Dilip Kumar Singh

Scientist
ICAR-CIFE, Kolkata Centre
 Introduction
 Aquaculture currently represents the fastest growing food
producing sector of agriculture
 Feed manufacturing plays a significant role in the growth of aqua
industry
 In the early days, most of aquafeed formulated from animal based
products particularly trash fish
 But, this commodities are becoming increasingly expensive and
scares particularly in the developing part of world (Reason fish
meal trap/competition with other livestock's)
 However, in order to make aquaculture viable and sustainable, feed
costs must be reduced and shift towards plants sources
 At present, mostly aquafeed ingredients are used in bulk quantity
from plant sources and contains harmful/toxic substances
 Toxic substances reduce the nutritional value of aquafeed
Antinutrients/Antinutritional factor:
Those substances which either by themselves or through
their metabolic products, interfere with feed utilisation and affects the
health and production of animals

Why antimetabolites are synthesized in plants ?

 Unlike animal, plants lack immune system

 So, nature endowed all plants with the capacity to synthesize a

wide variety of chemical substances for protection against
attacking organisms

 This substances exert a deleterious effect when ingested by

animals
 General characteristics of antinutrients
 It is a products of secondary metabolism:
 Non infectious
 Non communicable
 Non transmittable
 Non-volatile
 It is a found in virtually all plants to some degree
 Characteristics:
 Bitter
 Colored
 Poisonous
 Odor
 Antinutritive
 Immunosuppresive
 Effects on Fish
 Depressions in growth performance and animal health,
due to a variety of mechanisms including reducing
protein digestibility, binding to various nutrients or
damaging the intestinal wall, thereby lowering digestive
efficiency
 Causing nutritional deficiencies
 Poor feed conversion
 Hormonal interference
 Organ damage (Pancreatic hypertropy/hyperglycemia
and liver damage)
 Death
 Protein level
Plant source Antinutritional factors
(% DM)
Protease inhibitors, lectins, phytic acid, saponin,
Soyabean meal 40
phytoestrogens, antivitamins, allergens

Rapeseed meal 36 Protease inhibitors, glucosinolates, phytic acid , tannins

Lupin seed meal 34 Protease inhibitors, saponin, phytoestrogens, alkaloids

Protease inhibitors, lectins, phytic acid, saponin, tannins,

Pea seed meal 21
cyanogens, antivitamins,

Sunflower oil cake 32 Protease inhibitors, saponin, arginase inhibitors

Phytic acid, phytoestrogens,

Cotton seed oil cake 43
gossypol,antivitamins,cyclopropenic acid.

Leucaena leaf meal 29 Mimosine

Mustard oil cake 36 Glucosinolates,tannins

Seasame meal 43 Protease inhibitors, phytic acid,

Alfalfa leaf meal 21 Protease inhibitors, saponin, phytoestrogens, antivitamins

 Main non-nutrient compounds and their main adverse effects

Compounds Adverse effects Main source

Protease
Growth inhibition Soya, Cereals
inhibitors

Amylase
Decrease starch digestion Cereals
inhibitors
Lectins Decrease nutrient absorption Beans

Phytates Decrease bioavailability of minerals Soya

Bran cereals and shredded

Oxalates Decrease bioavailability of minerals
wheat cereals

Tannins Astringent taste, decrease food intake Sorghum, Rapeseed

Lignans Growth inhibition Linseed

Saponins Bitter taste, decrease food intake Soybean, Groundnut

 Classification of Antinutritional factors

On the basis of

1. Chemical composition/nature

2. Activity/way of action

3. Heat stability
According to their chemical nature : 4 classes

I. Proteins :protease inhibitor, haemagglutins

II. Glycosides : goitrogens, cyanogen, saponins, oestrogens,

phytoestrogens

III. Phenols : gossypols ,tannins

IV. Miscellaneous : antimetals, antivitamins, antienzymes,

food allergens, microbial/plant origin carcinogens, toxic
amino acids etc
According to their activity/way of action : 5 classes
I. Factors affecting protein utilization and digestion: protease
inhibitors(trypsin and chymotrypsin inhibitors, elastase
inhibitors), tannins, lectins

II. Factor affecting mineral utilization: phytates, gossypols,

oxalates, glucosinolates

III. Factor affecting carbohydrate utilization: α-amylase

inhibitor

IV. Antivitamins: antivitamin A, D, E, and B12, thiaminase and

antipyridoxine(linatine)
V. Miscellaneous: mycotoxins, mimosine, cyanogens,
alkaloids, phytoestrogens and saponins
According to heat stability : 2 classes

I. Heat labile factors: protease inhibitors, goitrogens,

antivitamins, haemagglutinins, glucosinolates

II. Heat stable factors: saponins, nonstarch polysaccharides,

antigenic proteins , estrgens, and some phenolic
compounds(gossypols ,tannins)
Remediation measures by mainly : two methods
1. Physical processing
2. Bioprocessing and biotechnology

Physical processing
 Fundamental of heat processing
 Moist heating- blanching, cooking, steaming or
autoclaving(20 min at 115°C)
 Dry heating- roasting and toasting
 Extrusion cooking
 Dehulling, Milling
 Soaking treatment with chemical and water
 Oil extraction and ultrafiltration
 Nutrient supplementation
 Irradiation
Bioprocessing and Biotechnology :
 Selective breeding
 Genetic engineering
 Enzyme processing
 Germination
 Fermentation
Common Antinutritional factors
1. Protease inhibitors
2. Lipase Inhibitors
3. Glycosides
4. Saponins
5. Alkaloids
6. Phytic acid
7. Tannins
 Protease inhibitors
 are proteinaceous in nature and mainly found in plant
seeds
 Inhibit the actions of • Trypsin • Pepsin • Other proteases
 Mostly in leguminous plant like Soya
 Trypsin inhibiter is more common in leguminous plant
 It inhibit proteolytic enzymes by binding to the active sites of
the enzymes
Detoxification
 Autoclaving
 Microwave radiation
 Soaking followed by cooking
 Fermentation
Tolerable limit
 Germination
• In soyabean, 2-6 mg/g (avg. 4 mg/g).
• In most of the cultured species tolerable limit is below 5 mg/g.
Mode of Action and Pathology
Trypsin inhibitor
inhibits

Chymotrypsin & Elastase Trypsin

(less extent) Formation of
stable complex

Decrease in protein digestibility

After some time deficiency in sulphur
Trypsin secretion amino acid (cystein)
activates (not sufficient)
Hyper secretion of
Hypertrophy of
cholecystokinine
pancreas
Lipase Inhibitors
 Interfere with enzymes, such as Pancreatic lipase,
that catalyze the hydrolysis of some lipids,
including fats.
 For example:
 Anti-obesity drug orlistat causes a percentage of
fat to pass through the digestive tract undigested.
Lipase Inhibitors
 Recently, protamine, purothionin and histone,
which belong to a class of basic proteins, were
reported to inhibit lipase
 These substances inhibit lipase in substrates
containing phosphatidylcholine
 From these results, it was suggested that these
compounds inhibit lipase activity by interaction
with triolein- phosphatidylcholine
 PhyticAcid
 is the hexaphosphate of myo-inositol a cyclic compound
 Found in oilseed meal/cake, legumes and cereals
 Phytic acid has been shown to have a negative effect on
divalent ions absorption
 It is also formed complex with protein (Phytin) and starch
resulting reduced digestibility of this nutrient
Detoxification
 Fermentation
 Exogenous phytase supplementation
 E-beam irradiation
 Zn-supplementation

Tolerable Limit
In most of the cultured species tolerable limit is below 5 g/kg fish feed
 Mode of action and pathology
Phytic Acid (Inositol hexaphosphoric acid)

Fish do not poses enzyme to effectively hydrolyze the esterified

phosphorus

In digestive tract

Chelate Ca+2,Mg+2, Mn+2 , Fe+2 , Co+2

, Zn+2

Interfere protease enzymes

activity by chelate co-factor of
enzymes

Decrease the bioavailability of divalent metals

and phosphorus and cause water pollution

Source: Soybean Meal, Pea Seed Meal , Sesame Meal

 PhyticAcid
 Phytic acid reduces pepsin and trypsin activity thus interfere
with protein digestibility

 Reduced feed conversion efficiency & PER

 Growth depression

 Cataract formation (due to Zn deficiency)

 Reduced thyroid function and abnormality of kidney and

alimentary tract morphology

 Hypertrophy of pyloric cecae

 Glucosinolate
 are glycosides containing β-D-thioglucose
 They are always produce by myrosinase in the plants but this enzyme are
found in different cell components

 When raw material are crushed or ground the enzyme release and come
in contact of glucosinolate and yield toxic metabolites includes goitrin,
isothiocyanates, thiocyanate ions and nitrile
 Glucosinolate as such is less harmful whereas its metabolites are more
harmful
 This compound mainly found in Brassica family (Crucifera) and are
present in all part of the plant

Tolerable Limit
It is recommended that dietary glucosinolate level should be below
0.2 & 0.4 mg/g diet for Coldwater salmonid and warm water
fishes respectively
Enzymatic hydrolysis of glucosinolate

Creates lack of appetite

Hyperplasia of
thyroid/Goitre

Inhibits attachement
of iodine to thyroid
hormone

Goitrine
 Reduced FCR & growth depression
 Histopathological changes in thyroid gland
Detoxification
 Autoclaving

 Micronization

 Fermentation

 Hot water extraction

 Extraction by dilute alkali

 Organic solvent extraction

 Gossypol
 are yellow coloured polyphenolic pigment contained in the
pigment glands of certain plants (Genus Gossypium)

 During processing free gossypol released from pigment

gland and bound to iron and protein (lysine) make non-
availability and sometime methionine

 Though bound gossypol reduces protein quality but it is non-

toxic to the fish but free gossypol is toxic

 Histo-pathological changes in intestine, liver (necrosis) and

kidney (thickening of glomeruli or complete damage of
kidney)
Tolerable limit: Free gossypol: below 0.01%
 Necrotic liver in trout Thickening of glomerular
basement membrane in trout
 Reproductive problem with reduction of sperm motility,
abnormal histology of testes
Detoxification
 Autoclaving, extrusion cooking
 Supplementation: Lysine, methionine &cysteine
 Solvent extraction
 Solid State Fermentation
 Iron bound to free gossypol----less toxicity
 Tannin
 are polyphenolic compound and categorized into two major
groups i) hydrolysable ii) condensed tannin
 Effects:
 binding to digestive enzymes (trypsin and amylase)-reduce
digestion process
 binding to protein, carbohydrate or binding to minerals (Ca,
P, Mg) -reduced bio-availability
 reduce absorption of vit B12
 Condensed tannin decrease feed intake and nutrient
(especially protein) utilization
 Hydrolysable tannins easily degraded in biological system
forming smaller compounds that can enter into the blood
stream and cause toxicity to the organs e.g., liver and
kidney and lead to death of animal.
Limit: Less than 2%
 Oilseed meal/cake: rapeseed meal/mustard oil cake,
Sunflower meal, copra meal, soybean meal, sesame seed
cake, mango seed kernel, Jatropha kernel meal
 Legumes: lima bean, lupin, pea seed meal, pigeon pea,
chick pea
 Cereals: Sorghum grain
Detoxification
 Autoclaving, extrusion cooking
 Soaking
 Germination
 Fermentation
 Dehulling
 Roasting/Toasting
 Tannin binding agent: activated charcoal, Polyvinyl
Polypyrrolidone (PVPP), Compound Polyethylene Glycol (CPEG)
 Saponin
 are steroidal glycosides and saponin containing plants are
such as Lentil, Alfalfa, Soybean, Leucerne( Medicago sativa)

 they are highly toxic to fish, damage to the respiratory

epithelium of the gills by the detergent action
 Have distinctive foaming characteristics
 traditionally used as fish poisons, like mahua oil cake
containing saponin
 Reduced feed intake (bitter taste-deterrent effect)
 Form indigestible protein saponin complex
 Inhibit the activity of trypsin & chymotrypsin

Tolerable limit: Less than 1 g/kg diet

 Inhibit active nutrient transport, thus reduced nutrient
absorption
 Lower feed efficiency & growth performance
 It is considered as surface active component and acts on
biological membrane
 Reduction of blood & liver cholesterol
 Erythrocytes haemolysis
 GIT erosion
Detoxification
 Water soaking: 12-24 h soaking
 Ethanol extraction:
 Iso-propanol extraction
 Supplementation of cholesterol
 Lectins/Haemagglutinins
 are glycoproteins and found in many legume seeds (faba
bean, Lupin seed meal, pea seed meal, kidney bean, Lentil,
lima bean, Soyabean meal)
 bind to free sugar or sugar residues of polysaccharides,
glycoprotein, glycolipids in either free or bound form such as
in the cell membrane
 cause agglutinations of RBC
 Growth depression
 Lower insulin level in blood
 Depletion of lipid and glycogen
 Inhibit carbohydrate and protein digesting enzyme activity
 Degenerative changes in intestine, liver & kidney
 Impair immune system of animal
 Allergic reaction
Detoxification
 Heat treatment: aqueous heat treatment at 120°C for 10
min. or autoclaving or moist heating at 100° C for 10 min
 Cyanogens
 are cyanogenic glycoside which are not toxic but on
hydrlysis by β-glucosidase they yield toxic HCN along with
glucose, aldehyde or ketone

 Release by crushing of cells facilitate this conversion

 Linseed oil cake---Linamerin (cyanogens)---breakdown by
linamerase (glucosidase) to HCN, glucose & acetone

 Reduced energy metabolism by inhibiting cytochrome

oxidase enzyme of respiratory chain

 Supress natural respiration

 Cardiac arrest
 Oilseed meal/cake: Linseed meal
 Legumes: Chick pea/Bengal gram, Lupin seed meal, kidney
bean, lima bean, Jack bean, Cassava leaf meal, pigeon /red
pea, field pea

Detoxification
24 h Water soaking followed by sun drying
 Glycosides
 Generally bitter
 Structure: Contain CHO and non-CHO
groups(aglycone)
 Mode of action: Toxicity results from
aglycone release during enzymatic
degradation
 Sources: Linseed, sorghum, soyabean,
clover etc
 Non-starch polysaccharrides
 Present commonly in legumes &cereals
 They bind to bile acids or obstructing action against action of
digestive enzymes & movement of substrate in intestine

 NSP’s in soyabean - pectins , galactans, cellulose and lignin.

 NSP’s are more detrimental to growth of fishes than

oligosaccharides

Detoxification
 Removal-steam cooking
 Alkaloids
 are basic in nature, contain nitrogen in the heterocyclic
ring and are derived from amino acid precursor molecules
 They interfere with nerve functioning & also reduce the
feed intake
 Present in legume seeds &cereals, commonly found in
lupins
 mucosal lesions, abnormalities in villi, specific & non-
specific immune responses, abnormal movement in gut
 It can cause • Kidney damage • Pulmonary damage •
Liver damage
Detoxification
 Removal method-solvent extraction &alcohol extraction
 Anti-enzymes
 Anti-enzymes factors are a group of compounds having
an anti-enzymatic activity
 such as the amylase inhibitor, invertase inhibitor, and
arginase inhibitor etc.

 They are found in many plant-derived ingredients

(cereal, root tubers, legumes and oilseeds) and also raw
fish

Detoxification
 Remove by heat treatment
 Anti-vitamin
 Anti-vitamin are a group of compounds having an anti-vitamin
activity
 Anti-vitamin A - destroys carotene
 Anti-vitamin D - interferes with calcium and phosphorus absorption
 Anti-vitamin E - causes liver necrosis and muscular dystrophy
 Anti-thiamine- causes deficiencies in vitamin B1
 Anti-pyridoxine – interferes with amino acids metabolism
 Anti-vitamin B12- form complexes with vitamin B12 to reduce their utilization
rate
 Found in Soybean meal, sunflower meal, maize, rice bran, mustard
seed, cotton seed, linseed, raw fish etc.
Detoxification
 They are heat-labile and are readily destroyed by heat treatment
 Cyclopropenoic fatty acids
 are toxic fatty acids and include sterculic and malvalic
acid
 cause abnormalities in the reproductive processes,
disrupt the lipid metabolism, and impair the growth
 when associated with aflatoxins, CFA are suspected to
be carcinogenic to fish
 CFA are present in cottonseed oil and meal
Detoxification
 CFA are usually removed from the oilseeds during oil
extraction but residual levels may persist
 Erucic acid
 Erucic acid (cis-13-docosenoic acid) is a 22-carbon
monounsaturated fatty acid
 Erucic acid is found in rapeseed and mustard oils
 The reported signs are reduced growth, increased
mortality, and histopathology of skin, gills, kidney and
heart
Detoxification
 removed from the seed by oil extraction
 Mycotoxins
 is a group of toxin
 Mycotoxins are secondary metabolites of fungi
 Aflatoxins is produced by Aspergillus flavus and Aspergillus
parasiticus
 Cause pale gills, impaired blood clotting, anaemia, poor
growth rates or lack of weight gain, liver tumours, and
increased mortality
 Found in oil seed, rice bran, maize etc.
Detoxification
 Feeds should be stored in a cool and dry area
 Regular testing should be done by inspecting visually the
feed (with or without black light), or by the mean of
commercial detection kits
 Toxic amino acids
 Non-protein free amino acids can disrupt the proteins
synthesis by competing on the messenger RNA
Mimosine
 is a secondary metabolite synthesised against fungus
attack and stop cell multiplication and DNA replication
 Mimosine is found in the leaves of ipil-ipil (Leucaena
leucocephala)
 is an inhibitor of pyridoxal group containing transaminases,
tyrosine decarboxylase, several metal-containing enzymes
 Reduced growth and feed efficiency, and increased mortality
 heat-stable but can be removed by soaking in water
Thank You