**3. Prevention and mitigation of acrylamide**

staple food in Southern African and gets burnt during its preparation, there are indications of

The neurotoxicity of ACR in humans is well known from occupational and accidental exposures [79]. Owing to its low molecular weight and polarity, ACR is readily distributed and incorporated in mammals [80]. After ingestion, ACR is rapidly circulated throughout the whole body via the bloodstream [81] and can be found in the liver, kidney, thymus, brain, heart and human breast milk [82]. The conjugation of ACR to glutathione, and its epoxidation to glycidamide in the liver via cytochrome P450, is one of the major metabolic routes [83]. The formation of glycidamide is considered to be the critical step for the toxic effects of ACR and its metabolites. ACR and glycidamide, the latter at a much higher rate, can react with macromolecules such as haemoglobin and enzymes [80]. According to the European Food Safety Authority, ACR and its metabolite glycidamide have shown evidences of genotoxicity (DNA damage) and carcinogenicity [44]. Although evidence from studies on human exposure and possible causes of cancer is currently limited, epidemiological studies designed to target different populations and different organs in relation to cancer risks have been presented, with

Calleman [85] reported peripheral neuropathy symptoms of highly exposed workers in China. Characterized by numbness of hands and feet, ataxia and skeletal muscle weakness, ACR has been shown to be toxic to both the central and peripheral nervous system [86]. ACR induces nerve terminal degeneration [79] and has deleterious effects on the thalamus, hippocampus and cerebral cortex [79, 86]. A recent study demonstrated evidence of ACR neurotoxic effects of fried potato chips on rat postnatal development, causing cerebellar cortical defects and myodegeneration of the gastrocnemius muscle during the postnatal development of pups [87]. It has been postulated that neurotoxicity of ACR might be cumulative as the same neurotoxic effects can be seen at low and high doses of ACR with the low doses requiring longer exposure [86, 88]. In 1994, ACR was classified by the International Agency for Research on Cancer (IARC) as Group 2A, indicating that it is probably carcinogen to humans (Group 2A) [89, 90]. This was based on positive bioassay results in rodents, buttressed by evidence that ACR is transformed in mammalian tissues to a more reactive genotoxic metabolite (glycidamide) [8]. Evidence on experimental rodents indicates that ACR causes tumours in the skin, uterus, lungs, brain, thyroid and mammary gland [91]. The genotoxicity of ACR and glycidamide is also manifested as both clastogenicity and mutagenicity. ACR has proven to be genotoxic in vivo to the somatic and germ cells as well as to cell cultures [8] and mammalian cells [81]. As indicated by Rice [91], the oxidation of this contaminant to glycidamide is the prerequisite for genotoxicity of ACR. This is attributed to the higher reactivity of this metabolite (glycidamide) to form

For cancer-related effects, the margin of exposures (MOEs) of ACR have been estimated to range from 50 for high-consuming toddlers to 425 for average adult consumers. These numbers indicate concerns for public health [44]. Essentially, since any level of exposure to a genotoxic substance could possibly cause DNA damage and lead to cancer, no tolerable daily

a huge risk of ACR exposure to millions of individuals consuming this product daily.

**2.4. Toxicity of acrylamide**

160 Acrylic Polymers in Healthcare

adducts with DNA [83].

absolutely none reported from the African continent [84].

Agencies such as the FAO and WHO in collaboration with the academia and food industry have put forth strategies for reducing levels of ACR in food. In Europe, food manufacturers have collaborated with researchers and the academia through the Confederation of the Food and Drink Industries of the EU (CIAA) to produce series of strategies called the 'CIAA Acrylamide Toolbox' for decreasing ACR levels in different foods [93]. However, it should be noted that designing mitigation strategies is quite challenging, considering the fact that precautions must be taken to avoid compromising the nutritional, chemical, physical and microbiological quality and safety of the food. Accordingly, such measures must not result into the formation of other process contaminants nor detrimentally affect the organoleptic properties and acceptability of the final product [20, 94].

#### **3.1. Methods that interrupt reactions leading to acrylamide formation**

Several approaches have been successful at preventing ACR formation by preventing the key reactions responsible for generating it. Lowering the pH of foods blocks the nucleophilic addition of asparagine with a carbonyl compound, preventing the formation of the Schiff base, a critical intermediate in the formation of ACR [95, 96]. While this approach could be successful in lowering ACR levels in fried potato products, it may bring about undesirable taste to foods [21]. The use of organic acids and the addition of mono- and divalent cations (Na+ or Ca2+) to foods are other approaches of mitigating ACR by preventing the Schiff base formation [95–97]. The addition of proteins or free amino acids other than asparagine has also been investigated as a strategy for reducing ACR formation by causing competitive reactions and/or covalently binding ACR via Michael addition reactions [95, 96]. These additions however had low-to-moderate success at decreasing ACR levels in both cereal-based and potato foods [98].

#### **3.2. Treatments that reduce acrylamide precursor's levels**

As asparagine and reducing sugars are the major ACR precursors in foods, eliminating either of these substrates is a viable way to reduce ACR formation [94]. Procedures for achieving this include rinsing and blanching treatments, using asparaginase, fermentation and controlling storage conditions [21].

Rinsing, blanching and soaking treatments have been effective at reducing ACR formation in potato products [4, 21]. Soaking potato slices in water before frying resulted in over 50% reduction in ACR [21]. Further experiments by blanching slices in warm or hot water removed more glucose and asparagine than ordinary water immersion [4]. Changing the design of frying units to reverse the flow direction of the heated oil may alter the thermal load, which will reduce ACR levels in finished products [99]. Blanching and soaking treatments reduce ACR formation by leaching out asparagine and sugars from the surface of the slices [4]. Using asparaginase, an enzyme which hydrolyzes asparagine into aspartic acid and ammonia, has successfully reduced ACR levels in potato and bakery products [4, 20]. Asparaginase treatment of gingerbread dough resulted in a 75% decrease in free asparagine and a 55% reduction in ACR levels in the baked products [20]. To this effect, two commercial asparaginase preparations have been developed and are available in the market: Acrylaway® (Novozymes, Denmark) and PreventAse™ (DSM Food Specialties, Denmark), respectively, synthesized from *Aspergillus oryzae* and *Aspergillus niger*. They are generally recognized as safe (GRAS) ingredients [100].

Likewise, fermentation with yeast has been identified as a way to reduce ACR through the elimination of free asparagine [28]. A 2 h fermentation of rye and whole wheat dough caused a 77 and 87% reduction in ACR levels in rye and grain breads, respectively [101]. Yeast fermentation was observed to be more effective than sourdough fermentation in reducing the asparagine content of the dough [101]. Ingredients and additives may also increase ACR formation during baking of cereal-based products. In a study by Amrein et al. [20], baking agent and ammonium bicarbonate reportedly improved ACR formation in bakery products, possibly by creating more reactive carbonyl compounds. Using an alternative baking agent (sodium hydrogen carbonate), sucrose rather than honey or inverted sugar syrup can also reduce ACR content by more than 60% [20].

#### **3.3. Modifying processing/cooking conditions**

A reduction in cooking temperatures and times can decrease ACR levels in foods. However, loss of desirable colour, flavour and texture may occur, since the Maillard reaction which is responsible for ACR formation also guarantees desirable flavour and colour compounds in heated food [28]. Conditions that minimize ACR in French fries involve optimizing frying or baking processes to obtain a surface golden in colour and crispy texture [21]. Blanching, soaking, parboiling and washing treatments may be adopted, as these can leach the reducing sugar/asparagine reactants before the subsequent cooking step [102]. Overall, prolonged baking/frying and excessive browning should be avoided to minimize ACR formation in baked and fried products. Since a linear relationship exists between ACR formation and baking process, there is a need to ensure proper and optimum cooking endpoint to minimize ACR formation. This suggests that the degree of surface browning could be used as a visual indicator of ACR formation during cooking.

#### **3.4. Agronomic factors**

Selective crop propagation is a potential strategy for controlling ACR levels by decreasing levels of ACR precursors [94]. Since the first occurrence of ACR in foods, several researchers have demonstrated the significance of variety and cultivar selection on the formation of ACR [103]. Amrein et al. [29] found a 50-fold variation in total reducing sugars in the different potato cultivars the authors studied. Cultivars with low reducing sugars were more suitable for potato products, cooked or processed at high temperatures [29]. Konings et al. [28] established the significant impact of fertilizer application rate on ACR levels mainly due to differences in crude protein and asparagine and contents. A study by Claus et al. [38] demonstrated the effect of nitrogen-based fertilizers in causing high amounts of protein and amino acid. This resulted in increased ACR levels in breads, ranging from 10.6 to 55.6 μg/kg [38]. Producers should best adopt effective fertilizer application regimes that will subsequently yield suitable produce for processing, as this influence the levels of reducing sugars [29]. Tubers should be harvested at full maturity as selection of immature tubers for further processing increases the chances of ACR occurrence, because they have relatively higher reducing sugars and produce products with potentially higher ACR levels [104, 105]. Unfortunately, most African subsistence farmers harvest immature tubers to immediately obtain income for their needs. While this should be discouraged, effective handling, packaging and storage of produce must also be emphasized. Selection and use of crop varieties that are low in ACR precursors will most definitely help reduce ACR occurrence. Storage is also another component and practicable way of mitigating ACR in foods. While storage of potatoes at low temperatures is generally meant to minimize shrinkage and spoilage, studies have shown that low temperatures tend to increase sugar levels (an ACR precursor) [106]. Though for a short-term storage, hot temperature is desirable (this can however lead to sprouting, which can be controlled using suppressants); for long term, a minimum storage temperature of 6°C is desirable [97].

#### **3.5. Antioxidants and other phytochemicals**

Rinsing, blanching and soaking treatments have been effective at reducing ACR formation in potato products [4, 21]. Soaking potato slices in water before frying resulted in over 50% reduction in ACR [21]. Further experiments by blanching slices in warm or hot water removed more glucose and asparagine than ordinary water immersion [4]. Changing the design of frying units to reverse the flow direction of the heated oil may alter the thermal load, which will reduce ACR levels in finished products [99]. Blanching and soaking treatments reduce ACR formation by leaching out asparagine and sugars from the surface of the slices [4]. Using asparaginase, an enzyme which hydrolyzes asparagine into aspartic acid and ammonia, has successfully reduced ACR levels in potato and bakery products [4, 20]. Asparaginase treatment of gingerbread dough resulted in a 75% decrease in free asparagine and a 55% reduction in ACR levels in the baked products [20]. To this effect, two commercial asparaginase preparations have been developed and are available in the market: Acrylaway® (Novozymes, Denmark) and PreventAse™ (DSM Food Specialties, Denmark), respectively, synthesized from *Aspergillus oryzae* and *Aspergillus niger*. They are generally recognized as safe (GRAS) ingredients [100].

Likewise, fermentation with yeast has been identified as a way to reduce ACR through the elimination of free asparagine [28]. A 2 h fermentation of rye and whole wheat dough caused a 77 and 87% reduction in ACR levels in rye and grain breads, respectively [101]. Yeast fermentation was observed to be more effective than sourdough fermentation in reducing the asparagine content of the dough [101]. Ingredients and additives may also increase ACR formation during baking of cereal-based products. In a study by Amrein et al. [20], baking agent and ammonium bicarbonate reportedly improved ACR formation in bakery products, possibly by creating more reactive carbonyl compounds. Using an alternative baking agent (sodium hydrogen carbonate), sucrose rather than honey or inverted sugar syrup can also

A reduction in cooking temperatures and times can decrease ACR levels in foods. However, loss of desirable colour, flavour and texture may occur, since the Maillard reaction which is responsible for ACR formation also guarantees desirable flavour and colour compounds in heated food [28]. Conditions that minimize ACR in French fries involve optimizing frying or baking processes to obtain a surface golden in colour and crispy texture [21]. Blanching, soaking, parboiling and washing treatments may be adopted, as these can leach the reducing sugar/asparagine reactants before the subsequent cooking step [102]. Overall, prolonged baking/frying and excessive browning should be avoided to minimize ACR formation in baked and fried products. Since a linear relationship exists between ACR formation and baking process, there is a need to ensure proper and optimum cooking endpoint to minimize ACR formation. This suggests that the degree of surface browning could be used as a visual indicator

Selective crop propagation is a potential strategy for controlling ACR levels by decreasing levels of ACR precursors [94]. Since the first occurrence of ACR in foods, several researchers

reduce ACR content by more than 60% [20].

of ACR formation during cooking.

**3.4. Agronomic factors**

162 Acrylic Polymers in Healthcare

**3.3. Modifying processing/cooking conditions**

According to Kahkeshani et al. [107], the correlation between antioxidants and ACR can be considered from two different points of view, namely, antioxidants as exogenous additives and as endogenous secondary metabolites. According to these authors, lack of sufficient studies and discord in the results available from literature hinders a logical judgement about the effectiveness of phytochemicals against ACR. While some reports have reported their beneficial effect, some have been shown to facilitate ACR production [107]. While these compounds can react with asparagine to produce ACR, they could also possibly react with the amide group of the intermediates in Maillard reaction and block ACR formation [107–110]. The oxidation of polyphenols to corresponding quinones, which can react with 3-animopropionamide (3-APA), thus preventing the deamination of 3-APA to ACR, has also been proposed as a mechanism for ACR reduction by these compounds [111]. Nonetheless, studies demonstrating the effectiveness of these compounds towards the reduction of ACR have been presented in the literature. Fernandez et al. [112] recorded a 50% ACR reduction after the addition of a flavonoid spice mix to potato chips. Zhang and Zhang [108] reported a 76% reduction in ACR after French fries were dipped into extracts of bamboo leaves with antioxidant properties, while a 59% decrease in ACR was recorded when fried chicken wings were dipped into same extracts [111]. Further reports in the literature on this have been adequately reviewed by Kahkeshani et al. [107] and can be consulted for further reading. Interestingly, Africa is the home to a vast and diverse number of plants and other botanicals with rich phytochemicals. Extracts of these plants and herbs are used for various purposes prominently in traditional medicines. Application of such extracts would go a long way in the mitigation of ACR occurrence in foods.

#### **3.6. Genetic modification**

As defined by Key et al. [113], genetically modified crops/plants are those that have been genetically altered through the use of recombinant DNA technology. This may be to express a gene not native to the plants or to modify endogenous ones [113]. This issue has in the recent years attracted worldwide attention especially regarding its risk to the environment and human health. While it is widely accepted in many parts of the world, including parts of America and Asia, it still remains a controversial issue in Europe [35]. Most African continents follow the latter, as concerns regarding the immediate and long-term effects of genetically modified crops are major hindrances to adopting this technology. Although different authors, government agencies and international organizations have backed and supported genetic modification, there is still a stiff opposition against its acceptance. Inconsistencies in free sugar, amino acid and asparagine contents in crops of different cultivars (varieties) and genotypes however suggest that the varying concentration of these parameters is due to genetic variations [19, 29, 38, 114]. Consequently, fast tracking the natural breeding process through the use of genetic engineering to develop cultivars (varieties) with lower concentration of asparagine and reducing sugars should be possible and encouraged [35]. It has been shown that simultaneous silencing of the genes (StAst1 and StAst2) that encode for asparagine synthetase which is the enzyme that catalyse the formation of asparagine in potato, significantly reduced the levels of asparagine in the transgenic crop [115]. Another study demonstrated reduction in the concentration of reducing sugars, which also participate in reaction leading to ACR formation. For instance, since reducing sugar is accumulated during the cold storage of potato (cold-induced sweetening), silencing of the enzyme acid invertase resulted in potato with reduced concentration of fructose and glucose as well as low ACR concentration when processed into French fries [116]. While genetic modification continues to be controversial, the farmers, the food industry and other vital stakeholders should be proactive in the development of crop varieties that would yield lower ACR levels in food.
