0.97 aw, with the exception of P. roqueforti. However, at pH 6.0 practically no control was achieved with the recommended concentration of 0.3% treatment. Furthermore, when concentrations of calcium propionate or potassium sorbate were incorporated at a tenth of the concentration (0.03%; w/w) then stimulation of E. repens P. corylophilum and P. roqueforti was observed. This is supported by recent studies, which examined the effect of intermediate concentrations of both these preservatives on growth and ochratoxin A production by P. verrucosum. These studies showed stimulation of growth and toxin production at intermediate concentrations and were supported by molecular analyses which showed that a gene involved in the biosynthesis of the ochratoxin A (OTApks) showed a similar stimulation in expression (Schmidt-Heydt et al., 2007). Studies with Spanish sponge cakes treated with sodium benzoate or calcium propionate at up to 0.3% at 0.80 to 0.90 aw and pH 6 or 7.5 also showed that four Eurotium species were only effectively controlled at pH 6 and 0.80–0.85 aw. Over all other conditions growth was not significantly controlled (Guynot et al., 2002). More recent studies by Suhr and Nielsen (2004, 2005) of 0.003–0.3% of calcium propionate, potassium sorbate and sodium benzoate in fermented sourdough rye bread and more alkaline intermediate moisture sponge cakes (0.80–0.95 aw; pH 4.7–7.4), found that these were effective against a range of spoilage moulds, but not against Eurotium rubrum, P. roqueforti and P. commune. Growth of P. roqueforti was often stimulated. It can also grow under micro-aerophilic conditions and can thus cause problems in packaged bread products. Interestingly, calcium propionate was more effective than potassium sorbate and sodium benzoate.

© Woodhead Publishing Limited, 2012

602

Breadmaking

Weak acids are lipophilic in nature and they can thus penetrate the cell membrane in the undissociated form. When the undissociated acid enters the cell a higher pH environment is encountered and the molecule dissociates, resulting in the release of charged anions and protons which cannot cross the plasma membrane. The high solubility, low taste threshold and low toxicity of weak organic acids make them highly suitable for use in bread and baked product preservation systems (Ray and Bullerman, 1982; Davison and Juneja, 1990; Blackburn, 2006). The pH of the environment and the solubility of the acid often determine the foods in which these aliphatic acids may be effectively used (Ray and Bullerman, 1982). Indeed, because of their low pKa value (4.19–4.87), these compounds are effective anti-microbial agents, especially in low pH matrices because these conditions favour the uncharged, undissociated state of the molecule which is freely permeable across the cell membrane. The maximum pH for activity is around 6.0–6.5 for sorbate, 5.0–5.5 for propionate and 4.0–4.5 for benzoate (Liewen and Marth, 1985). Another aspect which is important in bread is the presence of salt (NaCl), which is an important ingredient and flavour component. However, because of the overall high dietary level of intake of NaCl by the general population in Europe and world-wide there is concerted action to try and reduce the content in bakery products. A reduction in salt, which also acts as an anti-microbial agent, may have an impact on shelf life (Bidlas and Lambert, 2008) and on the functional properties of the food product. Studies have recently been carried out to examine the substitution of NaCl with CaCl2, MgCl2, KCl and MgSO4 on the growth in vitro of P. roqueforti and A. niger and in white bread in challenge type tests (Samapundo et al., 2010). They found that the stability of the bread products was influenced by the replacement salt ingredient. Differential efficacy was observed against P. roqueforti and A. niger. In challenge tests, the growth of P. roqueforti was similar on natural bread containing NaCl to that with 30% less NaCl and substituting additional mixtures of other salts. Overall, CaCl2 appeared to provide more stability to the product in terms of microbiological control. These studies suggest that mould inhibition and shelf life will be influenced by both ingredients, as well as the presence or absence of organic acidbased preservatives and the type of moulds actually contaminating the bakery product. Ethanol has been used as a method of significantly increasing the shelf life of bakery products as it can be added to the surface or in the packaging. Normally it can be added at 2% v/w although efficacy against yeasts is variable (Seiler, 1984). Use of 1% ethanol released from an adsorbent pad extended the shelf life of packaged white bread to more than 60 days at 22°C. However, chalk moulds developed within 10 days, demonstrating that this approach was less effective against yeasts (Legan and Voysey, 1991). Smith et al. (1988) used commercial ethanol generators to show that efficacy against Saccaromyces cerevisiae was related to aw and concentration of ethanol. Ethanol vapours have also been shown to be very effective at controlling bacterial toxin production and growth of Clostridium botulinum (Daifas et al., 2000).

© Woodhead Publishing Limited, 2012

Mould prevention in bread

603

Alternatives to chemical preservation include destroying or damaging the mould spores that gain access to the cut surfaces of bread during cooling and wrapping processes. This is usually achieved by using UV light, IR or microwave irradiation. These types of procedures have also been used for sourdough breads in continental Europe (Seiler, 1984). However, consumer pressure for minimally processed and freshly baked bakery goods has meant that in some processes the use of such procedures has had to be eliminated. It should also be noted that while UV irradiation is very effective on surface contamination, it does not inhibit any mould spores inside the product. Other strategies that have been followed to limit the rate of mould growth, especially in ready-to-eat fresh baked goods (e.g. garlic bread; tomato and herb-based breads) include:

• • •

Reformulating recipes, e.g. by reducing the water availability but without adversely affecting the eating quality of the product or causing changes in volume, texture or shape. Using novel ingredients such as fruit juices (raisin, prune, apple juice concentrates) that inhibit fungal growth (Sanders, 1991) Using modified atmosphere packaging (MAP) techniques (Abellana et al., 2000; Blackburn, 2006).

MAP systems are based on the fact that moulds require O2 to grow and that they are sensitive to CO2 (Porter et al., 1989; Farber, 1991). Thus by changing the ratio of both (usually low O2 and elevated CO2) it is possible to retard aerobic germination and growth of moulds as well as other microorganisms. Packaging development has led to the growth of biopackaging, and packaging which may incorporate a preservative as an active material. In this case there is a slow diffusion of the preservative into the packaging environment. The rate of diffusion, the retention properties and the efficacy will all influence the effectiveness of such systems (Han and Floros, 1998). While organic acids and their salts are the most commonly used preservatives in bakery products, there is increasing pressure from the EU via food directives and from consumer organisations to reduce their use in food products. There is thus significant interest in the use of alternative anti-oxidants or natural plant extracts (essential oils) either alone or as mixtures, or in combination with packaging systems, to stabilise or extend shelf life. However, the evidence now available suggests that a reduction of the concentrations of organic acids/their salts may lead to more mould spoilage problems and significantly shorten the shelf life of bakery products and perhaps increase the risk from mycotoxigenic moulds and toxin contamination. This may become particularly evident with filamentous spoilage moulds in wrapped, cut, mass-produced bread and baked goods. However, there are only a limited number of approved compounds (see Table 24.1). Interestingly, although essential oils are approved for use as flavourings for baked products, they are not approved specifically as anti-microbial compounds. However, it is worthwhile examining work that has been carried out to evaluate such alternative compounds for use in bread and bakery products.

© Woodhead Publishing Limited, 2012

604

24.3

Breadmaking

Developing new methods for mould control

24.3.1 Synthetic anti-oxidants Phenolic-derived antioxidants have been screened for their possible antimicrobial efficacy. Some of those which have been screened include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propylgallate (PG) and tert-butylhydroquinone (TBHQ). Studies have been usually conducted in bread/ bakery products by incorporation and then challenge-testing. We have screened BHT, BHA, n-propyl gallate, propyl paraben, octyl gallate at 0.95 aw and pH 6 in bread analogues. Common bread spoilage fungi such as Cladosporium herbarum, Penicillium corylophilum, P. verrucosum and Aspergillus westedijkiae (= A. ochraceus) were completely inhibited by propyl paraben, BHA and octyl gallate at 500 ppm. These compounds can also increase the lag time prior to growth being initiated, and this can have a significant impact on the shelf life of baked products. Resveratrol, an extract of grape skin, has also been examined for efficacy as it is also used as an absorbent of free radicals. Since these are GRAS chemicals they can, of course, be used as part of the formulation of bakery products. Kubo et al. (2001) compared the anti-fungal activity of three gallates, propyl (C3), octyl (C8) and dodecyl (C12). They found that octyl gallate was the only active compound against fungal species from four genera with a minimum inhibitory concentration (MIC) of 25 ppm. Recent studies suggest that mycotoxigenic spoilage fungi such as Fusarium, Penicillium and Aspergillus species are significantly inhibited by both parabens and BHA, and able also to inhibit mycotoxin production. In some cases combinations of antioxidants have been found to be more effective, and at lower concentrations than individual ones, in some cases having a synergistic effect (Cairns and Magan, 2003; Hope et al., 2003). In more recent studies on wheat, where both antioxidants and ESOs were compared, the former were more effective, for example resveratrol, than the ESOs. This was so for both control of growth and production of ochratoxin A based on calculated ED50 values (Table 24.3). Because of their high pKa value (8.5), parabens are effective over a wider range of pH (3–8) than the organic acids discussed earlier. The anti-microbial activity of parabens is related to the length of the ester group of the molecule. As additives, parabens are applied as alkali solutions or as ethanol or propyl glycol solutions in fillings for baked goods, fruit juices, marmalades, syrups, preserves and pickled sour vegetables (Belitz and Grosch, 1999). 24.3.2 Essential oils (ESOs) Plant extracts have received significant attention in the last few decades as food preservatives because of consumer perceptions and pressure for the provision of preservative-free products. Thus more natural products, which can give the same effective control of microorganisms as existing organic acid-based ones, are being sought. Essential oils are mostly derived from spices, i.e. solvent extracts of dried

© Woodhead Publishing Limited, 2012

Mould prevention in bread

605

Table 24.3 The effective dose (ED50) concentrations (μg g−1) of essential oils and resveratrol required for control of (a) growth and (b) ochratoxin A (μg kg−1) production by Penicillium verrucosum on wheat grain under different water activity and temperature treatments. Best treatment is highlighted in bold (a) Growth effects P. verrucosum Temperature (°C)

15

25

Water activity Treatment Clove oil Cinnamon oil Thyme oil Resveratrol

0.90

0.95

0.995

0.90

0.95

0.995

250 200 260 10

230 220 235 40

320 280 320 360

220 210 260 40

190 185 185 20

260 380 385 380

(b) Effects on ochratoxin A Temperature (°C)

15

25

Water activity Treatment Clove oil Cinnamon oil Thyme oil Resveratrol

0.90

0.95

0.995

0.90

0.95

0.995

240 260 180 25

210 210 325 80

295 295 320 180

210 210 210 90

230 230 200 80

160 210 210 215

Source: Adapted from Aldred et al., 2008.

aromatic products, obtained from different parts of plants including leaves (e.g. rosemary, sage, thyme), flowers (e.g. clove), bulbs (e.g. garlic, onion) or fruit (e.g. pepper, cardamom) (Shelef, 1983; Deena and Thoppil, 2000). Extracts and essential oils of many of these plants are now been screened for their antimicrobial efficacy in vitro and in situ. Two approaches have been used: (1) direct efficacy of the ESOs in vitro and in situ by incorporation and (2) by examining the efficacy of the volatile components which can be included in packaging systems. Aspergillus flavus, one of the most toxigenic food-borne mould species that can contaminate flour used for bakery products, has been reported to be inhibited by some of these plant derivatives. For example, Dwivedi and Dubey (1993), studying the anti-fungal activity of several umbelliferous plant essential oils against Aspergillus species found good fungistatic effect of Trachyspermum seed essential oil at relatively low concentrations (<500 ppm). Azzouz and Bullerman (1982) established clove and cinnamon as the strongest anti-fungal agents against Penicillium and Aspergillus species. However, many of these studies did not take account or simulate aw and pH concentrations relevant to bread or baked products. In some cases (Salmeron et al., 1991) stimulation of growth of the same Aspergillus species was demonstrated when extracts of thyme or oregano were

© Woodhead Publishing Limited, 2012

606

Breadmaking

incorporated into nutritive media. Lopez-Malo et al. (2002, 2005) carried out dose–response curves for A. flavus in relation to vanillin, thymol, eugenol, carvacrol and citral or potassium sorbate/sodium benzoate. They showed that A. flavus has a higher sensitivity to many ESOs and to the salts of organic acids than to vanillin and citral at pH 3.4. Aw levels (0.99 and 0.95 aw) also affected lag times and relative growth rates. MICs varied from 200 ppm for the organic acids to 1800 ppm for citral. Lopez-Malo et al. (2007) examined cinnamon extract and sodium benzoate mixtures for controlling A. flavus growth. They determined MIC and found that for cinnamon extract at 200 ppm it was unaffected by pH (4.5, 3.5). In contrast, sodium benzoate MIC changed from 800 to 400 ppm. They found that mixtures of these two acted synergistically at pH 4.5 and only additively at pH 3.5. Screening a range of over 20 ESOs for activity against four spoilage moulds, A. westerdijkiae, C. herbarum, P. corylophilum and P. verrucosum on wheat flour-based medium at 25°C showed that at least 500 ppm was required for control of growth. An example of the effect of an ESO on growth of a range of mould species in shown in Figure 24.1. Indeed, only clove, thyme, bay and cinnamon completely inhibited growth of all the species studied. The lag phases prior to growth were also significantly increased in these studies. Similar studies with Eurotium, Aspergillus and Penicillium species isolated from Spanish bakery

Fig. 24.1 Effect of cinnamon oil on growth of spoilage fungi on bread-based substrate (pH 4.5, 0.97 aw) at 25°C (Arroyo, 2003). Key to fungi: Aw, Aspergillus westerdijkiae; Er, Eurotium rubrum; Pr, Penicillium roqueforti; Pc, P. corylophilum; Pv, P. verrucosum; Ch, Cladosporium herbarum.

© Woodhead Publishing Limited, 2012

Mould prevention in bread

607

products suggested that only cinnamon leaf oil, rosemary, thyme, bay and clove essential oils exhibited good efficacy (Guyenot et al., 2005). They also found an interaction between aw and pH. Only rosemary, thyme and bay were effective at pH 5 and at lowered aw levels (<0.85 aw). The efficacy in delaying germination/ growth is the best indicator of the potential for improving the shelf life of baked products to avoid visible mould growth at different temperatures and aw levels. These studies reflect the work being carried out for direct inhibition of germination/ growth of spoilage moulds by ESOs with a view to their use in bread and baked goods. The alternative may well be to use the volatile components of these ESOs, perhaps as part of the packaging system, especially for wrapped bread. Guynot et al. (2003) examined the volatile fractions of 16 ESOs against common bakery spoilage fungi including Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. They applied 50 μl on filter paper at different aw levels on wheat flour-based media. They found that in vitro cinnamon leaf, clove, bay, lemongrass and thyme essential oils totally inhibit all the fungi tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. This suggests that complex interactions may occur between ingredients of baked goods and essential oils. More recently Bluma et al. (2008) examined the vapour phase of five ESOs on maize-meal agar against species from the Aspergillus section Flavi group and this included anise, boldus, mountain thyme, polio and clove. They examined efficacy against strains of A. flavus and A. parasiticus. Overall, boldus was found to be the most effective when compared with the other ESOs for lag phase prior to growth, growth rates and aflatoxin production, regardless of aw used both in vitro and on maize grain. It is notable that while some work has been done on the potential of using mixtures of antioxidants, practically no work has been done to examine mixtures of antioxidants/ESOs or de-odorised ESOs. These may possibly be more efficacious at lower concentrations and need to be studied for potential use in bread and bakery products. 24.3.3

In situ control of moulds in bakery products using anti-oxidants and essential oils (ESOs) The efficacy of some anti-oxidants and ESOs has been tested for controlling mould spoilage in bread and baked goods. These studies have often shown that the concentrations required for inhibition of growth were generally higher than those found to be effective in vitro. For example, propyl paraben has little effect on growth of four filamentous spoilage moulds, even at 1000 ppm. This suggests that the active ingredients are probably being bound to ingredients, or dispersion in the product is not effective enough to enable contact with the spoilage moulds and thus inhibition or delayed growth. ESOs have been examined in two formats for control of spoilage moulds in bakery products. The volatile compounds produced by ESOs have been

© Woodhead Publishing Limited, 2012

608

Breadmaking

incorporated into packaging to allow slow release of the product into the package or direct incorporation of the ESOs into the bakery product to inhibit mould growth. For example Nielsen and Rios (2001) examined the efficacy of volatiles in MAP systems for control of spoilage moulds on rye bread. Mustard ESO in the volatile phase at 1–10 μg ml−1 was very effective against a range of spoilage moulds including Penicillium commune, P. roqueforti, Aspergillus flavus and Endomyces fibuliger. Later work supported the use of mustard oil volatile in active packaging for improved shelf life of both wheat and rye bread (Suhr and Nielsen, 2005). Cinnamon, garlic and clove also showed efficacy in controlling growth of these moulds on sliced bread. Interestingly, vanilla showed no inhibitory effects, although it is known to include cinnamic acid, a well-known anti-microbial active ingredient. A. flavus was particularly resistant to this ESO. Although, as has been mentioned previously, mixtures of cinnamon extract and sodium benzoate were found to be effective in controlling A. flavus (Lopez-Malo et al., 2007). More recently the incorporation of ESOs into packaging as ‘active packaging paper’ has been examined (Rodriguez et al., 2008). This approach involved incorporation of cinnamon ESO into solid wax paraffin as an active coating. The antifungal activity of the active paper was tested for control of Rhizopus stolonifer (black bread mould). They found that 6% (w/w) of the ESO was effective in situ, while 4% (w/w) was effective in vitro. They found that 6% cinnamon ESO completely inhibited growth on sliced white bread for three days. Extraction of the bread showed that the active ingredient, cinnamaldehyde, was present in the bread, and mainly responsible for the inhibition of the Rhizopus. An alternative approach has been examined with ESOs. Studies have been done to examine application of extracts of citrus peel ESOs on anti-microbial and sensory aspects of wheat-based bread (Rehman et al., 2007). They used cold extracts of two citrus fruit peels. They found that ESO addition to ingredients affected sensory characteristics, as well as character of the crust, crumb colour, crust colour, taste and texture. However, the ESOs were very effective in inhibiting and delaying mould growth on the bread. Maximum efficacy was achieved by spraying the ESOs onto the bread surface after manufacture and prior to packaging. The incorporation of ESOs directly as an ingredient appears to have less efficacy than expected from in vitro studies. For example, up to 1000 ppm of oregano ESO was necessary to completely inhibit growth of A. flavus in bakery products for 21 days (Basilica and Basilica, 1999), while only 100 ppm was required to inhibit Aspergillus niger and several Penicillium species over periods of six days in vitro. Chemically, ESOs consist of a mixture of esters, aldehydes, ketones and terpenes. The question arises as to which components actually have efficacy for controlling mould growth in bakery products. Often the ESOs used consist of a mixture of these and it is thus difficult to know exactly which are the active ingredient/s responsible for efficacy. Screening of ESOs (20) and antioxidants (5) for control of potentially mycotoxigenic fungi that can contaminate wheat used for flour and bakery product production has been evaluated against Penicillium verrucosum (ochratoxin A

© Woodhead Publishing Limited, 2012

Mould prevention in bread

609

(OTA) producer) and Fusarium culmorum and F. graminearum (deoxynivalenol (DON) producers) (Hope et al., 2003; Cairns and Magan, 2003; Aldred et al., 2008). Only three ESOs (bay, clove, cinnamon oil) and three antioxidants (propyl paraben, BHA, resveratrol) appeared to be effective in controlling growth in the range 50–200 ppm both at 15/25°C and at 0.995 and 0.95 aw. At 500 ppm clove oil and BHA effectively controlled growth and DON production. Similarly, effective control of P. verrucosum and OTA was achieved with <500 ppm. Table 24.3 shows the ED50 values of different compounds for growth and OTA control (Aldred et al., 2008). Direct incorporation of the best ESOs into bread showed that efficacy was often lower than on wheat flour-based media, with 50% control of E. repens by cinnamon and clove oils, and only about 25% control of A. westerdijkiae by thyme oil. Practically no control in bread was achieved by P. verrucosum and P. corylophilum, with any of the best treatments shown to be effective in vitro. Studies were subsequently carried out to examine the effects of the best ESO treatments on mycotoxin production on bread. Table 24.4 shows that they were not very effective for controlling OTA produced by strains of P. verrucosum. In many cases a stimulation of mycotoxin production occurred. Overall, incorporation of ESOs into bread or baked products may not be the most effective approach. For cakes, some ESOs are sometimes added, but predominantly for sensory purposes (e.g. orange, lemon, carrot cakes). Speciality bread production, e.g. tomato-based, olive-based breads, may contain basil and other herbs or herbal extracts. In this case a side effect may be an improvement in the shelf life of such products. However, no specific claims are usually made with regard to potential anti-microbial efficacy. The use of ESOs in packaging, either in the wrapping material directly, or as a sachet, for slow release of the volatile components may be the best and most effective method when used in conjunction with MAP systems. These could also be used in conjunction with lower concentrations of organic acids, especially if they may act synergistically. Table 24.4 Ochratoxin content (μg/g) of bread made with different antioxidants and essential oils (1000 ppm) inoculated with three strains of Penicillium verrucosum at 0.97 aw/pH 6 P. verrucosum strain

M453

M450

PV3

Control

4.5

6.0

7.0

BHA PP Clove oil Thyme oil Cinnamon oil Bay leaf oil

7.1 5.2 3.5 7.4 1.5 3.1

1.1 0.6 3.0 0.6 0.9 0.3

4.2 1.7 7.0 1.5 2.3 6.3

Source: Arroyo, 2003. Key to compounds: BHA: butylhydroxyanisole; PP: propyl paraben.

© Woodhead Publishing Limited, 2012

610

Breadmaking

24.3.4 Biopreservatives Because of consumer demands for more natural products biopreservation approaches have received much more attention in recent years. Microorganisms are already commonly used in the preparation of sourdough breads. Lactic acid bacteria (LABs) have been used for centuries as starter cultures in food processing. They are able to produce a wide range of GRAS bioactive molecules including organic acids, fatty acids, and bacteriocins. The anti-mould activity of LABs against moulds have been recently reviewed by Hassan and Bullerman (2008). Some studies have also shown that LAB strains in sourdough breads offer very good protection against a wide range of mould spoilage organisms including P. corylophilum, P. expansum, E. fibuliger, A. niger and F. graminearum (Lavermicocca et al., 2000). The latter study isolated a novel bacteriocin from Lactobacillus plantarum 21B, which could perhaps be applied to other bakery product systems. The shelf life of sourdough bread was extended by the inclusion of this bacteriocin. At present, nisin is the only bacteriocin legally approved for use as a food additive (Ganzle et al., 1999). Recently, a range of LABs have been screened and some found to produce anti-fungal compounds acetic and phenyllactic acids (Gerez et al., 2009). Incorporation of LABs as starter cultures allowed a reduction in the concentration of calcium propionate (CP) by 50%, while still maintaining a shelf life similar to that of traditional bread containing 0.4% CP (w/w). This suggests that LABs may produce useful anti-fungal compounds which could be used to substitute the existing fungistats with a similar level of efficacy.

24.4

Future trends

In the last two decades consumer pressure, especially in Europe, to reduce the number of so-called synthetic preservatives used in bread and bakery products has concentrated research efforts into the provision of natural and fresh baked products free of such preservation compounds or their substitution with more acceptable ones. Combinations of physical modifications in packaging systems in combination with the reduced use of organic acid preservatives has been one way forward. For sourdough-based bread the use of natural propionates in situ as preservatives by using mixed-culture fermentations has been a successful approach to extend mould-free shelf life of bread products (Javanainen and Linko, 1993, 1994; Linko et al., 1997). The use of nanoparticles and nanotechnology has received much attention in the food industry. This has provided opportunities for new approaches to formulations of food products, including baked goods. It may well be possible to use nanoparticles for the slow release of preservatives or alternative antimicrobial agents directly in the food product over time to enhance shelf life and mould-free storage time. This may well be combined with active packaging systems where coatings and the slow release of compounds can be combined to extend shelf life without compromising quality of the product for the consumer.

© Woodhead Publishing Limited, 2012

Mould prevention in bread

24.5

• •

611

Sources of further information and advice

Blackburn (2006): Food Spoilage Microorganisms. Samson et al. (2010): Food and Indoor Fungi.

24.6

References

ABDEL-HADI, A.

and MAGAN, N. (2009). Influence of environmental factors on growth, sporulation and ochratoxin A and B production of the new grouping of the A. ochraceus group. World Mycotoxin Journal, 2, 429–34. ABELLANA, M., RAMOS, A. J., SANCHIS, V. and NIELSEN, P. (2000). Effect of modified atmosphere packaging and water activity on the growth of E. amstelodami, E. chevalieri and E. herbariorum on a sponge cake analogue. J Appl Microbiol, 88, 606–16. ALDRED, D., CAIRNS-FULLER, V. and MAGAN, N. (2008). Environmental factors affect efficacy of some essential oils and resveratrol to control growth and ochratoxin A production by Penicillium verrucosum and A. westerdijkiae on wheat grain. J Stored Prod Res, 44, 341–6. ANON (1984). Bread and flour regulations 1984. Statutory Instrument, HMSO, London. ARROYO, M. (2003). Natural antifungal systems for prevention of mould spoilage in bakery products. PhD Thesis, Cranfield University, Cranfield, Bedford, UK. ARROYO, M., ALDRED, D. and MAGAN, N. (2008). Environmental factors and preservatives affect carbon utilization patterns and niche overlap of food spoilage fungi. Fungal Ecology 1, 24–38. AZZOUZ, M. A. and BULLERMAN, L. B. (1982). Comparative antmycotic effects of selected herbs, spices, plant components and commercial antifungal agents. J Food Prot, 29, 1298–1301. BASILICO, M. Z. and BASILICO, J. C. (1999). Inhibitory effects of some spice essential oil on Aspergillus ochraceus NRRL 3174 growth and ochratoxin production. Letts Appl Microbiol, 29, 238–41. BELITZ, H. D. and GROSCH, J. C. (1999). Food Chemistry. Springer. BIDLAS, E. and LAMBERT, R. J. W. (2008). Comparing the antimicrobial effectiveness of NaCl and KCl with a view to salt/sodium replacement. Int J Food Microbiol, 124, 98–102. BLACKBURN, C. DE W. (ed.) (2006). Food Spoilage Microorganisms. Woodhead, Cambridge, UK BINSTOCK, G., AMPOS, C., VARELA, O. and GERSCHENSON, L. N. (1998). Sorbate-nitrite reactions in meat products. Food Res Inter, 31, 581–5. BLUMA, R. V. and ETCHEVERRY, M. G. (2008). Application of essential oils in maize grain: Impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation. J Food Microbiol, 25, 324–334. CAIRNS, R. and MAGAN, N. (2003). Impact of essential oils on growth and ochratoxin A production by Penicillium verrucosum and Aspergillus ochraceus on a wheat-based substrate. In P. CREDLAND, D. M. ARMITAGE, C. H. BELL and P. M. COGAN. (eds), Advances in Stored Product Protection. Cabi International, pp. 479–85. DAIFAS, D. P., SMITH, J. P., TARTE, I., BLANCHFIELD, B. and AUSTIN, J. W. (2000). Effect of ethanol vapor on growth and toxin production by clostridium botulinum in a high moisture bakery product. Journal of Food Safety, 20, 111–25. DAVISON, P. M. and JUNEJA, V. K. (1990). Antimicobial agents. In A. L. Branen, P. M. Davison and S. Salminen (eds), Food Additives. Marcel Dekker, Inc., New York. DEENA, M. J. and THOPPIL, J. E. (2000). Antimicrobial activity of the essential oil of Lantana camara Fitoter, Fitotepapia, 71, 453–5. DWIVEDI, S. K. and DUBEY, N. K. (1993). Potential use of the essential oil of Trachyspemum ammi against seed borne fungi of Guar. Mycopathologia, 121, 101–4.

© Woodhead Publishing Limited, 2012

612

Breadmaking

FARBER, J. M.

(1991). Microbiological aspects of modified atmosphere packaging technology: a review., J Food Prot, 54, 58–70. GANZLE, M. G., WEBER, S. and HAMMES, W. P. (1999). Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int J Food Microbiol, 46, 207–17. GEREZ, C. L., TORINO, M. I., ROLLAN, G. and DE VALDEZ, G. (2009). Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control, 20, 144–8. GUYNOT, M. E., RAMOS, A. J., SALA, D., SANCHIS, V. and MARIN, S. (2002). Combined effects of weak acid preservatives, pH and water activity on growth of Eurotium species on a spnge cake. Int J Food Microbiol, 76, 39–46. GUYNOT, M. E., MARIN, S., SETU, L., SANCHIS, V. and RAMOS, A. J. (2005). Screening for antifungal activity of some essential oils against common spoilage fungi of bakery products. Food Sci Technol, 11, 25–32. GUYNOT M. E., RAMOS A. J., SETÓ L., PURROY P., SANCHIS V. and MARÍN S. (2003). Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products. Journal of Applied Microbiology, 94, 893–9. HAN, J. H. and FLOROS, J. D. (1998). Potassium sorbate diffusivity in American processed and Mozzarella cheeses. J Food Sci, 63, 435–7. HASSAN, Y. I. and BULLERMAN, L. B. (2008). Antifungal activity of Lactobacillus in a liquid culture setting. J Food Prot, 71, 2213–6. HOPE, R., JESTOI, M. and MAGAN, N. (2003). Multi-target environmental approach for control of growth and toxin production by Fusarium culmorum using essential oils and antioxidants. In P. Credland, D. M. Armitage, C. H. Bell and P. M. Cogan (eds), Advances in Stored Product Protection. Cabi International, pp. 486–92. JAVANAINEN, P. and LINKO, Y. Y. (1993). Mixed-culture pre-fermentation of lactic and propionic acid bacteria for improving wheat bread shelf-life. J Cereal Sci, 18, 75–88. JAVANAINEN, P. and LINKO, Y. Y. (1994). Mixed culture preferment of lactic and propionic acid bacteria for improving bread shelf-life. Prog Biotech, 9, 627–30. KILLIAN, D. and KRUEGER, J. (1983). Potassium sorbate spray eliminates returns due to mould. Bak Ind, 150, 54–5. KUBO, I., XIAO, P. and FUJITA, K. (2001). Antifungal activity of octyl gallate:structural criteria and mode of action. Bioorganic and Medical Chemistry Letters, 11, 347–50. LAVERMICOCCA, P., VALERIO, F., EVIDENTE, A., LAZZARONE, S., CORSETTI, A. and GOBETTI, M. (2000). Purification and characterization of novel antifungal compounds by sourdough Lactobacillus plantarum 21B. Appl Environ Microbiol, 66, 4084–90. LEGAN, J. D. (1993). Mould spoilage of bread: the problem and some solutions. Int Biodet and Biodegr, 32, 35–53. LEGAN, J. D. and VOYSEY, P. A. (1991). Yeast spoilage of bakery products and ingredients. J Appl Bacteriol, 70, 361–71. LIEWEN, M. B. and MARTH, E. H. (1985). Growth and inhibition of microorganisms in the presence of sorbic acid. J Food Protection, 48, 364–75. LINKO, Y. Y., JAVANAINEN, P. and LINKO, S. (1997). Biotechnology of bread baking. Trends Fd Sci Technol, 8, 339–44. LOPEZ-MALO, A., ALZAMORA, S. M. and ARGAIZ, A. (2002). Aspergillus flavus doseresponse curves to select natural and synthetic antimicrobials. Int J Food Microbiol, 73, 213–18. LOPEZ-MALO, A., ALTAMORA, S. M. and PALOU, E. (2005). Aspergillus flavus growth in the presence of chemical preservatives and naturally occurring antimicrobial compounds. Int J Food Microbiol, 99, 119–28. LOPEZ-MALO, A., BARRETO-VALDIVIESO, J., PALOU, E. and SAN MARTIN, F. (2007). Aspergillus flavus growth response to cinnamon extract and sodium benzoate mixtures. Food Control, 18, 1358–62. LUCK, E. and JAGER, M. (1997). Antimicrobial Food Additives. Characteristics, Uses and Effects. Springer verlag, Berlin.

© Woodhead Publishing Limited, 2012

Mould prevention in bread MAGAN, N.

613

(2007). Fungi in extreme environments. Chapter 6, In C. P. Kubicek and I. S. Druzhinina (eds), Environmental and Microbial Relationships, 2nd edition, The MYCOTA IV. Springer Velag, Berlin, pp. 85–103. NABIM (2009). UK Flour Milling Industry 2008. National Association of British and Irish Millers, London, UK. NIELSEN, P. V. and RIOS, R. (2001). Inhibition of fungal growth on bread by volatile components from spices and herbs, and possible application in active packaging, with special emphasis on mustard essential oil. Int J Food Microbiol, 60, 219–29. PORTER, S., PASCOT, B. and BENQUALID, K. (1989). Augmentation de la duree de conservation dun aliment a humidite intermediare, conditionne sous atmosphere modifee ou controle. Sci desi Aliments, 9, 701–12. RAY, L. L. and BULLERMAN, L. B. (1982). Preventing growth of potentially toxic moulds using antifungal agents. J Food Prot, 45, 953–63. REHMAN, S., HUSSAIN, S., NAWAZ, H., AHMAD, M. M., MURTAZA, M. A. and RIZVI, A. J. (2007). Inhibitory effect of citrus peel on microbial growth on bread. Pakistan J Nutrition, 6, 558–61. RODRIGUEZ, A., NERIN, C. and BATLLE, R. (2008). New cinnamon-based active paper packaging against Rhizopus stolonifer food spoilage. J Agric Food Chem, 56, 6364–9. ROESSLER, P. F. and BALLENGUER, M. C. (1996). Contamination of an unpreserved semisoft baked cookie with a xerophilic Aspergillus species. J Food Prot, 59, 1055–60. SALMERON, J., JORDANO, R. and POZO, R. (1991). Antimycotoc and antiaflatoxigenic activity of oregano and thyme. J Food Prot, 53, 697–700. SAMAPUNDO, S., DESCHUYFFELEER, N., VAN LARE, D., DE LEYN, I. and DEVLIEGHERE, F. (2010). Effect of NaCl reduction and replacement on the growth of fungi important to the spoilage of bread. Food Microbiol, 27, 749–56. SAMSON, R. A., HOUBRAKEN, J., THRANE, U., FRISVAD, J. and ANDERSON, B. (2010). Food and Indoor Fungi. CBS, Holland. SANDERS, S. W. (1991). Using prune juice concentrate in whole wheat bread and other bakery products. Cereal Food World, 36, 280–83. SAUER, F. (1977). Control of yeast and molds with preservatives. Food Technol, 31, 66–7. SCHMIDT-HEYDT, M. BAXTER, E. S., GEISEN, R. and MAGAN, N. (2007). Physiological relationship between food preservatives, environmental factors, ochratoxin and otapksPv gene expression by Penicillium verrucosum. Int J Food Microbiol, 119, 277–83. SEILER, D. A. L. (1984). Preservation of bakery products. Institute Food Sci Technol, Proc, 17, 31–9. SHELEF, L. A. (1983). Antimicorbial effects of spices. J Food Safety, 6, 29–44. SMID, E. J. and GORRIS, L. G. M. (1999). Natural antimicrobials for food preservation. In M. Shafiur Rahman (ed.), Handbook of Food Preservation. Marcel Dekker, Inc., New York. SMITH, J., KHANIZADEH, S., VAN DE VOORT, F., OORAIKUL, B. and JACKSON, E. (1988). Use of response surface methodology in shelf-life extension studies of a bakery product. Food Microbiology, 5, 163–76. SUHR, K. I. and NIELSEN, P. V. (2004). Effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and pH values. Int J Food Microbiol, 95, 67–78. SUHR, K. I. and NIELSEN, P. V. (2005). Inhibition of fungal growth in wheat and rye bread by modified atmosphere packaging and active packaging using volatile mustard essential oil. J Fd Sci, 70, 37–44.

© Woodhead Publishing Limited, 2012