10 The stability of vitamins during food processing P. Berry Ottaway, Berry Ottaway and Associates Ltd 10.1 Introduction Vitamins, by their definition, are essential to health and have to be obtained from the diet on a regular basis because, with the exception of vitamin D, they cannot be produced by the body. In terms of medicine and nutrition, our knowledge of vitamins is relatively recent. Although James Lind discovered an association between limejuice and scurvy in 1753, it was over 170 years later that vitamin C was eventually isolated. The understanding of vitamin B 12 goes back only to the 1950s and new roles for folates were still being discovered in the late 1990s. Man’s supply of vitamins is obtained from a varied diet of vegetables, cereals, fruits and meats and the quantities of vitamins that are present in the dietary sources can be affected significantly by the processing and storage of the food. 10.2 The vitamins Vitamins are a heterogeneous group of substances and are vital nutrients that must be obtained from the diet. Although a number of these were termed vitamins between the 1930s and 1950s, nutritional science now recognises only 13 sub- stances, or groups of substances, as being true vitamins. The 13 substances are divided into two categories, the fat-soluble vitamins of which there are four (vitamins A, D, E and K) and the water-soluble vitamins of which there are nine (vitamins C, B 1 , B 2 , B 6 , B 12 , niacin, pantothenic acid and biotin). They are listed in Table 10.1. Even within the two sub-categories, the vitamins have almost no common attributes in terms of chemistry, function or daily requirements. In terms of requirements some, such as vitamins C, E and niacin, are needed in tens of milligrams a day whilst others, such as vitamins D and B 12 , are only needed in single microgram amounts. It can be seen from these examples that there is no relationship between the form of delivery (i.e. fat or water soluble) and the daily requirements. The heterogeneity also applies to the chemical structure and the functions of the vitamins. Chemically, there are no similarities between the sub- stances. Some are single substances such as biotin, whilst others, such as vitamin E, are groups of compounds all exhibiting vitamin activity. 10.3 Factors affecting vitamin stability One of the very few attributes that the vitamins have in common is that none is completely stable in foods. The stability of the individual vitamins varies from the relatively stable, such as in the case of niacin, to the relatively unstable, such as vitamin B 12 . The factors that affect stability vary from vitamin to vitamin and the principal ones are summarised in Table 10.2. The most important of these factors are heat, moisture, oxygen, pH and light. The deterioration of vitamins can take place naturally during the storage of vegetables and fruits and losses can occur during the processing and preparation 248 The nutrition handbook for food processors Table 10.1 Vitamins and some commonly used synonyms Vitamin Synonyms Fat-soluble vitamin A retinol vitamin D 2 ergocalciferol vitamin D 3 cholecalciferol vitamin E alpha, beta and gamma tocopherols and alpha tocotrienol vitamin K 1 phylloquinone, phytomenadione vitamin K 2 farnoquinone, menaquinone vitamin K 3 menadione Water-soluble vitamin B 1 thiamin vitamin B 2 riboflavin vitamin B 6 pyridoxal, pyridoxine, pyridoxamine vitamin B 12 cobalamins, cyanocobalamin, hydroxocobalamin niacin nicotinic acid (vitamin PP) niacinamide nicotinamide (vitamin PP) pantothenic acid — folic acid folacin (vitamin M) biotin vitamin H vitamin C ascorbic acid of foods and their ingredients, particularly those subjected to heat treatment. The factors that affect the degradation of vitamins are the same whether the vitamins are naturally occurring in the food or are added to the food from synthetic sources. However, the form in which a synthetic source is used (e.g. a salt or ester) may enhance its stability. For example, the vitamin E (tocopherol) esters are more stable than the tocopherol form itself. With the increased use of nutritional labelling of food products, vitamin levels in foods have become the subject of label claims that can be easily checked by the enforcement authorities. This poses a number of problems for the food tech- nologist. When more than one vitamin is the subject of a quantitative label claim for a food, it is very unlikely that the vitamins will deteriorate at the same rate. If the amounts of these vitamins are included in nutritional labelling, the shelf life of the food is determined by the life of the most unstable component. In order to comply with the legal requirements of maintaining the label claim throughout the declared life of a food product, the food technologist needs to obtain a reasonably accurate estimation of the stability of each of the vitamins in the product. This has to be evaluated in the context of the food system (solid, liquid, etc.), the packaging and probable storage conditions and is achieved by conducting well-designed stability tests. 10.4 Fat-soluble vitamins 10.4.1 Vitamin A Nutritionally, the human body can obtain its vitamin A requirements from two sources: from animal sources as forms of retinol, and from plant sources from b-carotene and related carotenoids. Both sources provide a supply of vitamin A, but by different metabolic pathways. In terms of stability the two sources are different from each other. Vitamin A is one of the more labile vitamins and retinol is less stable than the The stability of vitamins during food processing 249 Table 10.2 Factors affecting the stability of vitamins Factor ? Temperature ? Moisture ? Oxygen ? Light ?pH ? Presence of metallic ions (e.g. copper, iron) ? Oxidising and reducing agents ? Presence of other vitamins ? Other components of food (e.g. sulphur dioxide) ? Combinations of the above retinyl esters. The presence of double bonds in its structure makes it subject to isomerisation, particularly in an aqueous medium at acid pH. The isomer with the highest biological activity is the all-trans vitamin A. The predominant cis isomer is 13-cis or neovitamin A which only has a biological activity of 75% of the all-trans isomer; and 6-cis and 2, 6-di-cis isomers which may also form during isomerisation have less than 25% of the biological activity of the all-trans form of vitamin A. The natural vitamin A sources usually contain about one-third neovitamin A while most synthetic sources generally contain considerably less. For aqueous products where isomerisation is known to occur, mixtures of vitamin A palmitate isomers at the equilibrium ratio have been produced commercially. Vitamin A is relatively stable in alkaline solutions. Vitamin A is sensitive to atmospheric oxygen with the alcohol form being less stable than the esters. The decomposition is catalysed by the presence of trace minerals. As a consequence of its sensitivity to oxygen, vitamin A is normally available commercially as a preparation that includes an antioxidant and often a protective coating. While butylated hydroxyanisole (BHA) and butylated hydrox- ytoluene (BHT) are permitted in a number of countries for use as antioxidants in vitamin A preparations, the recent trend has been towards the use of tocopherols (vitamin E). Both retinol and its esters are inactivated by the ultraviolet compo- nent of light. In general, vitamin A is relatively stable during food processing involving heating, with the palmitate ester more stable to heat than retinol. It is normally regarded as stable during milk processing, and food composition tables give only small differences between the retinol contents of fresh whole milk, sterilised and ultra high temperature (UHT) treated milk. 1 However, prolonged holding of milk or butter at high temperatures in the presence of air can be shown to result in a significant decrease in the vitamin A activity. A provitamin is a compound that can be converted in the body to a vitamin and there are a number of carotenoids with provitamin A activity. Carotenoids are generally found as naturally occurring plant pigments that give the charac- teristic yellow, orange and red colours to a wide range of fruits and vegetables. Some can also be found in the liver, kidney, spleen and milk. The provitamin A with the greatest nutritional and commercial importance is b-carotene. The sta- bility of the carotenoids is similar to vitamin A in that they are sensitive to oxygen, light and acid media. It has been reported that treatment with sulphur dioxide reduces carotenoid destruction in vegetables during dehydration and storage. A study with model systems showed that the stability of b-carotene was greatly enhanced by sulphur dioxide added either as a sulphite solution to cellulose powder prior to b-carotene absorption or as a headspace gas in containers of b-carotene. While it was found that the b-carotene stability was improved by increasing the nitrogen levels in the containers, the stability was even greater when the nitrogen was replaced by sulphur dioxide. Comparative values for the induction period were 19 hours for b-carotene samples stored in oxygen only, 120 hours in nitrogen and 252 hours in sulphur dioxide. 2 250 The nutrition handbook for food processors Investigations into the effect of sulphur dioxide treatment on the b-carotene stability in dehydrated vegetables have given varying results and it has been pos- tulated that the effects of the drying and storage conditions on the stability of the sulphur dioxide has a consequential effect on the stability of the b-carotene in dehydrated products. 3 Studies on the heat stability of both a-carotene and b- carotene showed that the b-carotene was about 1.9 times more susceptible than a-carotene to heat damage during normal cooking and blanching processes. 4 Products containing b-carotene should be protected from light and headspace air kept to the minimum. 10.4.2 Vitamin E A number of naturally occurring substances exhibit vitamin E activity, including the a, b, g and d tocopherols and a tocotrienols. Dietary sources of vitamin E are found in a number of vegetables and cereals, with some vegetable oils such as wheatgerm, sunflower seed, safflower seed and maize oils being particularly good sources. Both synthetic and naturally-sourced forms of vitamin E are available commercially. Whilst the natural sources of the tocopherols, which also have the highest biological activity, are in the d form, the synthetic versions can only be produced in the dl form. Both the d and dl forms are also commercially available as esters. There is a considerable difference in the stability of the tocopherol forms of vitamin E and the tocopherol esters. While vitamin E is regarded as being one of the more stable vitamins, the unesterified tocopherol is less stable due to the free phenolic hydroxyl group. Vitamin E is unusual in that it exhibits reduced stability at temperatures below freezing. The explanation given for this is that the peroxides formed during fat oxidation are degraded at higher temperatures but are stable at temperatures below 0°C and as a consequence can react with the vitamin E. 5 It has also been shown that a-tocopherol may function as a pro-oxidant in the presence of metal ions such as iron. a-Tocopherol is readily oxidised by air. It is stable to heat in the absence of air but is degraded if heated in the presence of air and is readily oxidised dur- ing the processing and storage of foods. One of the most important naturally- occurring sources of tocopherols are the vegetable oils, particularly wheat germ and cotton-seed oils. While deep-frying of the oils may result in a loss of vitamin E of around 10%, it has been found that the storage of fried foods, even at tem- peratures as low as -12°C, can result in very significant losses. dl-a-Tocopheryl acetate is relatively stable in air but is hydrolysed by mois- ture in the presence of alkalis or strong acids to free tocopherols. 10.4.3 Vitamin D Present in nature in several forms, dietary vitamin D occurs predominantly in animal products with very little being obtained from plant sources. Vitamin D 3 The stability of vitamins during food processing 251 or cholecalciferol is derived in animals, including man, from ultra-violet irra- diation of 7-dehydrocholesterol found in the skin. Human requirements are obtained both from the endogenous production in the skin and from dietary sources. Vitamin D 2 (ergocalciferol) is produced by the ultraviolet irradiation of ergosterol, which is widely distributed in plants and fungi. Both vitamins D 2 and D 3 are manufactured for commercial use. Both vitamins D 2 and D 3 are sensitive to light and can be destroyed relatively rapidly if exposed to light. They are also adversely affected by acids. Prepara- tions of vitamin D in edible oils are more stable than the crystalline forms, and the vitamin is normally provided for commercial usage as an oil preparation or stabilised powder containing an antioxidant (usually tocopherol). The prepara- tions are normally provided in lightproof containers with inert gas flushing. The presence of double bonds in the structure of both forms of vitamin D can make them susceptible to isomerisation under certain conditions. Studies have shown that the isomerisation rates of ergocalciferol and cholecalciferol are almost equal. Isomerisation in solutions of cholecalciferol resulted in an equilibrium being formed between ergocalciferol and precalciferol with the ratios of the isomers being temperature dependent. The isomerisation of ergocalciferol has been studied in powders prepared with calcium sulphate, calcium phosphate, talc and magnesium trisilicate. It was found that the isomerisation was catalysed by the surface acid of these additives. 6 Crystalline vitamin D 2 is sensitive to atmospheric oxygen and will show signs of decomposition after a few days storage in the presence of air at ambient temperatures. Crystalline cholecalciferol, D 3 , is also destroyed by atmospheric oxygen but is relatively more stable than D 2 , possibly due to the fact that it has one less double bond. The vitamin D 3 naturally occurring in foods such as milk and fish, appears to be relatively stable to heat processing. 10.4.4 Vitamin K Vitamin K occurs in a number of forms. Vitamin K 1 (phytomenadione or phyl- loquinone) is found in green plants and vegetables, potatoes and fruits, while vitamin K 2 (menaquinone) can be found in animal and microbial materials. The presence of double bonds in both vitamins K 1 and K 2 makes them liable to isomerisation. Vitamin K 1 has only one double bond in the side chain in the 3-position whereas in K 2 double bonds recur regularly in the side chain. Vitamin K 1 exists in the form of both trans and cis isomers. The trans isomer is the naturally occurring form and is the one that is biologically active. The cis form has no significant biological activity. The various forms of vitamin K are relatively stable to heat and are retained after most cooking processes. The vitamin is destroyed by sunlight and is decomposed by alkalis. Vitamin K 1 is only slowly decomposed by atmospheric oxygen. Vitamin K is rarely added to food products and the most common commer- 252 The nutrition handbook for food processors cially available form is K 1 (phytomenadione), which is insoluble in water. A water-soluble K 3 is available as menadione sodium bisulphite. 10.5 Water-soluble vitamins The water-soluble vitamin group contains eight vitamins collectively known as the B-complex vitamins plus vitamin C (ascorbic acid). 10.5.1 Thiamin (vitamin B 1 ) Thiamin is widely distributed in living tissues. In most animal products it occurs in a phosphorylated form, and in plant products it is predominantly in the non- phosphorylated form. Commercially it is available as either thiamin hydrochlo- ride or thiamin mononitrate. Both these salts have specific areas of application and their use depends on the product matrix to which they are added. A considerable amount of research has been carried out on the heat stability of thiamin and its salts, particularly in the context of cooking losses. Early work on thiamin losses during bread-making showed an initial cleavage of the thiamin to pyrimidine and thiazole. 7 The destruction of thiamin by heat is more rapid in alkaline media. Vitamin B 1 losses in milk, which has an average fresh content of 0.04 mg thiamin per 100 g, are normally less than 10% for pasteurised milk, between 5 and 15% for UHT milk and between 30 and 40% for sterilised milk. 7 Between 30 and 50% of the vitamin B 1 activity can be lost during the production of evaporated milk. Losses of thiamin during the commercial baking of white bread are between 15 and 20%. Part of this loss is due to the yeast fermentation, which can convert thiamin to cocarboxylase, which is less stable than thiamin. Thiamin is very sensitive to sulphites and bisulphites as it is cleaved by sulphite. This reaction is rapid at high pH, and is the cause of large losses of the vitamin in vegetables blanched with sulphite, and in meat products such as comminuted meats where sulphites and bisulphites are used as preservatives. Where the pH is low, such as in citrus fruit juices, the bisulphite occurs mainly as the unionised acid, and thiamin losses in such systems are not significantly different from those in prod- ucts not containing bisulphite. 8 Studies on the rate of sulphite-induced cleavage of thiamin during the prepa- ration and storage of minced meat showed that losses of thiamin were linear with sulphur dioxide concentrates up to 0.1%. The storage temperature did not have a significant effect on the losses. It has also been reported that thiamin is cleaved by aromatic aldehydes. Thiamin is decomposed by both oxidising and reducing agents. If it is allowed to stand in alkaline solution in air it is oxidised to the disulphide and small amounts of thiothiazolone. A range of food ingredients has been shown to have an effect on the stability of thiamin. In general, proteins are protective of the vitamin, particularly food proteins such as egg albumin and casein. When heated with glucose, either as a The stability of vitamins during food processing 253 dry mixture or in solution, a browning analogous to a Maillard reaction can occur. This reaction is similar to the reaction between sugars and amino acids and may be important in the loss of thiamin during heat processing. Work has shown that fructose, invertase, mannitol and inositol can actually retard the rate of destruc- tion of thiamin. 9 Thiamin is unstable in alkaline solutions and becomes increasingly unstable as the pH increases. The stability of the vitamin in low pH solutions such as for- tified fruit drinks is very good. In common with that of some other vitamins, the stability of thiamin is adversely affected by the presence of copper ions. This effect can be reduced by the addition of metal-chelating compounds such as calcium disodium ethylenediamine tetra-acetate (EDTA). The heavy metals only appear to influence thiamin stability when they are capable of forming complex anions with constituents of the medium. The enzymes, thiaminases, which are present in small concentrations in a number of animal and vegetable food sources, can degrade thiamin. These enzymes are most commonly found in a range of seafoods such as shrimps, clams and raw fish, but are also found in some varieties of beans, mustard seed and rice polishings. Two types of thiaminases are known and these are designated thia- minase I and thiaminase II. The former catalyses the decomposition of the thia- mine by a base-exchange reaction, involving a nucleophilic displacement of the methylene group of the pyrimidine moiety. Thiaminase II catalyses a simple hydrolysis of thiamin. A problem associated with the addition of vitamin B 1 to food products is the unpleasant flavour and odour of the thiamin salts. 10 The breakdown of thiamin, particularly during heating, may give rise to off-flavours, and the compounds derived from the degradation of the vitamins are believed to contribute to the ‘cooked’ flavours in a number of foods. However, both thiamin hydrochloride and mononitrate are relatively stable to atmospheric oxygen in the absence of light and moisture, and both are normally considered to be very stable when used in dry products with light and moisture-proof packaging. 10.5.2 Riboflavin (vitamin B 2 ) Riboflavin is the most widely distributed of all the vitamins and is found in all plant and animal cells, although there are relatively few rich food sources. It is present naturally in foods in two bound forms, riboflavin mononucleotide and flavin adenine dinucleotide. Plants and many bacteria can synthesise riboflavin and it is also found in dietary amounts in dairy products. Riboflavin is available commercially as a crystalline powder that is only sparingly soluble in water. As a consequence, the sodium salt of riboflavin-5￠-phosphate, which is more soluble in water, is used for liquid preparations. The most important factor influencing the stability of this vitamin is light, with the greatest effect being caused by light in the 420 to 560mm range. Fluorescent light is less harmful than direct sunlight, but products in transparent packaging can be affected by strip lighting in retail outlets. Riboflavin and riboflavin phos- 254 The nutrition handbook for food processors phate are both stable to heat and atmospheric oxygen, particularly in an acid medium. In this respect, riboflavin is regarded as being one of the more stable vitamins. It is degraded by reducing agents and becomes increasingly unstable with increasing pH. While riboflavin is stable to the heat processing of milk, one of the main causes of loss in milk and milk products is from exposure to light. Liquid milk exposed to light can lose between 20 and 80% of its riboflavin content in two hours, with the rate and extent of loss being dependent upon the light intensity, the temperature and the surface area of the container exposed. Although vitamin B 2 is sensitive to light, particularly in a liquid medium such as milk, it remains stable in white bread wrapped in transparent packaging and kept in a lit retail area. 10.5.3 Niacin The term ‘niacin’ is generic for both nicotinic acid and nicotinamide (niaci- namide) in foods. Both forms have equal vitamin activity, both are present in a variety of foods and both forms are available as commercial isolates. Niacin occurs naturally in the meat and liver of hoofed animals and also in some plants. In maize and some other cereals it is found in the form of niacytin, which is bound to polysaccharides and peptides in the outer layers of the cereal grains and is unavailable to man unless treated with a mild alkali. Both forms of niacin are normally very stable in foods because they are not affected by atmospheric oxygen, heat and light in either aqueous or solid systems. 10.5.4 Pantothenic acid In nature, pantothenic acid is widely distributed in plants and animals, but is rarely found in the free state as it forms part of the coenzyme A molecule. It is found in yeast and egg yolk, and in muscle tissue, liver, kidney and heart of animals. It is also found in a number of vegetables, cereals and nuts. Pantothenic acid is optically active and only its dextro-rotary forms have vitamin activity. Losses of pantothenic acid during the preparation and cooking of foods are nor- mally not very large. Milk generally loses less than 10% during processing, and meat losses during cooking are not excessive when compared to those of the other B vitamins. Free pantothenic acid is an unstable and very hygroscopic oil. Commercial preparations are normally provided as calcium or sodium salts. The alcohol form, panthenol, is available as a stable liquid but is not widely used in foods. The three commercial forms, calcium and sodium d-pantothenate and d- pantothenol, are moderately stable to atmospheric oxygen and light when pro- tected from moisture. All three compounds are hygroscopic, especially sodium pantothenate. Aqueous solutions of both the salts and the alcohol form are ther- molabile and will undergo hydrolytic cleavage, particularly at high or low pH. The compounds are unstable in both acid and alkaline solutions and maximum stability is in the pH range of 6 to 7. Aqueous solutions of d-panthenol are more stable than the salts, particularly in the pH range 3 to 5. The stability of vitamins during food processing 255 10.5.5 Folic acid/folates Folic acid (pteroylglutamic acid) does not occur in nature but can be produced commercially. The naturally occurring forms are a number of derivatives collec- tively known as folates or folacin, which contain one or more linked molecules of glutamic acid. Polyglutamates predominate in fresh food, but on storage these can slowly break down to monoglutamates and oxidise to less biologically avail- able folates. The folic acid synthesised for food fortification contains only one glutamic group. Most of the stability studies have been carried out with the commercially avail- able folic acid, which has been found to be moderately stable to heat and atmos- pheric oxygen. In solution it is stable at around pH 7 but becomes increasingly unstable in acid or alkali media, particularly at pH less than 5. Folic acid is decomposed by oxidising and reducing agents. Sunlight, and particularly ultra- violet radiation, has a serious effect on the stability of folic acid. Cleavage by light is more rapid in the presence of riboflavin, but this reaction can be retarded by the addition of the antioxidant BHA to solutions containing folic acid and riboflavin. 11 The stability of the folates in foods during processing and storage is variable. Folic acid loss during the pasteurisation of milk is normally less than 5%. Losses in the region of 20% can occur during UHT treatment and about 30% loss is found after sterilisation. UHT milk stored for three months can lose over 50% of its folic acid. The extra heat treatment involved in boiling pasteurised milk can decrease the folic acid content by 20%. Losses of around 10% are found in boiled eggs, while other forms of cooking (fried, poached, scrambled) give between 30 and 35% loss. Total folic acid losses from vegetables as a result of heating and cooking processes can be very high. A study carried out on the stability of folic acid in spinach during processing and storage showed major differences between water blanching and steam blanching, with a folate retention of 58% with steam blanching and only 17% with water. Frozen spinach was found to retain 72% folate after 3 months storage. 12 10.5.6 Vitamin B 6 (pyridoxine) Vitamin B 6 activity is shown by three compounds, pyridoxol, pyridoxal and pyridoxamine and these are often considered together as pyridoxine. Vitamin B 6 is found in red meat, liver, cod roe and liver, milk and green vegetables. The commercial form normally used for food fortification is the salt, pyridoxine hydrochloride. Pyridoxine is normally stable to atmospheric oxygen and heat. Decomposition is catalysed by metal ions. Pyridoxine is sensitive to light, particularly in neutral and alkaline solutions. One of the main causes of loss of this vitamin in milk is sunlight with a 21% loss being reported after 8 hours exposure. 7 Pyridoxine is stable in milk during pasteurisation but about 20% can be lost during sterilisa- 256 The nutrition handbook for food processors tion. Losses during UHT processing are around 27%, 13 but UHT milk stored for 3 months can lose 35% of this vitamin. Average losses as a result of roasting or grilling of meat are 20%, with higher losses (30–60%) in stewed and boiled meat. Cooking or canning of vegetables results in losses of 20–40%. 10.5.7 Vitamin B 12 The most important compound with vitamin B 12 activity is cyanocobalamin. This has a complicated chemical structure and occurs only in animal tissue and as a metabolite of certain microorganisms. The other compounds showing this vitamin activity differ only slightly from the cyanocobalamin structure. The central ring structure of the molecule is a ‘corrin’ ring with a central cobalt atom. In its natural form, vitamin B 12 is probably bound to peptides or protein. Vitamin B 12 is commercially available as crystalline cyanocobalamin, which is a dark red powder. As human requirements of vitamin B 12 are very low (about 1–2mg a day), it is often supplied as a standardised dilution on a carrier. Cyanocobalamin is decomposed by both oxidising and reducing agents. In neutral and weakly acid solutions it is relatively stable to both atmospheric oxygen and heat. It is only slightly stable in alkaline solutions and strong acids. It is sensitive to light and ultraviolet radiation, and controlled studies on the effect of light on cyanocobalamin in neutral aqueous solutions showed that sunlight at a brightness of 8.6 ￥ 10 4 lux caused a 10% loss for every 30 minutes of exposure, but exposure to levels of brightness 3.2 ￥ 10 3 lux had little effect. 5 Vitamin B 12 is normally stable during pasteurisation of milk but up to 20% can be lost during sterilisation, and losses of 20–35% can occur during spray drying of milk. The stability of vitamin B 12 is significantly influenced by the presence of other vitamins. 10.5.8 Biotin The chemical structure of biotin is such that eight different isomers are possible and of these only the dextro-rotatory or d-biotin possesses vitamin activity. d- biotin is widely distributed, but in small concentrations, in animal and plant tissues. It can occur both in the free state (milk, fruit and some vegetables) and in a form bound to protein (animal tissues and yeast). It is commercially avail- able as a white crystalline powder. Biotin is generally regarded as having a good stability, being fairly stable in air, heat and daylight. It can, however, gradually be decomposed by ultraviolet radiation. Biotin in aqueous solutions is relatively stable if the solutions are either weakly acid or weakly alkaline. In strong acid or alkaline solutions the biologi- cal activity can be destroyed by heating. Avidin, a protein complex, which is found in raw egg white, can react with biotin and bind it in such a way that the biotin is inactivated. Avidin is denatured by heat and biotin inactivation does not occur with cooked eggs. The stability of vitamins during food processing 257 10.5.9 Vitamin C Although a number of compounds possess vitamin C activity, the most important is l-ascorbic acid. Vitamin C is widely distributed in nature and can occur at rel- atively high levels in some fruits and vegetables and is also found in animal organs such as liver and kidney. Small amounts can be found in milk and other meats. Ascorbic acid is the enolic form of 3-keto-1-gulofuranolactone. The endiol groups at C-2 and C-3 are sensitive to oxidation and can easily convert into a diketo group. The resultant compound, dehydro-l-ascorbic acid, also has vitamin C activity. The d-isomers do not have vitamin activity. The l-ascorbic acid in foods is easily oxidised to the dehydro-l-ascorbic acid. In fresh foods the reduced form normally predominates but processing, storage and cooking increase the proportions of the dehydro form. Commercially, vitamin C is available as l-ascorbic acid and its calcium, sodium and magnesium salts, the ascorbates. It is also available as ascorbyl palmitate and can be used in this form as an antioxidant in processed foods. Ascorbic acid and the ascorbates are relatively stable in dry air but are unstable in the presence of moisture. Ascorbic acid is readily oxidised in aqueous solutions, first forming dehydro- l-ascorbic acid which is then further and rapidly oxidised. Conversion to dehy- droascorbic acid is reversible but the products of the latter stages of oxidation are irreversible. Ascorbic acid is widely used in soft drinks and to restore manufacturing losses in fruit juices, particularly citrus juices. Research has shown that its stability in these products varies widely according to the composition and oxygen content of the solution. It is very unstable in apple juice but stability in blackcurrant juice is good, possibly as a result of the protective effects of phenolic substances with antioxidant properties. The effect of dissolved oxygen is very significant. As 11.2 mg of ascorbic acid is oxidised by 1.0mg of oxygen, 75–100mg of ascorbic acid can be destroyed by one litre of juice. Vacuum treatment stages are normally added to the process to deaerate the solution to reduce the problem. It is also important to avoid sig- nificant head-spaces in containers of liquids with added ascorbic acid as 3.3 mg of ascorbic acid can be destroyed by the oxygen in 1 cm 3 of air. 14 Different pro- duction and filling processes can have a significant effect on the retention of vitamin C in drinks. For example, the ascorbic acid loss in a drink packed in a 0.7 litre glass bottle with a partial deaeration of the water and vacuum deaeration of the drink immediately before filling was 16% of the same product filled without any deaeration. Traces of heavy metal ions act as catalysts to the degradation of ascorbic acid. Studies on the stability of pharmaceutical solutions of ascorbic acid showed that the order of the effectiveness of the metallic ions was Cu +2 > Fe +2 > Zn +2 . 5 ACu +2 - ascorbate complex has been identified as being intermediate in the oxidation of the ascorbic acid in the presence of Cu +2 ions. Other work on model systems has shown that copper ion levels as low as 0.85 ppm was sufficient to catalyse oxi- dation, and that the reaction rate was approximately proportional to the square root of the copper concentration. 258 The nutrition handbook for food processors Work with sequestrants has shown that ethylenediamine tetra-acetate (EDTA) has a significant effect on the reduction of ascorbic acid oxidation, with the optimal level of EDTA required to inhibit the oxidation of vitamin C in black- currant juice being a mole ratio of EDTA to [Cu + Fe] of approximately 2.3. 15,16 Unfortunately, EDTA is not a permitted sequestrant for fruit juices in many coun- tries. The amino acid cysteine has also been found to inhibit ascorbic acid oxi- dation effectively. Copper and iron ions play such a significant part in metal catalysed oxidation of ascorbic acid that the selection of process equipment can have a marked effect on the stability of vitamin C in food and drink products. Contact of product with bronze, brass, cold rolled steel or black iron surfaces or equipment should be avoided and only stainless steel, aluminium or plastic should be used. The rate of ascorbic acid degradation in aqueous solutions is pH-dependent with the maximum rate at about pH 4. Vitamin C losses can occur during the frozen storage of foods, and work has shown that oxidation of ascorbic acid is faster in ice than in the liquid water. Frozen orange concentrates can lose about 10% of their vitamin C content during twelve months’ storage at -23°C (-10°F). 17 Light, either in the form of sunlight or white fluorescent light, can have an effect on the stability of vitamin C in milk, with the extent of the losses being depend- ent on the translucency and permeability of the container and the length and con- ditions of exposure. Bottled orange drinks exposed to light have been found to lose up to 35% vitamin C in three months. 7 The destruction of vitamin C during processing or cooking of foods can be quite considerable, with losses during pasteurisation being around 25%, sterili- sation about 60% and up to 100% in UHT milk stored for three months. Milk boiled from pasteurised can show losses of between 30 and 70%. Large losses of vitamin C are also found after cooking or hot storage of vegetables and fruits. The commercial dehydration of potatoes can cause losses of between 35 and 45%. Destruction of vitamin C during the processing of vegetables depends on the physical processing used and the surface area of product exposed to oxygen. Slicing and dicing of vegetables will increase the rate of vitamin loss. Blanching of cabbage can produce losses of up to 20% of the vitamin C, whilst subsequent dehydration can account for a further 30%. 10.6 Vitamin–vitamin interactions One of the least expected and less understood aspects of maintaining the stabil- ity of vitamins in foods is the detrimental interaction between vitamins. This can lead to the more rapid degradation of one or more of the vitamins in a food or beverage. These interactions should be taken into consideration when vitamins are used to restore or fortify products presented in the liquid (aqueous) phase such as soft drinks or fruit juices. Most of the work in the area of vitamin–vitamin interactions has been carried out by the pharmaceutical industry in relation to the development of liquid multivitamin preparations. Four of the thirteen vitamins The stability of vitamins during food processing 259 have been identified as having interactions with each other with deleterious effects. These are ascorbic acid (vitamin C), thiamin (vitamin B 1 ), riboflavin (vitamin B 2 ) and vitamin B 12 . The principal interactions are given in Table 10.3. Other interactions have been identified that can be advantageous, particularly in increasing the solubility of the less soluble vitamins in aqueous solutions. For example, niacinamide has been shown to act as a solubiliser for riboflavin and folic acid. 10.7 Vitamin loss during processing As already discussed, all vitamins exhibit a degree of instability, the rate of which is affected by a number of factors. Naturally-occurring vitamins in foods are sus- ceptible to many of these factors during the harvesting, processing and storage of the food and its ingredients. It is particularly important that the effects of pro- cessing are taken into consideration when assessing vitamin stability in foods, as the food may have been subjected to a number of adverse factors during pro- cessing. The most common factor during processing is the application of heat, which in some cases, such as canning, can be for a relatively long time. Most of the work on the stability of vitamins in fruits and vegetables during blanching and canning was carried out during the 1940s and 1950s. Although there have since been refinements both in processing and analytical techniques, many of the conclusions drawn from this research are still valid. 10.7.1 Blanching In terms of blanching it was found that a high temperature–short time water blanch gave a better vitamin retention than a low temperature–long time blanch and that, overall, steam blanching was superior to water blanching. The addition of sulphite to the blanching water has been shown to affect significantly thiamin levels in fruits and vegetables. Beta carotene was found to be the best survivor during blanching. Riboflavin had retentions in the range 80 to 95%; 260 The nutrition handbook for food processors Table 10.3 Principal vitamin–vitamin interactions Activator Increased instability Ascorbic acid Folic acid Ascorbic acid Vitamin B 12 Thiamin Folic acid Thiamin Vitamin B 12 Riboflavin Thiamin Riboflavin Folic acid Riboflavin Ascorbic acid vitamin C was in the range 70 to 90% under optimum conditions and niacin 75 to 90%. 19 10.7.2 Heat processing Studies on the heat processing of fruits and vegetables in both tin and glass con- tainers showed significant losses of both vitamin C and thiamin. In some cases, the vitamin C levels assayed immediately after the heat processing were between 15 and 45% of the fresh product and these values further reduced during storage. Thiamin reduced by about 50% during heat processing and further declined to between 15 and 40% of the original level after 12 months’ storage. Riboflavin losses were between 12 and 15% during processing but levels of about 50% of the original were observed after 12 months. Niacin was more stable with initial losses of 15 to 25% but with much less than riboflavin being lost during storage. Beta carotene was found to be relatively stable. In milk, the fat soluble vitamins A and D are relatively stable to the heat treat- ments used for the processing of milk, as are the water soluble vitamins riboflavin, niacin, pantothenic acid and biotin. Vitamin C, thiamin, vitamin B 6 , vitamin B 12 and folic acid are all affected by the heat processing of milk, with the more severe the process, the greater the loss. With the exception of vitamin C, vitamin losses are generally less than 10% after pasteurisation of milk and between 10 and 20% after Ultra High Temperature (UHT) treatment. Average losses following sterili- sation of milk are reported as 20% for thiamin, vitamin B 6 and vitamin B 12 and 30% for folic acid. Studies have shown that the stability of vitamin C during the processing of milk is also affected by the oxygen content of the milk. Average losses for the vitamin C were 25% after pasteurisation, 30% after UHT and 60% after sterilisation. However, vitamin C appears to be particularly well retained in condensed full cream milk. 10 B-vitamin stability during the heat processing and cooking of meats varies widely. Cooking conditions can have a marked effect on stability and the reten- tion of thiamin in beef and pork is related to roasting temperatures. If the vitamin content of the drippings is taken into consideration, it is generally found that riboflavin, niacin and vitamin B 12 are stable during the cooking of meat. Pan- tothenic acid losses in cooked meat are usually less than 10% although high losses of folate (both free and total) of over 50% have been found in pork, beef and chicken that had been boiled for 15 minutes. Post-mortem ageing of beef can result in up to a 30% loss of niacin over seven days, although the remaining niacin is relatively stable on cooking. The baking of bread can induce losses of about 20% for thiamin, up to 17% for vitamin B 6 and up to one third of the natural folate content. Niacin and pantothenic acid are normally stable during baking. 10.7.3 Freezing Although most of the vitamins are stable in frozen fruits and vegetables for periods of up to a year, losses of vitamin C have been found to occur at tem- peratures as low as -23°C. The stability of vitamins during food processing 261 10.7.4 Dehydration Studies on the dehydration of blanched vegetables show that the dehydration process can result in additional losses. The dehydration of blanched cabbage (unsulphited) gave an additional 30% reduction in vitamin C content, 5 to 15% in the niacin content and about 15% of the thiamin. 10.7.5 Effect of irradiation on vitamin stability in foods The use of ionising radiation (irradiation) as a sterilisation technique for foods has been accepted in a number of countries, including the European Union. In many countries the foods and ingredients that are allowed to be irradiated are restricted by law and the process is normally only used for foods at risk of high levels of microbiological contamination. It has been shown that vitamin levels in a food can be affected by irradiation and the losses can, in general, be related to the dose. At low doses (e.g. up to 1 kilogray), the losses for most vitamins are not significant. At higher doses (3– 10 kGy) it has been shown that vitamin losses can occur in foods that are exposed to air during the irradiation and subsequent storage. At the highest permitted radiation doses, care has to be taken to protect the food by using packaging that excludes the air and by carrying out the irradiation process at a low temperature. There is evidence that the fat-soluble vitamins A, E and K and the water- soluble thiamin are the most sensitive to irradiation, whereas niacin, riboflavin and vitamin D are relatively stable. There is conflicting evidence for vitamins with some foods showing significant losses and others almost none. If it is intended that nutrition claims are to be made for irradiated foods, it is essential that studies are carried out on the content and stability of the vitamins after the treatment with the ionising radiation. 10.8 Vitamins and food product shelf-life As the tendency to include nutritional information on the labels of food products has increased, so have the liabilities of the manufacturers. For many, if not most, foods the inclusion of nutrition information is optional but any statements made on the label come under the force of law. A company making an inaccurate vol- untary nutritional declaration can be subject to prosecution. Within a nutritional information statement, vitamins are the main category of declared nutrients where the quantities can significantly decrease during the shelf life of the food. The vitamin content of processed foods can decrease during storage and it has already been pointed out that losses of vitamin C can occur in frozen vegetables stored at -23°C (page 259). If declarations of vitamin levels are required on the label, whether voluntary or statutory, the manufacturer needs to carry out suitable stability trials to deter- 262 The nutrition handbook for food processors mine the stability of each vitamin claimed on the label over the duration of the declared shelf life. The actual procedures used for the study will depend on the composition of the food, the processing and the form in which it is presented and stored. The type of packaging can have a significant effect on vitamin stability and the quality of the barriers to oxygen, moisture and light is very important. A requirement for label claims for vitamins can influence the selection of the form of packaging. The need to retain the vitamins often means that a compromise has to be achieved between the length of required shelf life and the barrier quality of the packaging. Due to the wide variety of products, processes and packaging, it is not pos- sible to give specific procedures for the determination of the shelf life of vita- mins in a food. However, guidelines have been established for the determinations and predictions of shelf life. 20 The determination of the vitamin levels at each stage of the shelf life study should be built into the protocol. As the degradation of most of the vitamins follow ‘first order’ or ‘zero order’ kinetics, it is possible for shelf life predictions to be made using a classical Arrhenius model on the assumptions that the model holds for all the reactions being studied; that the same reaction mechanism occurs throughout the temperature range of the study; that the energy of activation is between 10 and 20 kcal/mole and that the effects of moisture at ambient temperature are equivalent to maintaining the same relative humidity at the higher temperatures. 18 Where it is possible to add vitamins to a food either to restore losses during processing or to fortify the food, it is common practice to add an amount above the label claims to compensate for losses during storage. This additional amount is called an overage and is normally quoted as a percentage of the claimed level. For example, if a label claim is made for 60 mg/serving of vitamin C and it is determined that a 10% overage is required to achieve a stored shelf life of one year, the input of vitamin C would be 110% of label claim, or 66 mg/serving. The amount of overage added should be reasonable and well within any safety con- cerns for the vitamin. 10.9 Protection of vitamins in foods For all products for which claims for vitamins are intended, it is essential that all stages of the processing, handling and storage of the product are evaluated to minimise the degradation of the vitamins. This can be accomplished by keeping residence times at high temperatures to a minimum and reducing or eliminating exposure to light and oxygen. For example, during the processing of fruit juices, fruit squashes and fruit drinks, the deaeration of the solution can have a protec- tive effect on the vitamin C levels in the product by reducing or eliminating the oxygen. Commercial sources of vitamins for addition to foods can be obtained in forms that have been encapsulated or coated to improve their stability. The stability of vitamins during food processing 263 10.10 References 1 McCance and Widdowson’s The Composition of Foods (1991), 5 ed. (eds: Holland et al), London, The Royal Society of Chemistry/MAFF 2 baloch a k, buckle k a and edwards r a (1977), ‘Stability of beta carotene in model systems containing sulphate’, J. Food Technol., 12, 309–16 3 baloch a k (1976), The stability of beta carotene in model systems, PhD Thesis, University of New South Wales, Kensington, Australia 4 baloch a k, buckle k a and edwards r a (1977), ‘Effect of processing on the quality of dehydrated carrot’, J. Food Technol., 12, 285–7 5 institute of food science and technology uk (1997), Addition of Micronutrients to Food, London, IFST 6 de ritter m (1982), ‘Vitamins in pharmaceutical formulations’, J. Pharm. 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Berry Ottaway P), Glasgow, Blackie Academic and Professional Press 19 mallette m f, dawson c r, nelson w l and gortner w a (1946), ‘Commercially dehydrated vegetables, oxidative enzymes, vitamin content and other factors’, Ind. Eng. Chem., 38, 437–41 20 lenz m k and lund d b (1980), ‘Experimental procedures for determining destruc- tion kinetics of food components’, Food Technol., 34(2), 51–5 264 The nutrition handbook for food processors