This article is the third in a series about shelf life, where we predominantly focus on the microbiological part of spoilage of bakery products. Moulds, bacteria and to a lesser extent yeasts were covered in part 1. Part 2 handled the internal factors influencing shelf life and in this part, we will cover the external factors in relation to microbiological spoilage: temperature, humidity, atmosphere and bacterial competition.
3.1 Temperature of the environment
All bakery products are stored for a short or longer period of time. The temperature at which this is being done varies from -21°C to ambient temperatures (sometimes 10°C and sometimes 55°C). The effect this temperature has towards effects such as staling, is mostly topic of concern, where we will focus here on the microbiological part.
Microorganisms, as shown in part 1, have each their own specific range of temperature where growth is accomplishable. The most important requirement is that water should be in liquid state and thus available to support microbial growth. In general, microorganisms are not able to grow at temperatures below -8°C and above 100°C. No single organism is capable of growth of whole of this range. Bacteria are normally limited to a temperature span around 35°C and moulds around 30°C, as can be seen in Table 1.
Figure 1: Influence of temperature on multiplication of bacteria
In microbiology four major groups of microorganisms are classified based on their temperature ranges for growth:
Thermophilic microorganisms are only capable to grow in warm environments, psychrophilic microorganisms in cold environments, while mesophilic microorganisms can grow at moderate temperatures, thus almost everywhere. Psychotropic microorganisms can grow at low temperatures but have their optimum at moderate temperatures, sometimes considered as a subgroup of the mesophiles. Table 1 shows the temperature ranges for minimal, optimal and maximum growth of these groups of microorganisms5.
Table 1: Temperature ranges of four major groups of prokaryotic microorganisms (adapted from ICMSF 2012)6
|Group||Minimum temperature||Optimum temperature||Maximum temperature||Moulds||Bacteria|
|Thermophiles||40 - 45°C||55 - 75°C||60 - 90°C||-||Campylobacter ssp.|
|Mesophiles||5 - 15°C||30 - 45°C||35 - 47°C||Aspergillus ssp., Fusarium ssp.|| Salmonella
|Psychrophiles||-5 - 5°C||12 - 15°C||15 - 20°C||-|| Listeria monocytogenes,
Clostridium botulinum type E
|Psychotrophs||-5 - 5°C||25 - 30°C||30 - 35°C|| Rhizopus stolonifera (bread mould)
Bacillus cereus (ropy bread)
Mesophiles, with temperature optimal around 37°C include many of the common foodborne pathogens such as Salmonella, Staphylococcus aureus and Clostridium perfingens. There are only a few species of microorganisms that are psychrophilic. Examples are Listeria monocytogenes and Clostridium Botulinum Type E.
Mesophilic and psychotropic are in general the most import groups of microorganisms. Mesophiles grow generally more quickly at their optima than psychotrophs and so spoilage of perishable products stored in the mesophilic growth range is more rapid than spoilage under chill conditions5.
In Figure 2 the effect of temperature on the general growth curve of microorganisms is displayed. The asymmetric shows that the growth rate of microorganisms decreases slower below optimum temperature than above. The effect that microbiological growth stops at low temperatures is being hypothesized by a slowing down of the reaction rates of the present enzymes, this is however not supported by evidence at the time. A verified cause is that the transport mechanisms4 in the celmembranes of the spoiling microorganisms are interfering due to solidification of fats, blocking partially or fully5.
Figure 2: Effect of temperature on the general growth rate of microorganisms
At high temperatures, structural cell components become denatured and inactivation of heat-sensitive enzymes occurs. This is an irreversible process and it explains why the growth rate increases with increasing temperature, the rate tends to decline rapidly thereafter, until the temperature maximum is reached. In Table 2 this is effect is displayed.
|MFSL (days) at 27°C||MFSL (days) at 21°C|
Table 2: Effect of temperature on mould free shelf life (MFSL) of cakes
3.1.1. Baking & Cooling
Most bakery products are exposed to high temperatures during baking apart from e.g. crèmes, jams and fruits that are added after the baking process. During baking, almost all microorganisms will be eliminated. Only highly heat resistant toxins are possibly produced in an early stadium of the production process can survive.
Although the baking process kills all to nearly all living microorganisms, one comes across every now and then a product that is been contaminated although no man has touched it. A cause can be by the spores that are/ remain present on the product in combination with the right conditions (moisture, temperature) sometimes a more rapidly growth can be explained on your product(s). Another cause can be due to cross contamination due to airborne microorganisms.
When the temperature is increased above the maximum for growth, cells are injured and killed as key cellular components are destroyed and cannot be replaced. This occurs at an increasing rate as the temperature increases. The generally accepted view is that thermal death is a first order process at a given lethal temperature, the rate of death depends upon the number of viable cells present5.
Chilled storage implies temperatures near, but above the freezing point, approximately 0 - 5°C. Chill storage can change both the nature of spoilage and the rate at which it occurs. Low temperatures exert a selective effect preventing the growth of mesophiles and leading to a microflora dominated by psychotrophs. As these psychotrophs are not in their optimum temperature range, the growth is slow, delaying further spoilage.
In this respect temperature changes within the chill temperature range can have pronounced effects. The ability of organisms to grow at low temperatures appears to be particularly associated with the composition and architecture of the celmembrane.
Though mesophiles cannot grow at chill temperatures, they are not necessarily killed. Chilling will induce a phenomenon known as cold shock which causes death and injury in a proportion of the population but its effects are not predictable in the same way as baking. The principal mechanism of cold shock appears to be damage to membrane caused by phases changes in the membrane lipids, causing leaks for the microorganism to ‘bleed to death’.
The extent of a cold shock depends on several factors such as the type of micro-organisms, temperature differential and the rate of cooling is essential (in both cases the larger it is, the greater the damage)5.
During freezing, formation of ice crystals within the product changes the availability of water to participate in reactions. As the temperature is reduced and more water is converted to a solid state, less water is available to support deteriorative reactions. Table 3 shows the effect of freezing on the water activity of pure water and water-ice. In most cases approximately 10% of the water remains liquid if the product is frozen. As far as microbial safety is concerned, the reduction of water activity during freezing has only significant influences on shelf life at freezing temperatures where microbial growth is possible, above - 8°C. The temperatures used in frozen bakery storage are generally less than -18°C. At these temperatures, no microbial growth is possible, although residual microbial or endogenous enzyme activity such as lipases can persist and eventually spoil a product9.
Table 3: Effect of freezing on the water activity of pure water)
Each phase of the freezing process affects micro-organisms. In cooling down to the initial freezing temperature, a proportion of the population will be subject to cold shock.
Initially ice forms mainly extracellularly, intracellular ice formation being favoured by more rapid cooling. This may mechanically damage cells and the high extracellular osmotic pressure generated will dehydrate the micro-organisms. Changes of the water phase because of freezing will also disrupt the structure and function of numerous cell components and macromolecules which depend on these factors for their stability. Cooling down to the storage temperature will prevent any further microbial growth once the temperature has dropped.
Survival rates after freezing will depend on the precise conditions of freezing, the nature of the food material and the composition of its microflora, but have been variously recorded as between 5 and 70%. Bacterial spores are overall unaffected by freezing and some bacteria are more sensitive than others (so called Gram-negative bacteria)5. This also means that yeasts are slowly killed and explains that the functionality of regular baker’s yeast is drastically reduced after approximately 7 days in freezer storage.
Thawing of frozen foods is a slower process than freezing. With high thawing temperature, mesophiles may be growing on the surface of a product while the interior is still frozen. Slow thawing at lower temperature is generally preferred. It does have some lethal effect as microbial cells experience adverse conditions in the 0-10 ̊C, but it will also allow psychotrophs to grow.
Although freezing as a preservation process generally has relatively small effect on product quality, the freezing process and storage conditions influence quality. For a lot of bakery products rapid freezing is required to ensure formation of small ice crystals within the product structure and minimal damage to the product structure. The freezing rate or time allowed for the product temperature to be reduced from above to below the initial freezing temperature will influence product quality and thus product shelf life.
Table 4: Influence of different freezing temperatures on shelf life of some bakery goods adapted from Singh (2001)9
|Product|| Shelf life (months)
| Shelf life (months)
|Cakes (cheese, sponge, chocolate, fruit,etc.)||15||24|
3.2 Relative humidity of the environment
The relative humidity of the environment interacts with the relative humidity inside the product, which we also know as water activity. Moisture in bakery products and its immediate environment will always transfer from high to low relative humidity: this is not necessarily moisture. When bakery products have a low water activity and are stored in high relative humidity atmospheres, water will transfer from the gas phase into the product.
Condensation of water on product surfaces will result in local regions with an increased water activity level. Subsequently, spores of e.g. fungi that were not able to germinate before, at the stadium of lower water activity, will grow and moulds will become visible. Furthermore, it is shown that the product of the respiration of micro-organisms is water, and thus the water activity will raise if micro-organisms are multiplying.
Relative humidity is therefore a very important parameter that needs to be considered in food storage. Even microbiological stable products like biscuits can be spoiled in an environment with a high relative humidity level.
There is a strong relationship between the external factors of temperature and relative humidity. In most conditions, a high temperature relates to a low humidity and vice versa. Variations in temperature during storage and transport of packed bakery products can result in condensation and so enhance microbial growth of the packaged product.
It is possible to (partially) control the relative humidity within the packing material by modified humidity packaging (MHP). Although, this is a rarely used method in the bakery industry and is more applied in products where dehydration causes the main quality losses, such as leafy vegetables e.g. lettuce and endive8. Packaging films are however measured for the transportation mechanisms of oxygen and water; allowing you to choose the right quality for your shelf life.
3.3. Gaseous atmosphere
The average composition of air in the atmosphere is normally: 78% nitrogen, 20.9% oxygen, 0.9% argon gases, 0.03% carbon dioxide and 0.17%of other gases. This gaseous atmosphere makes it possible to breathe for human beings and thus is vitally important for human life likewise for microorganisms.
Gaseous atmosphere is also an external parameter that influences an internal parameter, namely the redox potential, discussed in Part 2 of the Shelf Life series. Oxidizing radicals generated by O3 and O3 are highly toxic to anaerobic bacteria like Bacillus species which can cause ropy bread.
3.3.1 Carbon dioxide
Another internal factor that is influenced by the gaseous atmosphere is the acidity level as a function of the concentration of carbon dioxide. When carbon dioxide dissolves in water it results among other things in carbonic acid. Carbonic acid is a weak acid but can produce a considerable drop in pH. Distilled water in equilibration with CO2 in normal atmosphere will have a pH of about 5. Therefore, carbon dioxide can act in a directly way to inhibit the growth of microorganisms by penetrating the membrane and acidifying the cell’s interior.
Inhibitory effects of carbon dioxide can be influenced by several intrinsic factors as water activity, pH, salt content of the aqueous phase, and fat content. Increasing salt concentrations (relating to lower water activity as well) decreases among other things the solubility of carbon dioxide and suppresses the antimicrobial effects of the gas.
Moulds and gram-negative bacteria are most sensitive for high concentrations of carbon dioxide. Gram positive bacteria like lactobacilli tend to be resistant.
Packaging can be a means to control gaseous atmosphere and to some extend relative humidity. Packaging materials act in a barrier and thus influencing the time in which changing composition of the surrounding gasses can enter the product.
By changing the atmosphere inside the packaging, we influence the growth rate of the microorganisms. Modified Atmosphere Packaging (MAP) or ‘gas flushing’ is a frequently used method for bakery goods with high water activity, e.g. highly perishable products. The method is that we flush the oxygen out the packaging, reducing the growth rate of aerobic microorganism (such as moulds) and risk lipid oxidation, especially in high fat products7.
Figure 3: Interaction of food deterioration mechanisms between the food and environment through the packaging wall (adapted from Singh, 2001)9
The gasses of choice are carbon dioxide and nitrogen, which are used product depended in varying ratio’s. Generally, carbon dioxide is the most effective gas, however when used without support from nitrogen there is a risk of pseudo-vacuum. The carbon dioxide in this case migrates into the product causing a packaging collapse. Table 5 shows some general guidelines on applied compositions of nitrogen and carbon dioxide used in MAP packaging.
Table 5: MAP packaging influencing shelf life of bakery goods
|Product||% nitrogen||% carbon dioxide||Shelf life in air (days)||Shelf life with MAP (days)|
|Bread||20 - 80||20 - 70||4||28|
|Non-dairy cakes||40||60||14||28 - 84|
|Dairy cakes||100||-||14||28 - 84|
Other options used as alternatives for MAP are controlled atmosphere packaging (CAP), where the proportion and type of gas mixture is controlled over the whole storage period. Additionally, vacuum packaging is also adapted in bakery products (e.g. pitas and tortillas), where the product is sealed in a low-gas-permeable pack after partial or full vacuumation.
A method being very effective in combination with nitrogen are oxygen scavengers. This is slightly more expensive than MAP or CAP, but the incorporated chemicals (like iron/ iron oxide forming rust) do not have the potential negative flavour (acidic) effect that dissolved carbon dioxide can have in e.g. cakes.
Figure 4: Vacuum packed bagels and pita´s
The use of edible coating or spraying of certain flavours (often high in propyleenglycol and/ or ethanol) are other methods, however due to costs or religion are not chosen often. These methods are considered as a delaying technique which can be used complementary to MAP/CAP/scavengers10.
3.4 Competitive microorganisms
A not very frequently used method is introducing the natural enemies of the most possible occurring spoiling microorganisms. This method is like the application of antibiotics. As antibiotics is under controversy, due to the potential development of resistant organisms and human intolerance to them, another method is developed.
By applying bacteriophages, which are viruses that infects a specific (group of bacteria) without damaging other desirable microorganisms. Bacteriophages are attacking the concerned bacterial cell wall (Figure 5) , where after it releases its DNA, which is covered in its protein shell (the head), into the bacteria and neutralising it.
Figure 5: Bacteriophages that attack the cell of a bacteria
Phage’s have been killing roughly half of all bacteria over hundreds of millions of years, and as different bacterial species evolved over time, so did their respective phage’s. Over time, it has been perfected by targeting the essential parts of the bacterial cell wall – which do not mutate – making the development of resistance far less likely than with antibiotics.
Examples of phage’s that are developed for the food industry nowadays are LISTEX (against listeria monocytogenes) and SALMONELEX (against salmonella). In the future, there will be probably phage’s developed especially for bacteria that cause serious problems in bakery products like Bacillus species that can cause ‘ropy’ bread.
3.5 Inactivation by technology
The design of the total manufacturing process influences the shelf life of bakery products as most microorganisms and spores are airborne, contamination during cooling, buffering and pre-packaging handling can easily occur. When working on a continuous line, with minimal handling of men between mixing and finished packaging one of the techniques could be controlling the air flow. By controlling the flow of the air, the direction (creating over- and under pressure rooms) the factory can organise that air always flows towards more polluted areas (such as warehouse, maintenance, etc). When using a high filtration before allowing the air flow over cooling products, risks are reduced10.
By using radiation techniques, the air and if exposed to the vicinity of the product are sterilised. Radiation techniques focus on specific energy regions of the electromagnetic spectrum. In particular UV11 and Infrared12 are reported to have a positive influence on shelf life. Infrared radiation is widely used in bread applications in the UK and certain Cheesecake productions in mainland Europe. UV is predominantly used in cleanroom technology and very often used in high perishable goods as dairy based cakes and pies (as are cheesecakes).
Infrared is distinguished in three working ranges:
Figure 6: The electromagnetic spectrum
Depending on the length of exposure and its distance to the product, this results in that approximately the first mm’s are sterilised12. Coatings tend to perform less in this system as it partly can reflect the radiation.
Ultraviolet (UV) can kill a wide range of microorganisms, but only the so-called UV-C range is considered to most effective: ranging from wavelengths 100-280 nm. By absorbing the radiation, the reproductive character of the microorganisms is altered; at a wavelength of 254 nm its germicidal action is optimal.
Both Infrared and UV have a low penetration rate and need to be close to the product, leaving almost no room for humans to intentionally get in touch with the radiation11,12.