Food Additives, Toxicology And Safety Lecture Notes PDF

FOOD ADDITIVES, TOXICOLOGY AND SAFETY FST 507 / Food Science & Technology PROF. F. O. HENSHAW and Dr. Celestina OMOHIMI 1 Naturally Occurring Toxins 2 Lecture One...

FOOD ADDITIVES, TOXICOLOGY AND SAFETY FST 507 / Food Science & Technology PROF. F. O. HENSHAW and Dr. Celestina OMOHIMI 1 Naturally Occurring Toxins 2 Lecture One 3 ❖ Natural toxins are naturally produced by wide variety of plants. May be low-molecular-weight endogenous toxins or products of secondary metabolism. Are usually metabolites produced by plants for defense against various threats: bacteria, fungi, insects and predators. Are not harmful to the organisms themselves. Inappropriate/inadequate processed food can be potentially harmful to human health, causing food poisoning. May be growth inhibitors, neurotoxins, carcinogens, and teratogens 4 ❖ They are found in different crop plants and in different parts of a plant. ❖Parts such as: foliage, buds, stems, roots, fruits and tubers ❖ Common examples: (i) Glycoalkaloids in potatoes (ii) Glucosinolates (iii) Cyanogenic glucosides in bamboo shoots, bitter apricot seed, cassava root (iv) lectins in green beans, red kidney beans, white kidney beans (v) Muscarine in Mushrooms 5  Glycoalkaloids Are secondary metabolites produced in the leaves, flowers, roots, and edible parts (sprouts and skin) of the plants of Solanaceae family All Solanacea plants; tomatoes, potatoes and eggplant - contain glycoalkaloids Are bitter components of plants Most commonly found glycoalkaloids in plants; α-solanine and α- chaconine α-solanine is the more toxic of the two In potatoes, the major glycoalkaloids are α-solanine and α-chaconine. In tomato - tomatine 6 ❑ Generally present in low levels but higher concentration in potato sprouts, bitter-tasting peel and green parts, also in green tomatoes ❑ The toxins are produced in response to stresses like bruising, UV light, microorganisms, and attacks from insect pests and herbivores. Cooked potatoes that contain high level of solanine have a bitter taste and cause a burning sensation in the throat Washing, soaking or cooking (baking, boiling, frying, microwaving) does not significantly reduce the toxin level 7 Sprouted root Greened root Bruised and insect infested8  Toxicity Amount of glycoalkaloids usually found in edible plants that are fresh and undamaged do not normally cause toxicity. Toxic effects can be produce at higher doses Acute Toxicity Ingestion of greened, damaged or sprouted potatoes as a consequence of high levels of glycoalkaloids Acute toxicity syndromes - levels of more than 2.8 mg/kg body weight Onset of symptoms – from minutes to 2 days after ingestion 9 Symptoms – acute gastrointestinal upset with diarrhoea, vomiting and severe abdominal pain. In more severe cases - neurological symptoms (drowsiness and apathy, confusion, weakness, and vision disturbances, unconsciousness, and in some cases death) Chronic Toxicity No-observed adverse effect level (NOAEL) has not been assessed, and a tolerable daily intake (TDI) for humans has not been determined – due to lack of chronic toxicity data JECFA intake of 3-6 mg/kg body weight (bw) considered a potentially lethal dose for humans 10  Cyanogenic Glucosides They are amino-acid-derived constituents of plants produced as secondary metabolites. Are chemical compounds in foods that release hydrogen cyanide (HCN) when chewed or digested Occur in at least 2000 plant species Approximately 25 cyanogenic glycosides are known Found in the edible parts of plants - apples, apricots, cherries, peaches, plums, quinces, seed of such fruits. Almonds, stone fruit, pome fruit, cassava, sorghum, bamboo roots, linseed/flaxseed, lima beans, coco yam, chick peas, cashews 11  Different for different plants e.g. taxiphyllin in bamboo shoots, linamarin in cassava Linamarin and lotaustralin - the two most commonly found Level of cyanogenic glycosides - dependent on the age, variety of the plant, and environmental factors Function dependent on activation by beta-glucosidases (hydrolytic enzyme) to release: (1) toxic volatile HCN (2) ketones or aldehydes to fend off herbivore and pathogen attack 12 Toxicity Dependent on the release of HCN on ingestion Becomes toxic due to its ability to link with metals (e.g. Fe++ and Mn++) Intoxication – dependent on body weight Acute Toxicity Clinical signs in human - rapid respiration, drop in blood pressure, rapid pulse, dizziness, headache, stomach pains, vomiting, diarrhoea, mental confusion, stupor, cyanosis with twitching and convulsions followed by terminal coma Death occurs – cyanide level exceeds limit individual is able to detoxify Acute lethal dose - 0.5-3.5 mg/kg bw World Health Organization (WHO) – Safe level for cassava flour is 10 ppm 13 Chronic Toxicity Uncommon to cause chronic diseases However, individuals with underlying dietary deficiencies – Inadequate protein Inadequate iodine intake (goiter and cretinism) Neurological diseases such as: Konzo - an upper motor neuron disease characterised by irreversible but nonprogressive symmetric spastic paraparesis that has an abrupt onset Tropical ataxic neuropathy (TAN) - sore tongue, optical atrophy, neuro sensory deafness, and sensory gait ataxia 14  Glucosinolates (GLS) ❖They are Secondary plant metabolites of vegetables Are modified amino acids, carrying an S-glucose functional group and a variety of different side chains ❖Are peculiar of vegetables belonging to Brassicaceae family (rapeseed, cabbage, cauliflower, Brussels sprouts, turnip, broccoli and Chinese cabbage) Also present in few other species (capers, papaya and moringa) ❖Types and Concentration in food are highly variable, These are dependent on factors: genetics, cultivation site, cultivar, growth conditions, developmental stage, plant tissue, post-harvest handling, and food preparation methods 15 ❑ More than 120 different GLSs have been identified – few are present in plants in high quantity ❑ Are chemically stable and biologically inactive Tissue damage through pests, harvesting, food processing or chewing initiates contact with the endogenous enzyme Myrosinase ❑ Interaction between GLS and myrosinase – induced by chewing, tissue damage during freezing, thawing, and chopping of edible plants A complex variety of breakdown products are formed Breakdown products - nitriles, isothiocyanates (ITCs), thiocyanates, epithionitriles, indole and vinyl oxazolidinethiones. Isothiocyanates (ITCs) is the most important 16 ▪ GLS enzymatic degradation – development of pungent flavour and bitter taste of raw edible plant Flavour and taste intensity – dependent on GLC concentration and degree of hydrolysis ❖ Role of breakdown products 1. Act both as poisons and deterrents to generalist insects/herbivores 2. Attract specialist insects/herbivores laying their eggs or feeding on that specific plant 17 Effect on Human Health 1. Increase in goitre (enlargement of the thyroid gland) - thiocyanates and oxazolidinethiones Interfere with iodine uptake (thiocyanates) and the synthesis of the thyroid hormones T3 and T4 (oxazolidinethiones) 2. Subsequent reduction in the fertility of male and female animals 3. Irritation of the gastro-intestinal mucosa followed by local necroses, and hepatotoxicity 18 Clinical Signs of GLS growth retardation, reduction in performance (milk and egg production), impaired reproductive activity, and impairment of liver and kidney functions (attributed to nitriles) Calculations of dietary exposure are limited - due to shortage of reliable data 19 SAFETY ASSESSMENTS Normal dietary levels of intake are not known Safety in the human diet has not been assessed Cooking by boiling will reduce exposure through leaching of GLS into water However, steaming, microwaving or stir-frying - insignificant decrease in concentrations No safety limits have been set for glucosinolates 20 Lecture Two 21 ANTI NUTRITIONAL FACTORS (ANF) ❑ Secondary metabolites that serve as Protection against attack by herbivores, insects, and pathogens ❑ Are generated by natural metabolism through different mechanisms ❑ Compounds which reduce maximum utilization of nutrients ▪ Are frequently related to plant-based, raw or vegan diets 22  Present in different food substances in varying amounts – depending on: ✓Type of food ✓Mode of propagation ✓Chemicals used in growing the crop ✓Chemicals used in storage and preservation 23  Induce undesirable effects in humans if their consumption exceeds an upper limit Some ANF may possess beneficial health effects if present in small amounts. ❖Anti-nutritional factors must be inactivated or removed Classification of ANF ❖Heat-stable - resist and can be maintained at high temperature e.g. Phytic acid, Condensed Tannins, Alkaloids, Saponins ❖Heat labile - are sensitive to standard temperature and lost at high temperature e.g. lectins, Cynogenic Glycosides, Protease inhibitors and Toxic amino acids 24 Classification of Anti-nutrients in Plants Proteins Protease inhibitors Trypsin inhibitors Chemotrypsin Hemagglutinins Food allergens Toxic amino acids Glycosides Saponins Cyanogens Estrogens Goitrogens Phenols Gossypol Tannins Others Anti-minerals Phytic acid Oxalates Anti-vitamins Anti- enzymes 25 Adverse Effects of Some Anti-nutrients Anti-nutrients Effects on body Phytic acids (Phytates) i. Forms complexes with metal ions and inhibit their absorption ii. Reduce minerals (Ca & Fe) bioavailability iii. A key component of crops that cause zinc deficiency Oxalates Reduce Ca absorption, encourage kidney stone formation Cyanide Respiratory inhibitors Lectins (Hemagglutinins) Prevent absorption of digestive end products in the small intestine. Protease inhibitors Substances reduce protein digestion. Phenol Compounds They reduce bioavailability of some minerals (especially zinc). They may negatively affect pH mechanism, Reduce protein digestion. Enzyme inhibitors Retardation of growth Reduce protein digestibility Saponins i. High concentrations cause many health problems such as growth impairment ii. Inhibit digestive and metabolic enzymes 26 iii. Reduces the absorption of Vits A and E, and lipids 1. Saponins Non-volatile, soap-forming surface active secondary metabolites Found principally in plants Consist of combination of polar and non-polar structural element – reason for their soap-like behaviour in aqueous solutions General characteristics: (i) give a bitter taste, (ii) foam when treated with various solutions, and (iii) cause haemolysis (breakdown) in red blood cells 27 ❖ Their ability to reduce the surface tension of blood in cold-blooded animals makes them extremely toxic ❖In high concentration, they impart bitter taste and astringency in dietary plants ❖ Bitter taste is the major factor that limits its use Saponins are steroids or triterpenes : i. Triterpenoid saponins - usually found in legumes (soybean, peanuts, chickpeas, broad beans, and lentils), sunflower seeds, spinach leaves, tea leaves, quinoa seeds, sugar beet i. Steroid saponins - present in oats, yucca, tomato seeds, fenugreek seeds, asparagus, aubergine and yam 28  Are not readily hydrolyzed by the human digestive enzymes, therefore gastrointestinal digestion can be severely impaired Adverse health effects of Saponins decreased liver cholesterol and overall growth rate reduce intestinal absorption or bioavailability of nutrients through binding to the small intestine cells decrease enzymes’ (trypsin and chymotrypsin) activity affects protein digestibility by inhibiting various digestive enzymes such as amylase, glucosidase, trypsin, chymotrypsin and lipase, which can cause indigestion-related health disorders Reduce absorption Vitamins ( e.g., A and E) 29  Note: Low levels of saponins in legumes may not be injurious to health but could become toxic when consumed at higher concentrations in the diet. Beneficial Effects possess hypocholesterolemic, immunostimulatory and anticarcinogenic properties reduce the risk of heart diseases in humans Cholesterol-reducing effect: bind with cholesterol and reduce its absorption preventing peptic ulcer, Osteoporosis 30 2. Phytates (or Phytic acids) Phytates are the salt form of phytic acids Occur naturally in plant kingdom Present in foods at various levels ranging from 0.1% to 6.0% It is a secondary compound, concentrated naturally in plant-based seeds - legumes, peanuts, cereals and oilseeds ❖ Seeds, grains, nuts and legumes store phosphorus as phytic acid in their husks in the form of phytin or phytate salt In Monocotyledons crops – phytates are present in the bran or aleurone layer and can be easily separated during milling 31  In Dicotyledons (legumes, oilseeds and nuts) - phytates are found in close association with proteins, hence, separation is difficult through simple processing method like milling ❖ Adverse health effects of Phytates 1. Hinders the activity of enzymes necessary for protein degradation in the small intestine and stomach 2. Causes the bioavailability of essential minerals to decrease and turn into insoluble compounds whose absorption and digestion is less in the small intestine 32 ❖Are generally a negatively-charged structure that binds with positively-charged metal ions (zinc, iron, magnesium and calcium) – Chelating property ❖This makes it to be the most effective anti-nutrient in foods. It chelate metal ions (calcium, magnesium, zinc, copper, iron and molybdenum) to form insoluble complexes Hence, prevents their absorption ❖Greatest effect of phytic acid on human nutrition is its reduction of zinc bioavailability ❖Has strong effect on infants, pregnant and lactating women, when consumed in large portion 33 Ways to Reduce Phytates: ❑Among the cooking treatments boiling appeared effective to reduce the phytate level (as high as 20% reduction) ❑During germination of seeds, some native enzymes are activated, which degrade phytic acid. Health Benefits ❖ It lowers the blood glucose response by reducing the rate of starch digestion and slowing gastric emptying ❖Regulate Insulin secretion ❖Reduces blood clots, Cholesterol, and Triglycerides - hence prevents Heart diseases ❖Used as a complexing agent for removal of traces of heavy metal ions 34  3. Tannins Are secondary compounds, which are formed in plant leaves, fruits and bark Are water soluble Phenolic compounds, which consist of molecular weights greater than 500Da Are formed in plants leaves, fruits and bark Are astringent bitter plant polyphenolic compound Are heat stable ❖Largely concentrated in beverages, pomegranate, berry fruits and cocoa bean, also found in cereals such (sorghum and barley) They accumulate mainly in the bran of legumes. ❖One of their properties: binds or precipitation of protein by forming reversible and irreversible tannin-protein complexes 35 Types of Tannins 1. Hydrolyzable (e.g. gallotannins and ellagitannins) - Leguminous forage and some seeds 2. Condensed (e.g. proanthocyanidins) – Peanut, millets, sorghum, faba bean, lima bean, sunflower seed meal ❖The two types differ in their nutritional and toxic effects ✓Condensed tannins – have a more profound digestibility-reducing effect ✓Hydrolyzable tannins – may cause varied toxic manifestations due to hydrolysis in rumen 36 ❖Toxicity level depends on: (i) Chemical structure (ii) Dosage Health Effects 1. Cause inactivation of many digestive enzymes (trypsin, chemotrypsin, amylase and lipase) 2. Decrease protein digestibility, leading to reduction of essential amino acids How??? ; By forming reversible and irreversible tannin-protein complexes between the hydroxyl group of tannins and the carbonyl group of proteins Note: Proteins which form complexes with tannins are relatively large and hydrophobic in nature 37 3. Increased excretion of endogenous protein (fecal nitrogen) 4. Interfere with dietary iron absorption 5. Form complex with Vit. B 6. Digestive tract malfunction 7. Toxicity of absorbed tannin or its metabolites 38  4. Oxalates Oxalates are soluble (potassium and sodium) or insoluble (calcium, magnesium, iron) salts or esters formed from oxalic acids Foods with high amount – Cruciferous/Leafy vegetables (cauliflower, broccoli, kale), spinach, parsley, beets, black pepper, chocolate, nuts, berries (Blue, black) and beans Can also be synthesised in the body Calcium oxalate is widely distributed in plants React with proteins to form complexes 39 ❖Health Effects: 1. When consume in large doses, can cause severe irritating of the lining of the guts; can prove fatal in large doses 2. Calcium oxalate adversely affects Ca absorption - rendering calcium unavailable for normal physiological and biochemical role (maintenance of strong bone, teeth, blood clotting process) Insoluble calcium oxalate precipitate in the Kidneys or Urinary tract – leading to formation of kidney stones 3. Reacts with protein to form complexes that has inhibitory effect in peptic digestion. 40 Ways to Reduce Oxalates ✓ Soaking and cooking foods high in oxalate will reduce soluble oxalate content by leaching, but not the insoluble fraction ✓Boiling affects the highest reduction (water has to be discarded) 41  5. Enzyme inhibitors i. Protease inhibitors Protease: enzymes cell-process-regulation enzymes Found in all cells and tissues Protease inhibitors: protein-based substances widely distributed within the plant kingdom Commonly present in raw cereals and legumes - Legumes contain high amounts (soybean), lower in cereals 42  Limit enzyme activity by binding to their target proteins reversibly or irreversibly - forming protein–protein interactions - by blocking the active site of the enzymes Inhibit the activity of proteolytic enzymes within the GIT of animals - Reducing protein digestion Examples of Protease Inhibitors 1. Serpins ✓molecular weight of 39 to 43 kDa ✓inhibit trypsin and chymotrypsin activities 43 2. Trypsin inhibitors ✓commonly found in soybean ✓Formation of irreversible condition known as enzyme-trypsin inhibitor complex ✓causes a drop in intestine trypsin and a decrease in protein digestibility ✓leading to slower animal growth - by reducing protein digestion and availability of amino acids ✓Not inactivated by heat treatment ❖ Health Effects of Protease Inhibitors (i) Poor food utilization, leading to Growth inhibition (ii) High levels of protease inhibitors lead to increased secretion of digestive enzymes by the pancreas 44 ii. Alpha-Amylase inhibitors α-amylase: regulates the breakdown of carbohydrate - polysaccharides into oligosaccharides Amylase inhibitors are found in almost all cereals and legume (pigeon pea) - based foods Known as starch blockers - prevent dietary starches from being absorbed by the body By bind to alpha amylases, making them inactive Active over a pH range of 4.5-9.5 Are heat labile 45 Health Effects: i. Decreased growth rate Beneficial effects: Have hypoglycemic effect – used as starch blocker tablet How??: Increase carbohydrate absorption time by delaying carbohydrate digestion Hence, used to control human diabetes (type II) Reduce dental caries 46  6. Anti-vitamin factors Exhibit anti-vitamin activity 1) Anti-vitamin A factor: present in soybeans - destroys carotene, not readily destroyed by heat 2) Anti-vitamin D factor: present in soybeans, interferes with calcium and phosphorus absorption destroyed by autoclaving 3) Anti-vitamin E factor: present in kidney beans, soybeans, alfalfa and field pea, causes liver necrosis and muscular dystrophy, destroyed by autoclaving, 47 4) Anti-vitamin K factor: present in sweet clover 5) Anti-thiamine factor (thiaminase): present in cottonseed, linseed, mung bean, and mustard seed 6) Antiniacin factor: present: in sorghum 7) Anti-pyridoxine factor: present in linseed, destroyed by water extraction and autoclaving 8) Anti-vitamin B 12 factor: present in raw soybeans 48 7. Contextual Antinutrients ❑ Some supplements or foods rich in certain nutrients can create reactions of an antinutrient nature: 1. Calcium-rich foods can impede iron absorption 2. Mutual antagonism between zinc and copper during the absorption process 3. Interference of some foods with medication absorption: food-drug interaction ❖High intakes of wines and peanut (resveratrol) could increase the risk of bleeding when consumed with anticoagulant drugs ❖Vitamin K-rich foods (e.g. broccoli, spinach) and Coumadin, an anticoagulant prescribed to thin the blood and prevent clots 49  DISABLING ANTINUTRIENTS Removing undesirable food components is essential to their quality improvement 1. Milling ✓Traditional method to separate the bran layer from the grains ✓Removes anti-nutrients present in the bran of grains - phytic acid, lectins, tannins, oxalates ✓Disadvantage - removes important minerals 50 2. Soaking One of the easiest physical processes to remove soluble antinutritional factors through leaching Research Findings: 6 h soaking reduced 27.9% and 24 h of soaking reduced 36.0% of phytic acid at room temperature Soaking in distilled water, 1% NaHCO3 and mixed salt solutions - total phenols, tannins and phytates reduction Soaking and sprouting grains, nuts, seeds and beans - excellent to deactivate enzyme inhibitors ▪ lectin is not affected by this method of deactivation 51 3. Fermentation Fermentation at varying temperature and time has shown significant reduction in ANF Provides optimum pH conditions for enzymatic degradation of ANF Research Findings: Protease inhibitors, phytic acids and tannins were reduced due to millet grain fermentation for 12 h and 24 h Fermentation by microorganisms significantly reduced antinutrients, as compared with spontaneous fermentation process. 52 4. Sprouting/Germination ✓ One of the most effective methods for antinutrients deactivation ✓It activates the enzyme phytase which degrade phytate, and hence the reduction of phytic acids concentration in food samples ✓Most frequently used for antinutrients reduction in Cereals Reduction of anti-nutrients (like tannin and phytic acid) in germinated cereals increases the bioavailability of several minerals 53 4. Heating: Autoclave and cooking Autoclave decreased tannins, phytic acid, hydrogen cyanide, trypsin inhibitors and oligosaccharides Protease inhibitors are easily denatured by heat treatment due to their protein nature Research Findings: Cooking sweet potato leaves with lemon reduced polyphenols with 56% and lowered the oxalate levels 54 5. Gamma radiation Reduction of Trypsin inhibitor, phytic acid and oligosaccharides of broad bean Research Findings: 2 kGy dose had no significant change in the tannin content of some maize cultivars. ❖Required radiation dose is dependent on the food sample and the type/concentration of antinutrient 55 Lecture Three 56  PHYCOTOXINS ❖ Are secondary metabolites, synthesized/produced by dinoflagellates and diatoms ❖Natural toxins found in seafoods ❖They accumulate in seafoods that feed on the microalgae Harmful algal blooms (HABs) pose public health hazard 57  Phycotoxins cause a number of human diseases when contaminated seafoods are consumed They are tasteless and odourless Not destroyed by cooking, freezing, drying or salting Hence, pose serious public health hazard Exposure of consumers is a function of: (i) amount of shellfish eaten and (ii) amount of toxin present in the shellfish Phycotoxin Classification 1. Based on their poisoning symptoms: (i) Paralytic Shellfish Poisoning (PSP), (ii) Amnesic shellfish poisoning (ASP), (iii) Diarrhetic shellfish poisoning (DSP), (iv) Neurotoxic shellfish poisoning (NSP), and (v) Ciguatera fish poisoning (CFP). 2. Based on their chemical structure: (i) Azaspiracids (AZAs), (ii) Brevetoxins (BTXs), (iii) Cyclic imines (CIs), (iv) Domoic acid (DA), (v) Okadaic acid (OA), (vi) Pectenotoxins (PTXs), (vii) Saxitoxin (STX), and (viii) Yessotoxins (YTXs) groups. 59  EXAMPLES OF PHYCOTOXINS 1. Paralytic Shellfish Poisoning (PSP) PSP is the result of exposure to toxin called SAXITOXINS and derivatives Due to the consumption of contaminated shellfish Produced by species of dinoflagellates belonging to the genera of Alexandrium, Pyrodinium and Gymnodinium Occurs in Clams, mussels, crabs, oysters and other seafoods that feed on these microalgae PSP TOXICITY Acute toxicity within 5-30 min. Symptoms: Mild: diarrhoea, nausea, vomiting, headache, tingling sensation around the lips, face, neck Moderately severe: incoherent speech, pickling sensation in the arms and legs, respiratory difficulty (shortness of breath) Extremely Severe: Muscular paralysis leading to respiratory difficulty Fatal case: Death 2. Diarrheic Shellfish Poisoning (DSP) Toxins; Okadaic acid and Dinophysistoxins Produced by dinoflagellates; Dinophysis spp. and Prorocentrum lima Occurs in shellfish; scallops, mussels, clams and oysters DSP TOXICITY Acute toxicity; less than 24 hours Symptoms: Nausea, vomiting, diarrhoea, abdominal pain Chills, headache and fever 3. Neurotoxic Shellfish Poisoning (NSP) Toxins: BREVETOXIN Produced by the dinoflagellate; Karenia brevis Occur in: mussels, oysters and scallops NSP TOXICITY Acute toxicity: 30 min – 24 hours Symptoms: Diarrhoea, nausea, vomiting Shortness of breath Cardiovascular arrhythmias Paraesthesia of mouth, lips tongue and throat Dizziness, reversal of hot and cold sensations Muscular aches 4. Amnesic Shellfish Poisoning (ASP) Toxin; Domoic acid Produced by diatoms; Pseudo-nitzschia spp Occurs in: Scallops, mussels, clams and oysters ASP TOXICITY Acute toxicity: less than 24 hours Symptoms: Diarrhoea, vomiting, abdominal pain Shortness of breath Reversal of cold and hot sensation Confusion, disorientation, memory loss, Seizure and coma Chronic toxicity; Amnesia 5. Azaspiracid Shellfish Poisoning (AZP) Toxin: Azaspiracid Produced by dinoflagellate; Protoperidinium spp. Occur in: clams and mussels Acute toxicity: less than 24 hours Symptoms: Diarrhoea, vomiting and abdominal pain 6. Ciguatera Fish Poisoning (CFP) Toxins; Ciguatoxin, Maitotoxin, Scaritoxin Produced by dinflagellates; Gambierdiscus toxicus, Ostreopsis spp., Prorocentrum spp. Occurs in large reef fish; barracuda, grouper, red snapper and amberjack CFP TOXICITY Acute toxicity: 2-6 hours post exposure Symptoms: diarrhoea, nausea, vomiting 3 hour post exposure; Symptoms: neurologic disorders, paraesthesia, reversal of cold and hot sensation, pain, weakness 2-5 days post exposure; Symptoms: cardiovascular disorders Lecture Four 68  MYCOTOXINS ❖Definition: natural products produced by fungi that evoke a toxic response when introduced in low concentrations to higher vertebrates and other animals via natural route ❖Toxic secondary metabolites ❖ low molecular weight ❖produced by filamentous fungi belonging to the phylum Ascomycota or moulds ❖Identified as cause of acute and chronic diseases 69 ❖Fungi - Aspergillus, Fusarium, Penicillium, Alternaria and Claviceps spp. ❖ 400 compounds identified as mycotoxin - 30 receive great attention, and they are considered a threat to human or animal health ❖ Most important mycotoxins 1. Aflatoxins (AFs) - B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and M1 (AFM1)) 2. Ochratoxins (OTs) - Ochratoxin A (OTA)) 70 3. Fumonisins (FBs) – Fumonisins B1(FB1), B2 (FB2) and B3 (FB3)) 4. Trichothecenes (TCs) – Type A [HT-2 toxin (HT2) and T-2 toxin (T2)] and Type B [deoxynivalenol (DON)] 5. Zearalenone (ZEN) Facts: ❖Cannot be detected by eye ❖Can be seen under ultraviolet (UV) light ❖Have no characteristic odour ❖Do not alter the organoleptic characteristics of foods 71 ❖Certain mycotoxins are produced by more than one fungal species ❖Some fungi are capable of producing more than one mycotoxin ❖Favorable climatic conditions cause more fungal and mycotoxin contamination in developing and tropical countries than in developed and temperate zones. 72  Groups of Fungi producing mycotoxin: 1. Feld fungi - infect crops before harvest (Fusarium graminearum, F. verticillioides, Aspergillus flavus) 2. Storage fungi - occur only after harvest (Penicillium verrucosum, A. flavus) Field fungi Storage fungi ▪ 70%–90% relative humidity, lower humidity ▪ temperature of 20–25 ◦C, higher temperatures ▪ Aw: > 0.85 for active growth, and 0.99 for optimal growth 73 Factors affecting micobial growth and the production of mycotoxins Temperature, humidity, pH, water activity (aw), nutrients, level of inoculation, nature of the substrate, physiological state and microbial interactions Most Aspergillus and Penicillium species: minimum aw of 0.75–0.85 and grow well (optimum) at aw 0.93–0.98 Aspergillus species: aw of 0.73 for active growth, temperatures of 30 – 40◦C Penicillium species: aw of at least 0.78–0.80, temperatures of 25–30◦C 74  Factors favouring development of fungi and formation of mycotoxins in developing countries: 1. Poor food quality control 2. Hot climate 3. Poor production technologies 4. Poor crop storage conditions 75 Agricultural commodities: peanuts, grapes and wines, grains, nuts, dried fruit, coffee, cocoa, spices, oil seeds, fruits, fruit juices, beer, and other foodstuffs and feed crops - both in the field and during transportation Mode of Exposure 1. directly through food consumption 2. indirectly through feed 3. Inhalation ❖Result of contaminated feed: leads to the presence of mycotoxins in animal foods (meat, eggs and milk), leading to contamination of the human plate 76 Mycotoxicosis is the disease that results from exposure to mycotoxins - ergotism, alimentary toxic aleukia, aflatoxicosis Toxicity effects in Human: Carcinogenic, Endocrine disorders, Teratogenic, Mutagenic, Hemorrhagic, Estrogenic, Hepatotoxic, Nephrotoxic and Immunosuppressive Strategies for Control: 1. Prevention of their production 2. Detoxification ❖ Conventional cooking processes cannot destroy all mycotoxins – most mycotoxins remain chemically and thermally stable during cooking, boiling, baking, frying, baking and pasteurizing 77 Regulatory limits Established by various authorities worldwide – FDA, WHO, FAO, European Food Safety Authority (EFSA) 1. Aflatoxins B1, B2, G1, G2 – ❖20 ug/kg for total aflatoxin by US FDA ❖2-12 ug/kg for AFB1 and 4-15 ug/kg for total by EU 2. Aflatoxin M1 – ❖ 0.5 ug/kg by US FDA ❖0.05 ug/kg in milk by EU ❖0.025 ug/kg in Infant formula and infant milk - by EU 78 3. Ochratoxin A – ❖2-10ug/kg by EU 4. Fumonisins B1, B2, B3 – ❖ 2000-4000 ug/kg by US FDA ❖ 2000-4000 ug/kg by EU 5. Zearalenone ❖ Not set by US FDA ❖ 20-100ug/kg by EU 79  1. Aflatoxins (AFs) Aflatoxins are difuranocoumarin derivatives Produced by by many strains of Aspergillus flavus, A. nomius and A. parasiticus - A. flavus in particular Warm and humid conditions favour their production - temperatures 22 - 35◦C and aw between 0.95 - 0.98 Are the best known among all mycotoxin – because of their serious impact on human and animal health 80  Are the first mycotoxins to be initially classified as toxic Over 20 known AFs, only 4 are widely studied - AFB1, AFB2, AFG1 and AFG2 Are named after the fluorescence displayed under UV light (B for blue and G for green) Presence of AFB1 is often the highest in the AF mixture 81 Mainly detected in: Cereals (barley, corn, rice, wheat, oat) and their derivatives (bread, flour, breakfast products, cornflakes and pasta) Nuts (almonds, pecans, pistachios, walnuts, cashews, and Brazil nuts) and peanuts Spices and herbs Edible vegetable oils Wines Sugarcane Cottonseed Dried fruits Animal food products (milk, eggs, cured meat and animal tissues) 82  Hydroxylated metabolites of AFB1 and AFB2 are aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) – Present in: (1) meat of animals that consumed aflatoxin contaminated feed (2) animal products such as eggs, milk and cheese Aflatoxin B1 is a carcinogenic substance - classification by the IARC as Category 1A AFM1 is a potentially carcinogenic substance – Category 2B Toxicity range - B1 > G1 > B2 > G2 AFB1 is considered the most potent carcinogenic toxin 83  Exposure to chronic hepatitis B virus infection and aflatoxin may increase liver cancer risk by up to 30 times compared to the risk in individuals exposed to aflatoxin only EU legal limit for AFB1 in processed cereal foods - 0.02 µg/kg Codex Alimentarius and the EU limit - 0.05 µg/kg AFM1 US and some Latin American countries - 0.5 µg/kg AFM1 84  Pre-harvest and post-harvest factors are related to its production Production of AFs depends on the aw interaction with temperature Aflatoxin diseases - Aflatoxicosis – in animals, pets and humans around the world They have carcinogenic, mutagenic (DNA damaging), teratogenic, and immunosuppressive effects ❖Symptoms of acute aflatoxicosis - vomiting, abdominal pain, jaundice, pulmonary edema, coma, convulsions and death ❖Chronic aflatoxicosis - cancer, immune system inhibition and liver damage 85 Factors determine Significant differences in species sensitivity ❑ Age ❑ Sex ❑ Weight ❑ Nutrition ❑ Metabolism ❑ Exposure to infectious agents ❑ The occurrence of other mycotoxins ❑type of toxin ❑mechanism of action ❑levels of intake 86  2. Ochratoxin A ❑ Among the ochratoxin categories A, B and C, OTA is the most abundant and harmful mycotoxin ❑ First identified from the fungus A. ochraceus ❑ Aspergillus and Penicillium are the two main genera of OTA producers ❑ Main producing species belong to the Aspergillus section Circumdati, Aspergillus section Nigri, P. verrucosum, P. thymicola, and P. nordicum ❑ Ochratoxin B which is much less toxic - sometimes co-occurs with OTA in food and feed 87 Ochratoxin Toxicity ❑ linked to immunotoxic, genotoxic, neurotoxic, carcinogenic, nephrotoxic and teratogenic effects ❑ Classified by the IARC as a possible human carcinogen (Group 2B) ❑ Provisional tolerable weekly intake (PTWI) of 112 ng/kg body weight (b.w.) was proposed by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) 88 Production condition for OTA: ✓aw range of 0.92–0.99 ✓ optimum temperature of 20◦C - significantly lower production at 30–37◦C ✓A. ochraceus - temperature range of 12–37◦C ✓P. verrucosum - 0–31◦C ✓ OTA can be produced in all agricultural areas of the world 89 Mainly detected in: Cereals Alcoholic beverages such as in wines Beer Dried vine fruits Coffee Cocoa and chocolate Meat Milk 90  Limits of OTA in food (by EU) ❖5 ng/g in raw cereal grains ❖3.0 ng/g in cereal-processed products, ❖10 ng/g in coffee and dried fruits, ❖2 µg/L in wine ❖0.5 ng/g in cereal-based baby foods ❑ At different stages of food processing such as baking, roasting, frying, brewing, canning, and peeling, OTA cannot be completely deactivated. 91  3. Fumonisins Belong to a large group of toxins referred to as Fusarium toxins Occur in cereals originating from pathogenic fungi, mostly Fusarium verticillioides and Fusarium proliferatum Group of 28 analogues of Fumonisins are divided into four main groups - : fumonisin A, B, C and P Fumonisin B (FB) occur in nature with the highest frequency FB analogues include FB1, FB2 and FB3 whereas FB1 is usually found at the highest concentrations 92 Mainly detected in: Cereals and their derivatives Maize and maize-based products Asparagus Grapes Raisins 93 Health effects ❑ IARC classifies FB1 and FB2 as possibly carcinogenic to humans ❑ cause leukoencephalomalacia in horses and pulmonary edema syndrome and hydrothorax in pigs ❑ JECFA PMTDI - 2 µg/kg b.w./day for FB1, FB2 and FB3 alone or in combination ❑European Union and US - Acceptable upper limits of 800– 4000 and 2000–4000 µg/kg FB1 and FB2, respectively in cereal-based products 94  4. Trichothecenes (TC) ❖ Produced by different species of Fusarium, Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys ❖ Are easily absorbed into the gastrointestinal membranes and are rapidly distributed to various organs and tissues of the body because of their low molecular weight ❖More than 200 different TCs are known - subdivided into four basic groups: A, B, C or D. ❖ Most toxic groups of trichothecenes are Type A (T-2 and HT-2) 95 4.1. Trichothecenes Type A (HT-2 Toxin and T-2 Toxin) ❑ Mainly produced by Fusarium langsethiae. Fusarium poae, Fusarium sporotrichioides, F equiseti, and F. acumninatum ❑ Found in oat, barley, wheat, maize and rice, as well as in cereal-based products and soybean ❑ Show toxic effects such as growth retardation, myelotoxicity, hematotoxicity, and necrotic lesions on contact sites ❑ TDI of 100 ng/kg b.w./day by EFSA 96 4.2. Trichothecenes Type B (Deoxynivalenol) ❑ produced by fungi of the Fusarium genus, mainly by Fusarium graminearum and F. culmorum ❑ Deoxynivalenol (DON) is the predominant trichothecene all over the world ❑ Among the least toxic of the trichothecenes ❑ Most frequently detected mycotoxin in cereal grains worldwide ❑ Very stable at 120◦C, moderately stable at 180◦C ❑Most common route of exposure to DON is through food 97 ❑Acute toxicity symptoms; vomiting, hemorrhagic diarrhea, and refusal of food ❑ Chronic toxicity symptoms; anorexia, suppression of body weight gain, hepatotoxicity, dermatological problems and altered nutritional efficacy ❑ Mutagenic and/or carcinogenic properties of DON are not established ❑FAO/WHO and JECFA - PMTDI of 1 mg/kg b.w. for the sum of DON and its acetyl derivatives PMTDI of 1 µg/kg b.w./day for DON and its metabolites ❑ Control in the field during cultivation is the best way to control 98  5. Zearalenone (ZEN) ❖ Produced by fungi of the Fusarium genus ❖Contamination is low in grains in the field, it increases in storage conditions with moisture of more than 30%–40% ❖ TDI of 0.25 µg/kg b.w./day established by EFSA ❖Maximum Permissible Limit (MPL) according to EU: ❖100–200 µg/kg in unprocessed cereals ❖75µg/kg for processed cereals ❖20µg/kg in processed cereal foods, ❖50µg/kg in cereal snacks 99 ❖ IARC classifies ZEA as a Group 3 carcinogen ❖ Affects conception, ovulation and fetal development at concentrations above 1 mg/kg ❖ Risk to human populations is minimal 100  Mycotoxin Control Strategies: Prevention and Decontamination/Detoxification in Foods ❖Pre-harvest strategies aim to avoid the development of toxigenic fungi ❖ Once mycotoxins are produced, detoxification of foods should be based on post-harvest practices Pre-Harvest Strategies: 1. Good agricultural practices (GAPs) 2. Good manufacturing practices (GMPs) 3. Appropriate environmental factors 4. Favorable storage practices 5. Use of biological control agents, such as antagonistic fungi - cereals, grapes and apples 101 Post-Harvest Strategies 1. Physical Treatment: ▪ Grading, sorting and the removal of the obviously affected parts ▪ Processing - can reduce the concentration of mycotoxins but can not completely destroy them Mycotoxins are thermally stable compounds, some conventional methods (baking, frying) at 100 ◦C above may reduce certain mycotoxins Storage - temperature and high humidity affect the overall growth of fungi and mycotoxins production 102  Radiation – ionizing radiation or non-ionizing radiation Can reduce or eliminate pathogenic microorganisms, but it partially removes mycotoxins Mycotoxin Binders - inhibit the absorption of mycotoxins by binding to mycotoxins, not allowing their entry into the bloodstream Absorbent materials: activated carbon, aluminosilicates, complex non-digestible carbohydrates and cholesterol 2. Chemical Control Bases (Ammonia, Hydrated Oxide) - (AFs, FBs, OTs) Chitosan - combined effects of chitosan and aw for controlling the fungal growth and mycotoxin production of FBs and DON Ozone Treatment - AFB1, AFG1, DON 103 3. Biological Control Bacteria – Flavobacterium aurantiacum B- 184 for detoxification of Afs Lactic acid bacteria (Lactobacillus (L.) casei and Lactobacillus reuteri) Yeast – Saccharomyces cerevisiae is a probiotic yeast which can significantly degrade DON Kluyveromyces marxianus were used to bind mycotoxins AFB1, OTA, or ZEA Fungi - non-toxic strains of A. flavus and A. parasiticus on maize, cotton, pistachio and peanuts Aspergillus, Rhizopus, Trichoderma, Clonostachys, and Penicillium spp 104  Food Fermentation - reduce and even eliminate mycotoxins 4. Enzymatic Detoxification Involves combination of the characteristics of chemical and biological processing does not cause toxicity to organisms Enzymes such as β-1,3-glucanase and chitinase - delay and decrease growth of spoilage fungi Inhibition of Penicillium simplicissimum, A. Niger complex, Penicillium nalgiovense, and A. flavus growth - spraying β-glucanase and chitinase Microbial manganese peroxide, oxidase enzymes, catalase and laccase enzymes - enzymatic detoxification of AFB1 105 Lecture Five 106  TOXINS FROM SOURCES EXTERNAL TO THE FOOD 107  TOXINS FROM MICROBIAL CONTAMINATION ❑Microorganisms (both beneficial and pathogenic) can be introduced to the crop or food animals during: i. Primary production (in the farm where plants are grown or animals are raised for food (pre-harvest or pre-slaughter stages), ii. At harvest and slaughter (food animals), and iii. At postharvest/post-slaughter (food processing, distribution and marketing, storage, preparation and serving). 108