Protective Role of Natural Antioxidants Against the Formation and Harmful Effects of Acrylamide in Food

INTRODUCTION

Food is important for the individual's life as it provides energy and several essential nutrients. Any edible food is a complex mixture of compounds, most of them are beneficial, protective and essential for life. Some are harmful components, which may interact with each other or with other food constituents. These harmful compounds are introduced to such edible food because of pollution by some environmental elements or as a result of heat treatment of foods to produce such compounds. Parts of them are important for the sensory properties such as color, flavor and aroma of the heated products due to maillard reaction which is a complex reaction between amino compounds (principally amino acids) and reducing sugars. Some of these compounds may not be beneficial or may be even toxic to humans like acrylamide. Acrylamide is traditionally known as an industrial chemical substance whose major use is to produce poly acrylamide, a coagulant used in drinking water and waste water treatment¹. Concern about the health of the tunnel workers encouraged specialists to conduct studies which led to find that acrylamide was carcinogenic in rats and a neurotoxin in occupationally-exposed workers. These findings led a group of scientists to develop a test that measured the presence of an acrylamide protein adduct in blood. The investigators not only found this adduct in the exposed tunnel workers, but also among members of the general population that had not known occupational exposure to acrylamide². Acrylamide has been classified as a group 2A carcinogen by the International Agency for Research on Cancer and a Category 2 carcinogen and Category 2 mutagen by the European Union. This finding has caused worldwide concern³. In 14 April, 2002 the Swedish National Food Administration (NFA) and researchers from Stockholm University reported that acrylamide is formed in many types of foods prepared or cooked at high temperatures. The highest levels were found in potato and cereal-based products subjected to heat processing such as frying, grilling or baking. Long-term exposure to acrylamide might cause damage to the nervous system both in humans and animals to a certain extent and acrylamide was also considered as a potential genetic and reproductive toxin^4-6. Since the subject of the study has a great concern at local and international levels. The aim of this study emerged through the expected identify the average of acrylamide intake among individuals around the world. Also, increase awareness of acrylamide dangers on health and how to reduce its formation in foods, improving food and nutritional concepts and behaviors in consumers.

DISCOVERY OF ACRYLAMIDE IN FOODS

In April, 2002, the Swedish National Food Administration (SNFA) and the University of Stockholm together reported that processed carbohydrate-rich foods that are fried or baked at relatively high temperature may contain considerable levels of acrylamide^7,8. Potato products (such as; potato chips, French fries and hash browns), bread, biscuits and cereal were among the food items containing the highest amounts of acrylamide⁷. Acrylamide levels rise very strongly with time duration of heat processing as the extent of cooking time affects the degree of formation as 10-20 fold increase in acrylamide levels which have been reported between cooked and over-cooked fried potatoes. In contrast, acrylamide formation has not been demonstrated at temperatures below 120°C as a temperature >100°C is required for its formation^9,10. This formation of acrylamide is primarily the result of the Maillard reaction between the amino acid asparagines and sugars such as; glucose and fructose¹¹. Foods that are rich in these two elements are considered precursors, which become major sources of acrylamide when cooked. They are derived from products of vegetable origin, such as potato, cereals, peanuts, lentils and asparagus, but apparently not from products of animal origin¹².

Acrylamide levels in food: Potato products such as French fries, chips, roasted and baked potatoes are characterized by high levels of acrylamide. In particular, french fries which may contain more than 2000 μg kg^–1 a level which is considered human carcinogen compound. Also, acrylamide contents in the same food differ according to cooking methods^13,14.

The European Food Safety Agency reported that acrylamide is formed during frying, roasting and baking but low contents are found in boiled or microwave foods¹⁵. The highest acrylamide levels have been found in fried potato products, bread, bakery wares, coffee, hazelnuts and almonds. On the other hand, acrylamide is found in foods not subjected to severe heating like olive and also dried fruits e.g., plums, pears and apricots^16,17. Animal derived heat treated foods such as; meat and fish, generally exhibit low or negligible levels of acrylamide¹⁵.

Ingredients play an important role in acrylamide formation as different ingredients have various amounts of free asparagine and reducing sugars available for the formation reaction. White wheat flour has a low content of free asparagine, but certain ingredients used in baking, such as germ and bran contain significant amounts^18,19. Therefore, different levels of acrylamide may be present in a wide range, depending on the product type and ingredients. However, in other dishes fried potato of containing breaded fish, hamburger or fried chicken may not reach such values of acrylamide. A possible explanation for this result may be the use of different potato varieties. The boiled rice contained less than 50 μg kg^–1 but was increased to 113 μg kg^–1, when various ingredients were added to produce risotto. Also values of less than 50 μg kg^–1 were reported for simple tomato sauce but it was increased to 124 μg kg^–1, when other ingredients such as olives and capers were added²⁰.

Possible mechanisms for acrylamide formation in foods: A number of theories have been proposed for the mechanism by which acrylamide (AA) is formed in food. Asparagine needs a carbonyl compound to be converted to acrylamide. The carbonyl can come from multiple sources²¹.

Due to the diversity of antioxidants in structure, properties and complexity of the reactions, different antioxidants are involved in different reactions during the process of Maillard reaction thus causing different effects towards acrylamide formation. It seems that whether the carbonyl is available in both A and B pathways depends on the functional group in the β-position to the nitrogen atom. The presence of a hydroxyl group favors the re-arrangement from azomethine yield to the decarboxylated Amadori product to afford acrylamide (pathway A). The α, β, γ, δ-diunsaturated carbonyl group and α-dicarbonyl groups may play a key role in pathway B. As for the carbonyl group, aldehydes are more reactive than ketones. Also, a pathway named acrolein pathway (pathway C) stating that acrolein and asparagine as well as acrylic acid (oxidized acrolein) and ammonia produced a significant amount of acrylamide. This pathway might be marginal, since the nitrogen atom of acrylamide comes from asparagine and other carbonyl compounds such as reducing sugars are way more reactive with asparagine that it's hard to spare nitrogen atom to acrolein in foodstuffs²².

Major acrylamide (AA) formation pathway: The major pathway for acrylamide formation in food is known as the asparagine route via Maillard reaction¹¹. This non-enzymatic browning reaction influences several aspects of food quality such as flavor, color and aroma formation. In this N-glucosides route, asparagine is converted to acrylamide through thermal decarboxylation and deamination, which necessarily needs the presence of a carboxyl compound (such as; a reducing sugar)²³. Fructose, which contained two α-hydroxycarbonyl groups increases acrylamide formation by about 2-fold compared with other reducing sugars such as glucose^24-26. Asparagine has been proved to provide the backbone chain of acrylamide by mass spectral studies demonstrated that all the three carbon atoms and the nitrogen atom are from asparagine. Although asparagines alone may release acrylamide by thermally initiated decarboxylation and deamination in the presence of reducing sugars, acrylamide formation from asparagine is significantly increased. Asparagine alone produced acrylamide (0.99 μg g^–1 of asparagine) upon thermal degradation, while glutamine produced 0.17 μg g^–1 under the same conditions. When asparagine was heated at 180°C with glucose, a large amount of acrylamide (1200 μg g^–1 of asparagine) was formed. The potential of generating acrylamide from suitable precursors has mainly been attributed to the concentration of asparagine, which directly provides the backbone of the acrylamide molecule²⁷. The type of sugar or in general the carbonyl compound may significantly affect the final amount of acrylamide generated through the Maillard reaction¹¹. For example that fructose was, by a factor of about 3, more efficient compared to glucose under the same pyrolytic conditions²⁸. The acrylamide formation was enhanced when the ratio of glucose to asparagine was greater than or equal²⁹ to 1.

Minor acrylamide formation pathway: Other minor reaction routes have been proposed for acrylamide formation, such as from ammonia and acrolein in the absence of asparagines, a pathway that was suggested to play a role in lipid rich foods²². Asparagine and glutamine are found abundantly in wheat, corn and oats contribute to non-enzymatic browning of these grains by release of ammonia. Both of these amino acids are abundant in potatoes^30,31. Acrolein can be formed by oxidative lipid degradation or from glycerol, leading to acrylic acid. Acrylic acid can react with ammonia to form acrylamide³². However, this pathway does not seem to be involved in acrylamide formation in fried potatoes¹⁰. It was also reported that oil degradation products such as glycerol, mono and diacylglycerols had no significant impact on acrylamide formation in a potato model system and French fries³³. Acrylamide formation has also been proposed from protein pyrolysis in dry heated wheat gluten³⁴.

SOURCES OF HUMAN EXPOSURE

Exposures to acrylamide may occur via the inhalation, oral or dermal routes. The potential routes of human exposure to acrylamide are ingestion, dermal contact and inhalation³⁵. Acrylamide can be absorbed through unbroken skin, mucous membranes, lungs and the gastrointestinal tract³⁶.

Human dietary intake of acrylamide: In the USA, most of dietary AA comes from potato chips, french fries, breads, cereals, biscuits/cookies, fried pastries and other salty snacks³⁷. In European countries, more than half of AA exposure comes from consumption of bread, crisp bread, rusks, coffee and potatoes³⁸. The resulting intake estimates ranged from 0.8 μg kg^–1 b.wt./day for the average consumer to 3 μg kg^–1 b.wt./day for the 95th percentile consumer and 6.0 μg kg^–1 b.wt./day for the 98th percentile consumer⁹. The general agreement of the several methods used to estimate exposure using well described food consumption data from Australia and Norway. The Netherlands, Sweden, USA and from the IARC EPIC study indicated a lower bound estimate of typical exposures in the range of 0.3-0.8 μg kg^–1 b.wt./day depending upon whether the average or median exposure is estimated and which age groups were evaluated. According to exposure assessments, the daily intake of AA of diet is approximately 0.4 μg kg^–1 b.wt., with a 90th percentile of 0.95 μg kg^–1 b.wt.³⁹. Median intakes in children and adolescents are higher due to lower body weight and different consumption patterns and worst-case intake scenarios of up to 6.9 μg kg^–1 b.wt./day have been discussed⁴⁰. It was found that the simulated daily exposure to acrylamide for males and females with mean exposures which were 0.052 and 0.064 μg kg^–1 b.wt./day, respectively⁴¹. In this scenario, females are exposed to slightly greater amounts of acrylamide. This may be due to the averaging of total acrylamide intake over respective body weights (i.e., males, in general, weigh more than females). The exposure level is significantly lower than that estimated when eating commercially produced French fries (0.27 and 0.20 μg kg^–1 b.wt./day for males and females, respectively as given in Cummins⁴². This is mainly due to the greater exposure levels and portion size for potato French fries compared to potato crisps. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) estimated the mean dietary acrylamide intake for general population including children, between 1 and 4 μg kg^–1 b.wt./day. It was noted that children have dietary acrylamide exposures at least twice as high as for adult consumers when expressed on a body weight basis⁴³. The EFSA also recently performed an exposure assessment based on acrylamide monitoring results from 2007-2008. The 95th percentile of acrylamide intake for adults (>18 years) and children (3-10 years) were estimated to range between 0.6-2.3 and 1.5-4.2 μg kg^–1 b.wt./day for adults and children, respectively⁴⁴. Foods contributing the most to dietary intake would differ from country to country, according to different dietary patterns and the way how foodstuffs are processed and prepared. The mean daily acrylamide consumption found was 0.534 μg kg^–1 b.wt./day, which is similar to that found for other European countries such as Germany, Norway, France, Sweden and Belgium in studies carried out in different population segments⁴⁰. The adolescents from a Canadian urban Center that median total daily intake of acrylamide was estimated at 0.29 μg kg^–1 b.wt./day and upper 97.5th percentile value was found to be 2.85 μg kg^–1 b.wt./day, which is approximately 5-fold greater than median intake⁴⁵. Average contribution of main food groups to total daily acrylamide intake as estimated with the 2-day food dairy were: deep-fried French fries had the highest contribution representing 50% of total daily acrylamide intake. The mean exposures as well as the 95th percentile were calculated for general population⁴⁶. The dietary acrylamide exposure in the Chinese population from each food group in the 12 provinces in 4 total diet study (TDS) regions. The acrylamide total intakes of 12 provinces ranged from 0.056-0.645 μg kg^–1 b.wt./day with overall average of 0.286 μg kg^–1 b.wt./day. Another study showed that the daily intakes corresponding to a 1 ×10^–5 upper-bound risk level are estimated to be 1 and 9 μg/person, respectively for high and average consumers (assuming a default body weight of 75 kg)⁴⁷.

EFFECT OF ACRYLAMIDE ON HEALTH

Neurotoxicity: Acrylamide is a well-known neurotoxic compound that produces central and peripheral distal axonopathy. Peripheral neuropathy and hemoglobin adduct formation have been seen in occupationally exposed humans and experimental animals to acrylamide^48-50. Neurotoxicity of acrylamide results from high levels of exposure and concerns workers occupationally exposed to acrylamide through inhalation or dermal absorption⁵¹. Longer exposure resulted in more severe symptoms, including cerebellar dysfunction followed by neuropathy³⁶. Long-term consumption of low levels of acrylamide contributed to the causes of Alzheimer’s or other degenerative diseases of the human brain. In rat and mice studies, No observable Effect Level (NOEL) for neurotoxic effect has been estimated ranging from 0.2-10 mg kg^–1 b.wt./day and is far above dietary exposure⁵¹. However, it has been postulated that neurotoxicity of acrylamide might be cumulative and thus that dietary exposure might not be negligible⁵². In vivo proteomic and in chemico studies demonstrated that acrylamide forms covalent adducts with highly nucleophilic cysteine thiolate groups located within active sites of presynaptic proteins and the resulting protein inactivation disrupts nerve terminal processes and impairs neurotransmission⁵³. In addition, since acrylamide can bind to sulfhydryl groups on cysteine residues of proteins and causes nerve terminal degeneration and inhibition of axonal transport, it is possible that acrylamide is associated with some neurodegenerative diseases⁵⁴.

Carcinogenicity: Several studies in rodents supported the evidence that acrylamide is a multi-organ carcinogen being able to cause tumors to many organs such as; lung, uterus, skin, mammalian gland and brain, etc⁵⁵. Although acrylamide is clearly carcinogenic in experimental animals, epidemiological studies on individuals exposed occupationally to acrylamide have not indicated any consistent dose-related increase in cancer at any organ site, with the possible exception of the pancreas ovaries and endometrium^55,56. It is widely accepted that the carcinogenicity of acrylamide would stem from its conversion in mammalians to glycidamide which has been shown to be mutagenic and genotoxic in vitro and in vivo test systems^57,58. The acrylamide-induced DNA adduction and consequent mutagenesis has been postulated as the key process in acrylamide carcinogenicity although the chain of events starting from glycidamide DNA adducts formation and leading to mutation has not been directly investigated (The first evidence of acrylamide and glycidamide inhibition of a mitotic/meiotic motor protein and speculated) that this could be an alternative mechanism to DNA adduction in the production of cell division defects and potential carcinogenicity^59,60. Also, a hormonal effect in rat endocrine (thyroid) and mammary glands has also been postulated for carcinogenicity of acrylamide. A prospective epidemiological study has found that increased dietary intake of acrylamide is associated with increased risks of post-menopausal endometrial and ovarian cancer particularly among neversmokers⁶¹. This was further supported by a large prospective cohort study among women in the U.S.⁵⁶. They observed that risk for endometrial cancer and possibly ovarian cancer was greater among high acrylamide consumers. In addition, a positive association between dietary acrylamide intake and renal cell cancer risk was observed in a prospective cohort study⁶². Moreover, one case-control study reported that consumption of French fries during preschool years was associated with a slightly increased risk of breast cancer later in life⁶³. There was a positive association between acrylamide-hemoglobin levels and estrogen receptor positive breast cancer⁶⁴. On the other hand epidemiological studies indicated that dietary acrylamide intake is not associated with other cancer risks, prostate cancer, gastrointestinal cancer, bladder cancer, thyroid cancer, brain cancer and lung cancer. Generally, epidemiological studies have provided valuable information regarding the associations between dietary acrylamide intake and cancer in human. It is suggested that more epidemiological studies are needed to substantiate the evidence^65-68.

Reproductive and developmental toxicity: Acrylamide has also been reprotoxic in rodents and mutagenic in somatic cells in vitro and in vivo as well as in germ cells in vivo^69-72.

The reproductive and developmental toxicity of acrylamide has been reported in laboratory animals. When mice or rats were treated with exogenous acrylamide, reproductive and developmental toxicity manifests as disrupted mating, reduced fertility rates, increased reabsorption of fetuses, reduced litter size in pregnant females, abnormal sperm and decreased sperm count in males⁷³. The molecular mechanisms of reproductive toxicity are associated with chromosomal damage, alkylation of SH groups, depletion of GSH and/or DNA damage⁵⁹. Nevertheless, there is no information as to the potential for reproductive and developmental effects of acrylamide in humans⁷³.

FACTORS AFFECTING THE ACRYLAMIDE FORMATION IN FOODS DURING PREPARATION

There are numerous factors affecting acrylamide formation whether by reducing or increasing its content in foods during preparation. It has been documented that most if not all the acrylamide found in foods is formed during preparation. Its intake is harmful to the body due to its carcinogenic effect. Therefore; it is crucial to avoid or reduce its content during preparation. Many effective methods regarding the reduction of acrylamide were found in the actual food matrixes. A number of mitigation strategies to reduce the acrylamide content in foods have been proposed and tested. They have been mainly focused on potato products and cereal-based products that are two of the major contributors to dietary exposure in most of the populations. Conversely, only very limited process options for coffee products are available for reducing acrylamide levels without affecting the final quality⁷⁴.

The mitigation approach referred to certain steps which include changing in recipes and formulations. This included for example selection of foods such as potato varieties and cereal species or varieties low in acrylamide precursors. Also, it includes addition of some nutrients or compounds such as proteins, amino acids like glycine, cysteine, organic acids and acidulants, calcium ions, cyclodextrin, natural antioxidants or antioxidant extracts etc, replacement of reducing sugars with sucrose and of ammonium bicarbonate with sodium bicarbonate. In addition variations in preparation can be followed like changing in process conditions and/or cooking methods including changing of time, temperature of frying or baking, changing the type of oven, prolonged fermentation of bakery products etc. Also, the removal of acrylamide after formation by means of vacuum has also been proposed, but its impact on manufacturing practices and food quality has not yet been clearly established⁷⁵. One of the most promising tools to control acrylamide content in heat treated foods is the addition of the enzyme asparaginase. The enzyme asparaginase (L-asparagine amidohydrolase) is an enzyme able to catalyze the hydrolysis of asparagine to aspartic acid and ammonia thus lowering the content of asparagines as a precursor. Asparaginase has been successfully applied at lab scale both to potato products and cereal-based products with percentage of reduction up to 85-90% with no effect on products taste and appearance¹¹. This has been used for some products at industrial scale. Some preliminary results achieved at lab scale highlighted that asparaginase pretreatment of green beans may represent a viable way to reduce acrylamide concentration in roasted coffee as well. Nevertheless, the high cost of the enzyme may represent a serious constraint on its application on a large scale^76-78.

It has been also emphasized that some mitigation strategies are associated with an increase in other risks or a loss in benefits. For example, replacement of ammonium bicarbonate with sodium bicarbonate as rising agent for fine bakery products results in an increase of sodium intake⁷⁹. Some of the mitigation strategies that have been proposed and bring about changes in organoleptic properties of foods like excessive browning as result of glycine addition, generation of off-flavors, insufficient browning as a result of changing in time temperature profile etc. This can dramatically affect the final quality and consumer’s acceptance^78,80.

Generally, all potential strategies to prevent acrylamide formation may be resumed in two major approaches, removal of the acrylamide precursors or interference with the Maillard reaction, which is essential for the desired and characteristic flavor and color formation of food products like potatoes⁸¹. Therefore, this constitutes the first challenge for food scientists on how to reduce acrylamide formation without affecting the final product specifications and its quality. Numerous researches have been performed which demonstrated the necessity of a farm-to-fork approach in order to reduce acrylamide like in fried potato products. Several relevant aspects regarding acrylamide formation should be considered at different stages of producing food products including methods of treatment and type of additives⁸¹.

Influence of food size: Acrylamide is formed in the surface layer of the food like potato product and therefore, the size and the cut shape of the product (surface-to-volume ratio) will influence the final acrylamide contents. Accordingly, thinner and smaller cut sizes result in increased acrylamide formation upon final frying⁸². Larger French fries (“pont-neuf”) 20×20 mm contained somewhat less acrylamide, which is probably related to the lower proportion of the crust on the total mass⁸³. These differences are compared to 7×7 mm French fries which are small, but larger pieces require longer frying (prolonged pre- frying). However, thinner pieces (such as 6×6 mm) are more difficult to fry to the adequate point, since few seconds of overtime are sufficient to initiate general browning and shooting acrylamide formation. Fine pieces from the outer part of the potato are a problem since they reach optimum frying earlier than the bulk rapidly getting brown and contain acrylamide in excess⁸³ of 1000 μg kg^–1.

Influence of washing and soaking in water: Washing and soaking the food may decrease acrylamide formation during heat treatment. There are quite significant changes that water soluble components, such as glucose and fructose are washed out from the external layer of the potato. Matthaus et al.⁸⁴ reported a reduction in acrylamide formation of 69% following soaking at 50°C for 15 min. The glucose content was decreased by 32% in potato slices soaked for 90 min and 25% for 40 min in distilled water. Other reducing sugars such as fructose and sucrose followed a similar trend. Asparagine content tended to remain unchanged even after a 90 min soak⁸⁵. Acrylamide formation was reduction by 50% in French fries following soaking in distilled water⁸⁶ for 1 h and frying at 190°C.

Influence of temperature: Temperature has an effect on acrylamide formation in foods as requires temperatures³ >120°C. In addition, acrylamide synthesis is mainly limited to the surface of the heated material⁸⁷. When beef patties (commercially available as frozen hamburgers) were fried (without addition of oil) at temperatures of 180, 200, 220 and 240°C for 3 min on each side, a significant dependence of acrylamide formation on temperature was demonstrated⁸. In raw hamburgers the acrylamide content was below the detection level (5 μg kg^–1). Acrylamide was absent (below the detection limit of 10 μg kg^–1) in fresh potatoes. Acrylamide content in potatoes that were oven-dried for 72 h at 50°C was only 50 μg kg^–1 of dry weight. In contrast, at all applied frying temperatures (120-230°C) acrylamide concentrations above 1000 μg kg^–1 were produced, with considerably higher levels in the grated potatoes (max. 18000 μg kg^–1). Accordance with studies (model reaction conditions)⁸⁸. Acrylamide is formed in the last moments of a frying, roasting or baking process. With an initial temperature of 180°C, optimum French fries were obtained after 4-5 min. After 6 min, the local browning at the ends spread over the whole pieces accompanied by a steep increase in the acrylamide content from about 80-320 μg kg^–1. At 170°C the comparable optimum product was obtained after 6 min. The acrylamide content was as low as 40 μg kg^–1. The general browning and the rapid increase in acrylamide content started only 2 min later⁸³. The large variations in mean concentrations of acrylamide in deep-fried French fries purchased in restaurants or home-prepared. Acrylamide contents were found to be higher with a 10 min cooking at around 190°C compared to a 2.5-3 min cooking⁴⁵ at 170-177°C.

Influence of time: The length of time affects acrylamide formation. The highest levels of acrylamide were present after heating for 150 sec (mean 4400 μg kg^–1 of heated product) in microwave. A heating time of 100 sec resulted in ~100 time’s lower levels⁸. Another study investigated the effect of time (20, 30, 40, 50 and 60 min) on the generation of acrylamide at a heating temperature of 170±3°C and found that 30 min were optimal in terms of formed acrylamide yield. The yield of acrylamide decreased with further increase in the duration of heat treatment⁸⁹.

Influence of cooking method: The level of acrylamide was below the detection level (5 μg kg^–1) of boiled beef-potato, boiled fish as well as in broth from the boiling tests. When heating carbohydrate rich foods relatively higher contents of acrylamide 150-1000 μg kg^–1 were measured. After microwave-heating of fish and potato no detectable levels of acrylamide were found in the fish. In French fried potatoes and potato crisps exhibited relatively high levels of acrylamide (median values of 424 and 1739 μg kg^–1, respectively)⁸. Toasting of soft bread produced a moderate formation of acrylamide (increasing from 13-53 μg kg^–1). Analyses of fried potato revealed acrylamide contents at 10-100 times higher levels than in the protein-rich foods. Frying as a method of cooking dramatically affect acrylamide levels of the products as they are eaten, since acrylamide is actually formed during this last step. Simultaneously to acrylamide formation, browning, texture and flavor development caused by the Maillard reaction equally occur during frying. Therefore, acrylamide formation is correlated to color development given that both are linked to the Maillard reaction in addition to applied frying conditions (time and temperature) that have the same effect in similar manner. Intense frying conditions (time and temperature) lead to darker fries and higher acrylamide contents^90,91. On the other hand, frying at lower temperatures (below 140°C) results in increased frying time and enhances fat uptake⁸². Thus, frying time and oil temperature should be controlled in order to avoid high acrylamide levels meaning that they should not exceed 170-175°C. Lower temperatures towards the end of the process may reduce acrylamide formation. So, the product/oil ratio may influence a drop of initial frying temperature and therefore longer frying periods would be needed but these results in higher acrylamide contents. The real oil temperature towards the end of frying depends on the amount of potato added to a given amount of oil. Most acrylamide is formed in the last moments of the frying process, when the surfaces are sufficiently dried and heated. The cool potato and water evaporation extract a large amount of heat from the oil. On adding 100 g potato to 1 L of oil (10%), the temperature typically drops by 20-35° depending on the fryer⁸³. The heating immediately starts but is unable to substitute the consumed energy i.e., the regulation of the oil temperature fails in the moment that is most relevant for acrylamide formation. With 200 g L^–1 (20%) temperature usually drops from 170 to below 130°C with the effect that French fries tend to be soft and oily. The relevant temperature for acrylamide formation depends on the initial oil temperature, thermal mass of the fryer, its heating capacity, heat transfer characteristics of the oil and the amount of potato related to the amount of oil. The type of oil used for frying was investigated on acrylamide formation in a potato model system and French fries. Some studies indicated that palm oil relatively to rapeseed and sunflower oils generated higher acrylamide contents⁹². Other authors reported that the oil type did not influence acrylamide in the final product^84,93. Moreover, consideration to oil oxidation and hydrolysis products which have been proposed as possible acrylamide precursors, but they are negligible in acrylamide formation in fried potato products³³. Temperature and time of frying process have been shown to be significant factors affecting the amount of acrylamide formed in potato foods as well as the development of desirable aroma components. The use of low temperature of frying (under 180°C) reduces the concentration of acrylamide produced, but quality characteristics of the product like texture, color and oil contents are negatively affected and consequently the acceptability of the product by the consumer is deeply reduced¹⁴.

Influence of protein source: In a previous study, samples of fish fillet (cod), lean beef, lean pork, chicken fillet and soy flour (soaked in water) were heated in a fry pan for 2.5 min on each side at 220°C without addition of oil⁸. The results showed that different protein-rich foods exhibited different acrylamide concentrations ranging between 5 and 50 μg kg^–1 with lower levels in fish. The adding of different relative amounts (0-100%) of cod fish meat mixed with grated potato and then heated in a frying pan or oven revealed a decrease in acrylamide content by 70% at equal amounts of potato and fish. This reflected a protective action of protein by elimination of forming acrylamide via reaction with nucleophilic groups (-SH,-NH₂) on the amino acid side-chains⁹⁴.

Effect of pH: At pH of ~ 8, the α-amino group of asparagines is protonated to a lower degree than is the case for the other amino acids (glutamine, glycine, lysine and alanine). At relatively high pH values, asparagines are more prone to react with electrophiles and aldehyde groups in carbohydrates resulting in Schiff base formation. Which is the initial step in the formation of acrylamide. This explained the comparatively high levels of acrylamide obtained upon addition of asparagines or carbohydrates. When citric acid was added to homogenized potato (Cv. Rosor) prior to heating in the oven at 180°C for 25 min, the acrylamide content was reduced⁸⁷.

ADDITION OF NATURAL SOURCES OF ANTIOXIDANTS

Antioxidants are presented in this section in some details as in this study they are related to the section concerned with trials to reduce the acrylamide content in foods during preparation and also in the section concerned with the biological experiment.

Eggplant peels: Eggplant (Solanum melongena L.) a common vegetable grown in the subtropics and tropics is consumed throughout the world not only as a kind of tasty food but also with underlying medical value. The whole eggplant fruit possessed antioxidant activities and is ranked amongst the top 10 vegetables in terms of antioxidant capacity due to its high content of anthocyanin, which is a major phenolic in eggplant^95,96. This compound is the most important antioxidants with a variety of physiological functions such as anti-mutagenesis, anticancer and vision improvement^97,98.

Antioxidant activities and phenolic compounds are found also in both the pulp and skin of eggplant⁹⁹.

Green tea: Green tea extract has been used in various food applications such as bread, dehydrated apple products and biscuits as well as various meats products^100-103. Green tea extract effectively reduced the formation of acrylamide and achieve a maximum reduction rate of (74.3%) due to its content of flavonoids which have superior antioxidant properties largely due to the phenolic hydroxyl groups in their chemical structures^104,105. The lowest concentration of acrylamide was obtained by submerging the potatoes in green tea extract before frying, achieving a 62% reduction of the toxin. The fried potatoes treated with the green tea extract presented the lowest concentration of acrylamide (118 mg kg^–1) indicating a reduction of 62%, which was significantly different from that in the control potatoes. Also the concentration obtained with the potatoes treated with ascorbic acid which showed a reduction of 61%. These results are encouraging since there are currently no reports on the use of green tea extract to decrease acrylamide in fried potatoes. However, some publications have already mentioned the effect of green tea on the formation of the toxin¹⁰⁶. For example, the acrylamide level was a reduction in bread by adding the aqueous extract of green tea at different concentrations achieving a reduction of 72.5% with a concentration of 0.1 g kg^–1 of the tea. Using green tea in the soaking water of potato chips reduced 14, 14 and 31% of the acrylamide formation after 20, 40 and 60 min, respectively^107,108.

Onion peels: Onion skins contained significantly higher content of flavonoids than the edible portion by around 2-10 g kg^–1. Onion peel contained flavonoid levels that are 48 fold on that in its flesh and cell-based investigations found that the peel has a greater capacity for controlling lipid peroxidation than the flesh does¹⁰⁹.

Further, dry onion skin has a different configuration of quercetin derivatives compared to fleshy scales were as much as 53% of total quercetin is present in the free form¹¹⁰. The major flavonoids found in dry peel of onion that has usually been considered as waste contain large amounts of quercetin, quercetin glycoside and their oxidative product which are effective antioxidants against the lethal effect of oxidative stress^111,112. Also, they reported that they have a liver protective effect, immune enhancement potential, anti-infection, anti-stress, anti-cancer and other pharmacological properties¹¹³.

Rosemary: Rosemary, a spice with a known antioxidant property had been proposed as one effective inhibitor of acrylamide. It was shown that acrylamide contents were lowered in fried potato slices when rosemary herb was added to either the corn or olive oil¹⁰. It was found that the added of aqueous rosemary extract, rosemary oil and dried rosemary leaves to a bread model and found that the content of acrylamide was reduced by 62, 67 and 57%, respectively compared to bread without rosemary¹¹⁴. Also, the added of rosemary extract to the frying oil significantly reduced the acrylamide content by up to 38%¹¹⁵. That rosemary extract has exceptional carry-over protective effects for deep-fried food, because of its high heat stability. The effect of rosemary extract and the other antioxidants on the reduced formation of acrylamide during frying might be ascribed to different mechanisms although mainly to the radical scavenging activity that prevents hydroperoxide formation and thus secondary oxidation products formation by stabilizing radicals formed during the induction and propagation steps of lipid oxidation. Using rosemary in the soaking water of potato chips reduced 11, 48 and 24% of the acrylamide after 20, 40 and 60 min, respectively¹⁰⁸.

Sage: The main antioxidant effect of Sage has been reported to relate to the presence of phenolic diterpenes (such as carnosic acid and carnosol) and especially rosmarinic acid¹¹⁶. The addition of sage retarded lipid oxidation in Chinese-style sausage^117,118. Sage leaves can be considered a source of powerful antioxidants which can be used as a substitute for synthetic additives in food production process in order to prevent or delay oxidative deterioration of food products¹¹⁹. Sage has been reported to have excellent activities in scavenging radicals, reducing metal ions and inhibiting lipid peroxidation¹²⁰. The phenolic compounds such as carnosol, carnosic acid and rosmarinic acid in the plant may account for the antioxidant activity of sage. Some researchers have reported that sage or sage extracts can effectively retard lipid oxidation of muscle foods¹²¹. Adding sage to the soaking water of potato chips reduced 7, 14 and 36% of acrylamide after 20, 40 and 60 min soaking, respectively¹⁰⁸.

Black berry: Black berry (Morus nigra) contains large amounts of flavonoid pigments (anthocyanins) that give blackberries their characteristic red to blue color¹²². Many studies have demonstrated the antioxidant activities and health benefits of the anthocyanins occurring in various fruits and vegetables^123,124. Berry flavonoids are best known not only for their anti-oxidant and anti-inflammatory action, but the research also has shown that their action extends to impacting on cell signaling mechanisms¹²⁵. Polyphenolics included several classes of phenolic compounds that are secondary plant metabolites and an integral part of both human and animal diets. Interest in food phenolics has increased greatly because of their antioxidant capacity (free radical scavenging and metal chelating activities) and possible beneficial roles in human health such as; reducing the risk of cancer, cardiovascular disease and other pathologies¹²⁶. Acrylamide increased thiobarbituric acid reactive substances (TBARS) while decrease the activities of GSH and antioxidant enzyme (Catalase, SOD, GST and GPX) in liver, kidney, brain, lung and testes in rats while the oral intake of black berry decreased the risk of acrylamide^108,127.

Bioactive milk peptides and lactic acid bacteria: Peptides generated from the digestion of various proteins are reported to have antioxidant activities. Studies with peptides contained histidine have demonstrated that these peptides can act as metal-ion chelators, active-oxygen quencher and hydroxyl radical scavenger. The ability of protein hydrolysates to inhibit deleterious changes caused by lipid oxidation appears to be related to the nature and composition of the different peptide fractions produced, depending on the protease specificity¹²⁸. Antioxidant peptides contained of 5-18 amino acid residues. Antioxidative peptides from foods are considered safe and healthy compounds with low molecular weight, low cost, high activity and easy absorption. They have some advantages in comparison to enzymatic antioxidants, that is with simpler structure and having more stability in different situation and no hazardous immunoreactions¹²⁹. The peptides generated from the digestion of milk proteins are reported to have antioxidant activity. These peptides are composed of 5-11 amino acids including hydrophobic amino acids, proline, histidine, tyrosine or tryptophan¹³⁰. Treatment with milk bioactive peptides improved activities of antioxidant enzymes (catalase, superoxide dismutase, reduced glutathione, glutathione-S-transferase and glutathione peroxidase) in healthy and diabetic rats¹³¹. Casein hydrolysate exerted (Tyr-Phe-Tyr-Pro- Glu-Leu) scavenging activity towards the superoxide anion, hydroxyl radical and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. It is suggested that the casein-derived Glu-Leu sequence is important for this radical scavenging action thus the primary structure of the protein plays a role in determining the antioxidant activity¹³². Also, hydrolysates of WPI have been shown to possess AO activity. About 5 h digestion with alcalase produced a hydrolysate with strong reducing power (Ferric reducing antioxidant power FRAP). When fractionated on the basis of molecular mass, the low molecular weight fraction (0.1-2.8 kDa) was most potent¹³³. A corolase PP digest of β-Lg produced the most potent peptide (f19-29, Trp-Tyr-Ser-Leu-Ala-Met-Ala- Ala-Ser-Asp-Ile). Synthetic β -Lg f19-29 had a higher radical scavenging ability than BHA (2.62 μmol Trolox/μmol peptide vs. 2.43 μmol Trolox/μmol BHA). The Antioxidant (AO) activity was attributed to the presence of tryptophan (Trp), Tyr and Met residues in the peptide. The radical scavenging ability of another β -Lg peptide (f42-46, Tyr-Val- Glu-Glu-Leu) was compared to an equimolar mixture (Tyr + Val + 2(Glu) + Leu) of amino acids and the peptide was more potent (0.8μmol Trolox/μmol peptide vs. 0.4 μmol Trolox/μmol amino acid mixture). This suggested that in some instances the peptide bond or structural conformation of the peptide can enhance AO activity¹³⁴. Lactic acid bacteria convert reducing sugars in vegetables to lactic acid thus lowering pH. Previous studies have shown that lactic acid fermentation of potato reduces sugar levels amounts of Maillard products including acrylamide, consequently burnt taste and color of deep fried potato products. Several lactic acid bacteria (LAB) (e.g., Lactococcus lactis, Lactobacillus helveticus) have been reported to release bioactive peptides by the process of fermentation. Despite of the important potential of this process strategy in acrylamide reduction, its application is not very occurred. This system consisted of a number of distinct intracellular peptidases including endo-peptidases, amino-peptidases, di-peptidases and tripeptidases. From these studies it can be concluded that, bioactive peptides from dairy product could be reduce the harmful effect of acrylamide in human body¹³⁵.

EFFECT OF NATURAL ANTIOXIDANTS OF ACRYLAMIDE FORMATION

Antioxidants could inhibit acrylamide formation in high-temperature processed foods through three ways. The first one is to destruct the formed acrylamide by their oxidized products. The second one is forming the quinones or carbonyl compounds such as vitamin C which then reacts with the main precursor of acrylamide and asparagine. The inhibition effect depends on that they are easily oxidized and the rate of oxidation and their oxidized products react with asparagine. The third one is inhibiting production of carbonyl compounds produced from frying oil¹³⁶.

Previous study, examined the capacities of 15 vitamins in reducing the formation of acrylamide. Inhibitory activities of the water-soluble vitamins were tested in both chemical models containing acrylamide precursors (asparagines and glucose) and a good model system (fried snack products) while the activities of fat soluble vitamins were examined only in the latter model. Biotin, pyridoxine, pyridoxamine and L-ascorbic acid exerted a potent inhibitory effect (>50%) on acrylamide formation in the chemical model system. Using the food model, it was shown that water-soluble vitamins are good inhibitors of acrylamide formation. On the other hand, only weak inhibitory effects were observed with fat-soluble vitamins. Effects of pyridoxal, nicotinic acid and L-ascorbic acid were further examined using fried potato strips. Nicotinic acid and pyridoxal inhibited acrylamide formation in fried potato strips by 51 and 34%, respectively. Thus, certain vitamins at reasonable concentrations can inhibit the formation of acrylamide⁸⁸.

Also, a similar phenomenon occurs in ascorbic acid cases at low concentrations (0.2, 0.5, 0.8 and 1.2%). Ascorbic acid inhibited acrylamide formation with a highest inhibitory rate of 58%, but the increase of ascorbic acid concentration to 1.5% promoted acrylamide formation¹³⁷ by more than 90%. Such findings suggested that the effect of an antioxidant towards acrylamide is not only determined by the type of the antioxidants but also its concentration, which makes the case more complicated. But, this may partly explain the different behaviors of the same antioxidant among studies, since the applied concentrations were mostly different. Acrylamide level was significant reduction following the addition of caffeic and ferulic acid¹³⁸. On the same side, several antioxidant compounds, including tert-butylhydroquinone (TBHQ), butylated hydroxyl anisole (BHA), butylated hydroxytoluene (BHT), ferulic acid and vitamin C showed a reduction or elimination of acrylamide¹³⁶. Also using of eggplant peel in the soaking water of potato chips before frying reduced 24, 46 and 28% of the acrylamide after soaking for 20, 40 and 60 min, respectively¹⁰⁸.