The following point highlight the four main chemical preservatives used for prevention of micro-organisms from foods. The preservatives are:- 1. Organic Acids and Esters 2. Nitrite 3. Sulfur Dioxide 4. ‘Natural’ Food Preservatives
Chemical Preservative # 1. Organic Acids and Esters:
The most important organic acids and esters that are used as food preservatives are listed in Table 4.12 along with their E-numbers (EC codes for food additives used throughout the European Community). Their structures are presented in Figure 4.11.
Both are produced microbiologically, although food-grade acetic acid derived petro-chemically is also sometimes used as an alternative to vinegar. They can be an added ingredient in formulated products such as pickles and sauces, or they can be generated in situ in the large range of lactic-fermented products.
They differ from the other acids and esters described here in that they are usually present in amounts sufficient to exert an effect on flavour and on product pH, thus potentiating their own action by increasing the proportion of un-dissociated acid present.
Benzoic acid occurs naturally in cherry bark, cranberries, greengage plums, tea and anise but is prepared synthetically for food use. Its antimicrobial activity is principally in the un-dissociated form and since it is a relatively strong acid (pKa 4.19) it is effective only in acid foods.
As a consequence, its practical use is to inhibit the growth of spoilage yeasts and moulds. Activity against bacteria has been reported but they show greater variability in their sensitivity.
Inhibition by benzoic acid appears multifactorial. The ability of the un-dissociated molecule to interfere with membrane energetics and function appears to be of prime importance since growth inhibition has been shown to parallel closely the inhibition of amino acid uptake in whole cells and membrane vesicles.
Some inhibition may also result from benzoic acid once it is inside the cell as a number of key enzyme activities have also been shown to be adversely affected.
Parabens (Para-hydroxybenzoic acid esters) differ from the other organic acids described here in the respect that they are phenols rather than carboxylic acids. They are much weaker acids with pKa values of 8.5 and so are predominantly uncharged even at neutral pH.
This means that they can be used effectively in non- acidic foods. Their antimicrobial activity increases with the length of the ester group carbon chain, although this also decreases their water solubility and may lead to poor performance in some foods where partition into the fatty phase may occur.
Some Gram-negatives are resistant to the higher homologues and this has been ascribed to the cell’s outer membrane acting as a barrier.
Parabens appear to act mainly at the cell membrane eliminating the ΔpH component of the proton motive force and affecting energy transduction and substrate transport. In contrast to other weak acid preservatives, there is little evidence suggesting that parabens interfere directly with specific enzymic activities.
Sorbic acid is an unsaturated fatty acid, 2,4-hexadienoic acid, found naturally in the berries of the mountain ash. It has a pKa of 4.8 and shows the same pH dependency of activity as other organic acids.
It is active against yeasts, moulds and catalase-positive bacteria but, interestingly, is less active against catalase-negative bacteria. This has led to its use as a selective agent in media for Clostridia and lactic acid bacteria and as a fungal inhibitor in lactic fermentations.
As with the other weak acids, the membrane is an important target for sorbic acid, although inhibition of a number of key enzymes of intermediary metabolism, such as enolase, lactate dehydrogenase and several Krebs cycle enzymes, has been shown.
In contrast to its use as a selective agent for Clostridia, some studies have shown that sorbic acid inhibits the germination and outgrowth of C. botulinum spores. At one time this attracted some interest in the possibility that sorbic acid could be used as an alternative or adjunct to nitrite in cured meats.
Propionic acid (pKa 4.9) occurs in a number of plants and is also produced by the activity of propionibacteria in certain cheeses. It is used as a mould inhibitor in cheese and baked products where it also inhibits rope-forming bacilli.
Objections to the use of preservatives led, in the late 1980s, to the increased use of acetic acid in the form of vinegar as an alternative to propionate but the complete omission of a rope inhibitor has had serious consequences for the public on at least one occasion.
Chemical Preservative # 2. Nitrite:
The antibacterial action of nitrite was first described in the 1920s though it had long been employed unknowingly in the production of cured meats where it is also responsible for their characteristic colour and flavour.
In early curing processes nitrite was produced by the bacterial reduction of nitrate present as an impurity in the crude salt used, but now nitrate, or more commonly nitrite itself, is added as the sodium or potassium salt.
Nitrite is inhibitory to a range of bacteria. Early workers showed that a level of 200 mg kg 1 at pH 6.0 was sufficient to inhibit strains of Escherichia, Flavobacterium, Micrococcus, Pseudomonas and others, although Salmonella and Lactobacillus species were more resistant.
Of most practical importance though is the ability of nitrite to inhibit spore-forming bacteria such as Clostridium botulinum which will survive the heat process applied to many cured meats. To achieve this commercially, initial levels of nitrite greater than 100 mg kg-1 are used.
The mechanism of its action is poorly understood partly due to the complexity of the interaction of several factors such as pH, salt content, presence of nitrate or nitrite and the heat process applied to the cured meat. Descriptive mathematical models of these interactions have however been produced which quantify the precise contribution of nitrite to safety.
Bacterial inhibition by nitrite increases with decreasing pH suggesting that nitrous acid (HNO2, pKa 3.4) is the active agent. In the case of spores, it appears that nitrite acts by inhibiting the germination and outgrowth of heated spores and by reacting with components in the product to form other inhibitory compounds.
The latter effect was first noted in the 1960s by Perigo who observed that when nitrite was heated in certain bacteriological media, the resulting medium proved more inhibitory to Clostridia than when filter-sterilized nitrite was added after heating. Clostridia are very sensitive to these ‘Perigo factors’ which differ from nitrite in displaying activity that is independent of pH.
However, they do not seem to be formed in meat and their effect in bacteriological media could be removed if meat was added. The presence of ‘Perigo-type factors’ has been reported in heated cured meats but these are only produced by severe heating and have minor antibacterial activity.
Studies into the nature of Perigo and Perigo-type factors have looked particularly at the production of Roussin’s salts; complex salts of iron, nitrosyl and sulfydryl groups. Although these compounds have not been shown to be present in cured meats in sufficient quantity to cause the inhibition observed, their formation may give an indication of the way nitrite itself interferes with bacterial metabolism.
It has been proposed that the biochemical mechanism of inhibition involves nitrite reacting with iron and sulfhydryl groups of key cell constituents. Iron-containing proteins such as ferredoxins are very important in electron transport and energy production in Clostridia. For example the phosphoroclastic system is used by Clostridia to generate additional ATP by substrate-level phosphorylation.
Pyruvate, produced by glycolysis, is oxidized to acetate via acetyl-CoA and acetyl phosphate which phosphorylates ADP to produce ATP (Figure 4.12). Ferredoxin acts as a carrier for the electrons removed in the oxidation step and which are ultimately used to reduce hydrogen ions to hydrogen gas.
Support for this hypothesis has come from the observation that nitrite addition leads to an accumulation of pyruvate in C. sporogenes and C. botulinum.
The use of nitrite and nitrate in food has attracted scrutiny since it was discovered in the 1950s that N-nitrosamines, formed by the reaction of nitrite with secondary amines, especially at low pH, can be carcinogenic.
Concern developed that they may be present in food or formed in the body as a result of ingestion of nitrate or nitrite with food.
Surveys have indicated that cured meats, particularly fried bacon, beer and, in some countries, fish, make the most significant contribution to dietary intakes of nitrosamines, although a US survey made the point that a smoker inhaled about 100 times the amount of volatile nitrosamines per day as were provided by cooked bacon.
Dietary intake of nitrite is low, generally less than 2 mg NaNO2 day-1, and comes mainly from cured meats, although it is also present in fish, cheese, cereals, and vegetable products.
Nitrate is also of concern since it can be reduced to nitrite by the body’s own microflora. Cured meats are not a significant source; vegetables contribute more than 75% of the dietary intake of nitrate, although water can be an important source in some areas.
Awareness of the problem has led to changes in production practices for cured meats such as the use of low levels of nitrite in preference to nitrate and the increased use of ascorbic acid which inhibits the nitrosation reaction. These measures have produced significant reductions in nitrosamine levels.
Mention should also be made here of the other contributions made by nitrite to the quality of cured meats. Reduction of nitrite to nitric oxide produces the characteristic red colour of cured meats. The nitric oxide co-ordinates to the haem ferrous ion in the muscle pigment myoglobin converting it to nitrosomyoglobin (Figure 4.13).
When raw cured meats such as bacon are cooked this pigment decomposes to produce nitro-sylhaemo-chrome which has the pink colour also seen in cooked cured hams. Only small quantities of nitrite are required to produce the cured meat colour: theoretically 3 mg kg-1 is sufficient to convert half the myoglobin present in fresh meat, but because of competing reactions, 25 mg kg– 1 are required to give a stable colour.
Nitrite also contributes to the typical cured meat flavour. Taste panels can distinguish cured meats where nitrite has not been used but the precise reasons for this are not known. It is thought that nitrite acts as an antioxidant to inhibit lipid degradation in the meat although this may only be part of the story.
Chemical Preservative # 3. Sulfur Dioxide:
Sulfur dioxide (SO2) has long enjoyed a reputation for its disinfecting properties and its earliest use in the food industry was when sulfur candles were burnt to disinfect the vessels used to produce and store wine. Nowadays, it is also used as an antioxidant to inhibit enzymic and non-enzymic browning reactions in some products.
Sulfur dioxide is a colourless gas that readily dissolves in water to establish a pH- dependent equilibrium similar to CO2.
Sulfurous acid (H2SOs) is a dibasic acid with pATa values of 1.86 and 6.91.
The unionized forms of SO2 which can readily penetrate the cell have the greatest antimicrobial activity. It has been reported that they are between 100 and 1000 times more active than the bisulfite anion. Since the unionized forms predominate at low pH values, it follows that SO2 is used to best effect in acidic foods.
At neutral pH, SO2 is present as a mixture of the relatively inactive bisulfite (HSO3–) and sulfite (SO2-3) ions, although salts of these anions prove the most convenient way of handling the preservative in the food industry.
SO2 is a reactive molecule and can disrupt microbial metabolism in a number of ways. As a reducing agent, it can break disulfide linkages in proteins and interfere with redox processes. It can also form addition compounds with pyrimidine bases in nucleic acids, sugars and a host of key metabolic intermediates.
One disadvantageous consequence of this reactivity is its ability to destroy the vitamin thiamine in foods and the once widespread practice of using it in meat and meat products has now been prohibited, with the exception of British fresh sausage.
Sulfur dioxide is active against bacteria, yeasts and moulds, although some yeasts and moulds are more resistant. Gram-negative bacteria are most susceptible and in British fresh sausage where sulfite is permitted up to a level of 450 mg kg-1, the Gram-negative spoilage flora normally associated with chilled meats is replaced by one dominated by Gram-positive bacteria and yeasts.
In winemaking the tolerance of the wine yeast Saccharomyces cerevisiae to SO2 levels around 100 mg 1-1 is exploited to control the growth of wild yeasts and acetifying bacteria. Seasonal surpluses of soft fruits are also preserved by the addition of high levels of SO2 to permit jam production throughout the year.
Chemical Preservative # 4. ‘Natural’ Food Preservatives:
The uncertainty voiced by consumer organizations and pressure groups over the use of food additives including preservatives has already been referred to. One approach to reassuring the consumer has been recourse to methods of preservation that can be described as ‘natural’.
The whole area though is riddled with inconsistency and contradiction; it can be argued that any form of preservation which prevents or delays the recycling of the elements in plant and animal materials is unnatural.
On the other hand there is nothing more natural than strychnine or botulinum toxin. Smoking of foods might be viewed as a natural method of preservation. Its antimicrobial effect is a result of drying and the activity of wood smoke components such as phenols and formaldehyde which would probably not be allowed were they to be proposed as chemical preservatives in their own right.
The use of natural food components possessing antimicrobial activity such as essential oils and the lacto-peroxidase system in milk have attracted some attention in this respect. Attention has also been paid to the bacteriocins produced by food-grade micro-organisms such as the lactic acid bacteria.
Nisin is an already well-established example and its use can be extended by expedients such as inclusion of whey fermented by a nisin-producing strain of Lactococcuslactis as an ingredient in formulated products like prepared sauces.