Paper No. : 11

Paper Title: Food Analysis and Quality Control

Module-21: Analysis of anti-nutrients in foods

1.1 Methods for quantification of the antinutritional factors in foods

Antinutrients are found at some level in almost all foods for a variety of reasons. They are

natural or synthetic compounds that interfere with the absorption of nutrients present in the food.

Although they not necessarily toxic per se, are plant compounds which decrease the nutritional

value of a plant food, usually by making an essential nutrient unavailable or indigestible when

consumed by humans/animals. Several methods are used for the quantitative determination of

anti-nutritional factors in foods based on reports by different authors. These are: trypsin inhibitor

activities are determined according to Liener (1979); haemagglutinatin, Jaffe (1979); cyanogenic

glucosides (HCN), Bradbury et al (1999); oxalates, Fasset, (1996); phytates, Maga (1983);

tannin, Dawra et al. (1988); saponinn, Brunner (1984); and alkaloids, Henry (1973) etc.

There are other some new methods for quantification of antinutritional factors also due to recent

advances in the nutritional sciences. Principles of these methods are discussed as under.

1.1.1 Phytic acid

Several methods are available for determining phytic acid concentrations in products. There are

many papers that report different modifications to these methods, but the ideal methodology is yet

to be agreed upon. The existing methodology needs to be optimized and standardized. There are

many different techniques that can be used for the identification of phytic acid, but there are no

direct methods. There are no specific reagents that detect phytic acid or its various forms.

Moreover, phytic acid does not have a characteristic absorption spectrum in the UV or visible

light region. Most analytical methods are based on extraction or isolation of phytic acid.

Most convent iona l quantitative methods for phytate analysis have been based on the

procedure of Heubner and Standler (1914). These methods involve sample extraction with acid

and subsequent precipitation of the Fe (III)–phytate c o m p l e x following addition of

ferric chloride. Phytate is estimated either by determining the phosphorus (McCance and

Widdowson, 1935), Fe (Wheeler and Ferrel, 1971) or inositol (Oberleas, 1971) in the isolated

phytate complex, or indirectly based on the determination of the residual Fe in the solution

after precipitation of ferric phytate from a known concentration of ferric salt in an acid

solution (Young, 1936). Later, it was established that in addition to inositol hexaphosphate,

ferric ion will also precipitate myoinositol pentaphosphate and tetraphosphate in a dilute acid

solution, with the amount of IP5 and IP4 of the precipitate depending on the amount and

composition of wash solution (Oberleas, 1971; Frolich et al. 1986; Phillippy et al., 1986). Small

amounts of inorganic phosphate may also co-precipitate (Ellis et al., 1977). As the

stoichiometric ratio of phosphorus to Fe in Fe (III)–IP precipitates is affected by several

variables, the results are unreliable.

Harland and Oberleas (1977) introduced the use of an anion exchange resin column.

Phytic acid was eluted from the column separately from the lower inositol phosphates and

inorganic phosphate employing a stepped gradient system and quantified by measuring

the phosphate released after acid hydrolysis of the phytate fractions. Ellis and Morris

further modified the anion exchange column stage of the method (Ellis and Morris, 1986) and

it was accepted as an official method b y the AOAC in1986 (Harland and Oberleas, 1986).

The method of Harland and Oberleas (1977) has also been mo d i f i e d by other

w o r k e r s . Phytic acid content can be measured after elution from the anion exchange

column either based on the reaction between ferric chloride and sulfosalisylic acid

(Wade reagent) (Latta and Eskin, 1980 ; Fru Hbeck et al.,1995) or formation of the phytate–o-

hydroxyhydroquinone- phtalein–Fe(III) complex (Fujita et al., 1986). In the Plaami and

Kumpulainen‟s modification (1995) total phosphorus determination of phytic acid, after

either anion exchange column or ferric precipitation, was performed by inductively coupled

plasma atomic emission spectrometry (ICP-AES). March et al. (1995) liberated phosphorus

from phytic acid after anion exchange column by enzymatic hydrolysis and measured it

spectrophotometrically, according to the method of Uppstro m and Svensson (1980).

However, in the method of Uppstro m and Svensson (1980), phytic acid was calculated from

the difference between phosphorus content before and after enzymatic hydrolysis of the

sample without using anion exchange separation.

The AOAC anion-exchange method is one that has been used to estimate phytic acid content in

products. The results of the AOAC method and the method of Latta and Eskin (1980)

and Fujita et al. (1986) agree with those of the earlier Fe precipitation methods. Later it

was shown that the concentration of phytic acid determined by all these methods may be

systematically overestimated because lower inositol phosphates (IP3-5) and adenosine

triphosphate (ATP), if present, may be associated with IP6 (Phillippy et al., 1988; 36 Lehrfeld

and Morris, 1992).

Near-infrared spectroscopy methods for the determination of phytic acid have been developed

by De Boever et al. (De Boever et al., 1994). NMR methods are capable of measuring

phytic acid and myoinositols with a lower number of phosphate groups (Frolich et al.

1986, Erso et al., 1980).

Blatny et al. (1995) developed a method in which myoinositol h e x a p h o s p h a t e was

determined with iso- tacophoresis. De Koning (1994) determined phytic acid in food by gas

liquid chromatography. The early HPLC methods were capable of separation and

determination of IP6 only (Camire and Clydesdal, 1982, Lee and Abendroth, 1983). Newer

methods are capable of separating and determining the other IPs also. HPLC and detection

methods are described.

The high-performance liquid chromatography (HPLC) method is the primary means of separation and

quantification. HPLC is capable of separating phytic acid and inositol phosphates as separate entities. It

also has the sensitivity and reproducibility to measure low concentrations in products. However HPLC

method is also not without its share of problems. The reagents used in this method must be pure and free

from metals or it will cause distortion in the readings. There are many different modifications to the

HPLC method. The most common are the use of different columns, mobile phases, flow rates,

extraction solvents, and preparation techniques.

A strong anion exchange HPLC column has been used by Mathews et al. (1988) for

separation in food analysis. Rounds and Nielsen obtained better separation and sharper

peaks in plant, food and soil samples by gradient anion exchange HPLC instead of

the isocratic elution used by Cilliers and Van Niekerk (1986). The use of reverse phase

columns in ion-pair chromatography has also been presented in several papers (Sandberg and

Ahderinne, 1986; Sandberg et al., 1989; Lehrfeld, 1994; Rounds, and Nielsen, 1993) with food,

intestinal content and faeces samples.

Methods for measuring phytic acid have been reviewed by Oberleas and Harland (1986),

and phytic acid and other myoinositol phosphates more recently by Xu et al. (1992). For

food and nutrition studies, methods which can determine different IPs separately are an

appropriate choice.

1.1.2 Analytical techniques used in the determination of polyphenolic compounds from

foods

The most representative analytical methods mentioned in the literature for the separation and or

quantification of polyphenolic compounds found in foods shall be discussed here. In the first

place chromatographic techniques such as fine layer chromatography, gas, and in particular high-

performance liquid chromatography used for the determination of polyphenolic compounds shall

be discussed.

1.1.2.1 Thin Layer Chromatography (TLC)

Before the onset of chromatography, the analysis of polyphenolic compounds was an extremely

tedious task and perhaps the most difficult endeavor for those responsible for analytical

determination. The birth of paper chromatography revolutionized the analysis of organic

substances, and during the 1950s and 1960s paper chromatography was widely used for the

determination of polyphenolic compounds, especially when applied for flavonoids determination

(Robards and Antolovich, 1997).

In no time paper chromatography was substituted by thin layer chromatography (TLC). It was

considered a very simple and cheap technique that offered great versatility with respect to

simultaneous qualitative analysis of polyphenolic compounds in distinct samples through the

employment of adequate absorbents and specific reagents. The choice of stationary phase as well

as an adequate solvent depends on the studied polyphenolic structures. Consequently, the most

hydrophilic flavonoids were separated with TLC by employing stationary phases such as

polyamide and microcrystaline cellulose. On the other hand, a classical stationary phase made of

silicone gel has been used widely to separate more apolar flavonoids such as flavons and

isoflavonoids. Likewise, this technique has numerous applications in the analysis of

anthocyanins as confirmed by many bibliographical pilot studies. The detection, as is well

known, is carried out by close inspection of migratory spot under the ultraviolet light.

Furthermore, in the current chemical arsenal we dispose of an array of specific reagents that can

be applied to each compound, previously separated on the plate. Therefore, for the sake of an

example we may cite aluminum chloride, boron hydride, sodium, 190 and vanillin193 as the

most common reagents employed in TLC. Inasmuch as that, based on the ensuing reaction and in

virtue of the generated color, it is possible to accomplish identification of determined

compounds, or at least the involved species of polyphenolic family. Thus, for example, while

flavonoles and flavanones do not react with vanillin and HCl in the methanol medium, these

reagents nonetheless are capable of reducing flavanones giving off a red or violet color that

intensifies throughout reaction, allowing the identification of individual species from a complex

polyphenolic environment.

1.1.2.2 Gas Chromatography

One of the principal objections against this kind of chromatography had to do with difficulty by

which it quantifies flavonoids. In its beginnings, gas chromatography (GC) was used in an

attempt to facilitate the determination of polyphenolic compounds. However, due to the fact that

it lacks high volatility, it was necessary to resort to the derivation stage, which in practice

resulted in being too complicated for any useful application in the characterization of this type of

substance. A very representative example of its application may be found in the determination of

flavonoids contained in citric fruits, which after recovery in a polyamide column were derived

into esterified structures to be characterized by CG. The CG has been also employed in the

determination of flavones found in orange skin oil by using open tubular capillary columns. On

the other hand, CG coupled with mass spectrometer (MS) has been employed in the

determination of previously derived and hydrolyzed citric juice flavanones. The GC-MS

combination has been also used for the analysis of fruit flavones, flavonols, flavanones, and

chalcones without the obligatory derivation step.

1.1.2.3 High-Performmance Liquid Chromatography (HPLC)

High-performance liquid chromatography is, without doubt, the most useful analytical technique

for characterization of polyphenolic compounds. The foregoing affirmation is fully justified in

view of great volume of published studies made available in the last decade.

The most common stationary phases are prepared with chemically modified silicone containing

hydrocarbon chains, where the denominated C8 have been used to the lesser extent than C18. On

the other hand, the employed elution modality, whether isocratic or gradient, depends on the

polyphenolic composition present in the samples. The isocratic elution has been employed in

those samples whose polyphenolic composition is constituted by the same group or structural

family. Therefore, by this method it is possible to conduct the determination of flavonols

(quercetin, miricetin and kaempferol) in wine samples, methoxylated flavones, cinamic acids

(caffeic, chlorogenic, ferulic, and cumaric), and flavonoids (naringin, hesperidin, and

neohesperidin) in citric fruit samples. Another example that illustrates this point is the separation

of antocyanins from different rosaceae fruits such as strawberries and raspberries by means of

acetonytrile and acetic acid at 5% as a mobile phase. The isocratic elution has been also

employed in certain vegetables and legumes. Likewise, isoflavonoids have been separated by

isocratic elution from soybean samples, more precisely in a C8 column with the help of

acetonitrile and a phosphate (pH 2.0) as a mobile phase.

On the other hand, it is necessary to indicate that the majority of published chromatography

studies certify the use of elution in mobile gradient phase. This fact should not surprise anybody

because we are dealing with complex samples that contain polyphenolic compounds that show a

different retention pattern. As a matter of fact, it is worth mentioning the chromatographic

separation of cinamic acids, flavanols, chalcones, and apple skin flavonols, plus flavones,

flavanones, and citric fruit flavonols from citric fruits. Also, it must be mentioned that majority

of chromatography experts have employed linear gradient under constant flow.

The published studies speak of using methanol, acetonitrile, and, to the lesser extent,

tetrahydrofurane (THF) as participating solvents in the mobile phase as well as of incorporating

into the medium small quantities of weak acids such as formic, acetic, or phosphoric.

Subsequently, under the mentioned conditions it was possible to solve many complex samples

originated from wines, citric fruits, rosaceae, and apples. In effect, a method of elution with

binary mobile gradient phase and constituted by AcH at 5% as low-grade eluting solvent in a

mixture constituted by aqueous acetonitrile, in the presence of the same acid modifier allowed

obtaining numerous peaks in wine samples. The said method required a time gradient of 150 min

due to a large number of polyphenolic compounds present in the sample. However, in order to

obtain a complete resolution, the gradient method was carried out by means of a ternary solvent

mixture, where the third solvent reaction also consisted on a dissolution of lighter acetic acid

(1%). Such would be the case of studies carried on bilberries sample, where 25 antocyianins

were separated in less than 40 min by means of a gradient elution consisting of methanol and

formic acid in a SuperPac column.

When it comes to temperature used in the separation, it can be said that in general it must never

be too high. Hence, for the analysis of wine and citric fruit samples some authors recommend

40°C, although as a rule, most of the separations were carried out under ambient temperature.

With respect to detection system used in high performance liquid chromatography suitable for

the derivation of polyphenolic compounds, it needs to be emphasized that the UV-VIS detection

is undoubtedly the most common. The fluorescence and the electrochemical detection systems

have been used to the lesser extent.

Thus, it can be observed that the immense majority of published studies rely on the detection of

polyphenolic compounds at column‟s exit by taking advantage of radiation absorption by these

compounds in the UV-VIS region of electromagnetic spectrum. The most frequently used

wavelength has been 280 nm, because at that wavelength it is known to absorb all the

polyphenolic compounds. Another employed wavelength, although to the lesser extent, has been

254 nm. Generally speaking, both wavelengths exhibit similar analytic sensibility; however, the

280 nm wavelength is used more frequently as the basis of absorption in the mobile phase,

especially when acetic acid is employed as an acid modifier. Nonetheless, some bibliographical

studies recommend employing different wavelengths with the purpose of achieving maximum

sensibility, and if possible an adequate selectivity depending on the type of sample and its

polyphenolic composition. Following this philosophy, it was possible to detect cinamic acids and

their hydroxylated derivatives at 325 nm, flavonol glycosides at 350 nm, and aglycones at 370

nm. Nonetheless, neither hydroxybenzoic acids nor flavan-3-ol exhibit absorption at the

previously mentioned wavelengths, and, consequently, they do not offer interference in the

chromatogram. An excellent example of the same argument is seen in the employment of visible

region wavelength of the spectrum for the identification of antocyanins. These structures possess

an intense absorption band sensitivity, generally above 500 nm, at which no other polyphenolic

structures absorb. This phenomenon allows detection of the mentioned structures in complex

samples without the interference of subjacent polyphenolic species.

On the other hand, the spectroscopic molecular UV-VIS absorption amounts to one of the most

powerful identification tools currently employed in the detection of polyphenolic compounds

when these are combined with chromatographic techniques. The usefulness of this technique has

been manifested by the incorporation of array diode detectors (DAD). It is well known that this

type of detector offers certain advantages with respect to the detection, for they secure

chromatograms at any wavelength, accompanied by the absorption spectrum of each eluted band.

The absorption spectrum can be combined with retention parameters for the possible

identification of an unknown compound and also to measure purity of the elution band in

question. This finding has gained in the last years enormous publicity due to its practical

application in the analysis of polyphenolic compounds, especially thanks to its usefulness in

chromatographic techniques when applied to quantitative sample analysis. The incorporation of

this type of detector has led to the publication of a series of relevant articles that deal with the

different possibilities of this type of detection in complex tea and wine samples, respectively.

The polyphenolic characterization of wine samples by direct injection into HPLC, without

subjacent treatment of the same, first suggested by Roggero was possible thanks to the

incorporation of diode detectors. Other researchers have studied different parameters that can be

evaluated by computer software that yielded useful information concerning identification of

polyphenolic compounds. Among these studies one can find a detailed description concerning

the isolation of determined procianidines structures quantified by absorption bands and by other

parameters secured by derivative spectroscopy.

Finally, it needs to be indicated that according to the bibliographical information, the serial diode

detectors have been essential in the characterization of polyphenolic compounds from all types

of foods, not only in drinks like tea or wine, but also in the detection of polyphenolic compounds

in fruits and vegetables. The fluorescence has been also employed for this purpose, although to

the lesser extent than UV-VIS detection with hope of improving sensitivity as well as selectivity

after the identification of the polyphenolic compound.

It is quite relevant to emphasize that one of the first investigations carried out with HPLC and

related to the study of nonvolatile orange and tangerine oils fractions suggests the fluorimetric

detection in conjunction with conventional UV-IVS detection. Thereafter, and also in citric fruits

samples, fluorescence detection was used to identify five principal methoxylated flavones in

orange juices. Similarly, after the determination of isoflavonoids in a large number of legume

samples, Frank et al. (1994) detected cumestrol by relying on this technique, given the superior

fluorescent character of this compound. With respect to the latter, it is worth mentioning that

electrochemical detectors were also employed in the characterization of polyphenolic compounds

such as isoflavonoids found in soybean or other polyphenols proper of wine and orange samples.

In the first case, the elution was conducted under an isocratic regime, while in the second the

elution was carried out under gradient routine. When it comes to the second case, 16 electrodes

connected in series at different potentials were subsequently employed. Finally, let us indicate

that HPLC coupled to mass spectrometer has allowed the resolution of many complex mixtures

of polyphenolic compounds.

1.1.2.4 Capillary Electrophoresis

From a technical point of view, the determination of polyphenolic compounds stored in

vegetables and produce does not seem to benefit from the use of this technique, although some

articles dealing with this subject can be retrieved easily from the bibliography. The model

separation involving this method was applied during the isolation of polyphenols from orange

juice, using sodium borate buffer at 35 mM with 5% of AcN and 21 kV voltage as electrode

potential. The developed method allowed the determination of flavonoids in alkaline medium

and the elimination of carotenoids by electrically induced osmotic flow.

The behavior of flavonoid migration in micelle electrokinetic chromatography has been studied

to a lesser extent. Some factors, such as applied voltage, capillary temperature, the concentration

and nature of the electrolyte (that is to say, complex or simple buffers), the concentration and

nature of surfactant agent responsible for micelles and organic modifiers have demonstrated an

influence on resolution and the selectivity of the separation. The addition of organic modifiers

such as methanol alters the interaction between analytes and miceller phase. Therefore, it was

possible to observe that the presence of this organic solvent triggers decomposition of peaks

corresponding to the most hydrophobic flavones.

Nonetheless, this phenomenon can be avoided if acetonitril is used as an organic solvent.

However, many scientists maintain that both capillary electrophoresis and HPLC are

indispensable analytical techniques, because in many cases they complement each other,

especially when it comes to secure general information about the presence of polyphenolic

compounds in certain foods. Even though analytical glitches may complicate HPLC, this can be

resolved through the employment of electrophoresis techniques.

1.1.2.5 UV-VIS Spectrophotometry

The spectrophotometric methods are not new to the field of analytical chemistry, as they are

often used to determine what in scientific terms is known as total polyphenols. The following

chemical mixture, Folin-Ciocalteu became the most frequently prescribed reagent for the

formation of colored compounds, crucial in polyphenolic determination. Basically, this method

consists of generating a certain color through the addition of the mentioned reagent into alkaline

medium replete with a liquefied sample. In most cases, the transformation is accomplished in the

presence of anhydrous sodium carbonate (75 g/L) with the subsequent spectrophotometric

evaluation at 750 nm. Swain and Goldstein (1964) have reviewed different spectrophotometric

methods currently available and based on the evidence they have strongly recommended Folin-

Ciocalteu as the most suitable reagent for spectrophotometric estimation of total polyphenols.

However, vanillin method seems to be more adequate for isolation of catechins when these are

suspected to constitute prevailing polyphenolic structures in a given sample.

Nonetheless, it needs to be stressed that Folin- Ciocalteu reagent, which was and still is used

with relative frequency, also reacts with other polyphenolic structures and, consequently, these

detractors ought to be eliminated in a stage previous to detection, or well calculated “a

posteriori” as the weight exerted on total polyphenolic fraction. As a matter of fact, other

reagents such as Prussian blue have been also employed for that purpose, albeit less frequently.

It is well known that spectrophotometric methods generally yield a gross estimation of the

polyphenolic content. Consequently, these methods were employed in the rough analysis of

polyphenolic compounds found in wines, legumes, and apple juice. Notwithstanding, they may

be useful in batch analysis or individual separation through continuous flow. Applying this

criterion Carmona et al. (1991) successfully determined total polyphenolic configuration of

tannins and Non-tannins in samples from common white and black beans varieties. In these

experiments extracts of ground beans mixed with MeOH and HCl at 1% were separated into two

fractions: tannins and non-tannins, after passing the substrate through Sephedex LH-20 column.

1.1.3 Analytical techniques used in the determination protease inhibitors/trypsin inhibitors

In a given plant food, a high content of protein with proportions of essential amino acids that are ideally

suited to satisfy the nutritional needs of humans does not necessarily ensure that the proteins will be

efficiently digested and absorbed by healthy people. This caveat is based on the possible co-presence of

protease inhibitors in the ingested plant, inhibitors that might reach the small intestine and block the

activity of proteases, such as trypsin, that normally catalyze the hydrolysis of dietary proteins, an

obligatory step in the process of protein digestion and absorption. For example, the Kunitz trypsin

inhibitor and the Bowman-Birk inhibitor in soybeans interfere with the digestion of protein in the

intestinal tract. Protease inhibitors prevent the complete breakdown and efficient absorption of dietary

protein.

Four main classes of proteolyic enzymes have been routinely utilized to describe proteases. The

serine proteases are probably the best characterized. This class of proteases includes trypsin,

chymotrypsin, and elastase. The cysteine protease class includes papain, calpain, and lysozomal

cathepsins. Aspartic proteases include pepsin and rennin. Metalloproteinases include thermolysin

and carboxypeptidase A. During isolation and characterization one or all four classes of

proteases may pose a threat to the fate of a protein.

Inhibitors of the digestive enzymes trypsin and chymotrypsin are present in many plant foods but most

notably in several varieties of legumes and seeds, soybeans and related beans, peas, jack beans and millet.

The leaves of some plants also contain protease inhibitors.

The most commonly employed procedure for determining the trypsin inhibitor activity of legume

products is based on the inhibition of the hydrolytic activity of bovine trypsin on the synthetic

substrate /V-alpha-DL-arginine-p-nitroanilide or casein, as originally proposed by Kakade et

al.(1969).This method has been subject to numerous modifications designed to increase its

accuracy and reproducibility (Kakade et al., 1974; Hammerstrand et al., 1981).

UV/Vis spectrophotometery

There are various methods which have been used by various researchers. According to the method

adopted from that of Dietz et al. (1974), known amount of dried weighed food sample (one hundred mg)

extracted with 5 ml of double-distilled water. Each sample is vortexed periodically for 8 hours and then

allowed to sit overnight at 40C. The samples are then sonicated for 30 sec at a setting of 3 in a Heat

Systems-Ultrasonics, Inc. sonicator and allowed to stand for 1 hour at 250C. The extracts are centrifuged

for 10 min at 12,000 x g using a superspeed centrifuge and filtered through No. 2 grade filter paper. The

supernatants are assayed for their antitrypsin content before and after boiling for five minutes.

The reagents consist of a 100 mM Tris buffer, pH 8.2, containing 20 mM CaCl2 which is prepared by

dissolving 12.1 g of Tris (hydroxymethyl)-aminomethane and 2.2 g of CaCl2 in distilled water. The pH is

adjusted to pH 8.2 with HCl and the volume is brought to 1 liter with distilled water. The α-N-benzoyl-

DLarginine-p-nitroanilide (BAPNA) substrate is prepared by dissolving 43 mg of BAPNA in 1 ml of

dimethyl sulfoxide, which is then brought to 1 liter using the Tris buffer. A stock solution of trypsin

enzyme is prepared by dissolving 10 mg of powdered bovine trypsin (Sigma) in 10 ml of 1 mM HCl. A

working solution of trypsin was made by adding 1 ml of the trypsin stock solution to 24 ml of Tris buffer.

A stock solution of the protein „control‟ is prepared by dissolving 4 g of bovine serum albumin in 100 ml

of buffer. An albumin working solution is prepared by diluting the stock solution 1:100 in Tris buffer.

Into 13 x 100 mm test tubes, 0.4 ml of each food extract and 0.4 ml of the trypsin working solution are

added. The mixture is incubated at 250C for 10 min. Concurrently, 1 ml of the BAPNA solution is

allowed to incubate in four test tubes in a 370C water bath. Each analysis is performed in triplicate. A 0.2

ml aliquot of the food extract/trypsin mixture is pipetted into 3 of the 4 test tubes containing BAPNA

solution. The contents of the three test tubes are vortexed and incubated for 10 min in a 370 C water bath,

after which the reaction is quenched with 0.2 ml of 30% (v/v) acetic acid. To the fourth test tube, 0.2 ml

of an albumin/trypsin (1:1) mixture is added to provide the control.

Absorbance is determined using a Perkin-Elmer UV/Vis spectrophotometer at 400 nm. From the

absorbances of the „test‟ and „albumin control‟ incubations, the extent of trypsin inhibition is calculated.

Assays are performed in triplicate and results are expressed as µg trypsin inhibited/mg dry weight of food

material.

Trypsin inhibitor activity has also been measured by using the modified method of Roy and Rao

(1974) in legumes. Reagents used are 0.1 M phosphate buffer (pH 7.6), 0.05 M phosphate buffer

(pH 7.0), Casein solution (2%), trypsin solution (5 mg/ml), 0.001 N HCl and Trichloroacetic acid

(5%). Known amount of food sample (1g) is mixed with 25 ml 0.05 M phosphate buffer, shaken

at room temp. For 3 h and centrifuged at 10, 000 rpm for 20 min. The following sets of

incubation mixtures are prepared.

Test Control Blank

Phosphate buffer (0.1 M, pH 7.6) 1.0 ml 1.1 ml 1.0 ml

Trypsin solution (mg/ml) 0.5 ml 0.5 ml 0.5 ml

HCl (0.001 N) 0.4 ml 0.4 ml 0.4 ml

TCA (5%) --- --- 6.0 ml

Casein (2%) 2.0 ml 2.0 ml 2.0 ml

Extract 0.1 ml --- 0.1 ml

Incubated at 370C for 20 min.

TCA (5%) 6.0 ml 6.0 ml ---

After incubation and addition of TCA contents were centrifuged at 10,000 rpm for 10 min TCA

soluble proteins in supernatant were determined by the method of Lowry et al. (1951).

Trypsin inhibitors units

One unit of trypsin is defined as the amount of enzyme which converted one mg casein to TCA

soluble components at 370C for 20 min at pH 7.6. One unit of inhibitory activity is that which

reduces the activity of trypsin by one unit under the assay conditions.

Other methods

Affinity chromatography using immobilized trypsin has also been suggested as a means of

avoiding interference from non-protein type of inhibitors (Roozen and de Groot, 1987; Roozen

and de Groot, 1985). The introduction of monoclonal antibodies directed specifically toward the

Kunitz and the Bowman-Birk inhibitors (Brandon et al., 1987; Brandon et al., 1988; Brandon et

al., 1989) should prove useful for the specific quantitation of these two inhibitors by an

immunochemical approach. It is important to note that most of the trypsin inhibitor assays

referred to above involve the measurement of the extent to which trypsin of bovine origin is

inhibited. This is frequently done despite the fact that the investigator may be interested in the

nutritional effects that may be expected in a completely unrelated species of animal. There are

differences in the response of various species of animals to the physiological effects of trypsin

inhibitors. Such differences have also been observed with respect to the in vitro inhibition of the

proteases in the pancreatic juice of different species of animals (Krogdahl and Holm, 1979;

Rascon et al., 1985). Another point to consider is the fact that not all protease inhibitors retain

their full activity after exposure to gastric juice. For example, the Kunitz inhibitor is readily

inactivated by human gastric juice, whereas the Bowman- Birk inhibitor retains its activity under

the same conditions (Krogdahl and Holm, 1981). For these reasons, any attempt to extrapolate

the results of in vitro assays for protease inhibitor activity to their true physiological effects in a

particular animal species must be viewed with caution.

1.1.4 Saponins

As a result of increased interest and intensive research activity in microcomponents of foods of

plant origin, knowledge concerning the saponins of foods increased substantially in the last few

years. Triterpenoid and steroid saponins occur primarily in legumes seeds, nevertheless a lot of

other foods and raw food materials contain small amounts of saponins.

The common methods for detection of saponins are colour reactions and haemolysis (Kerem et

al., 2005; Muetzel et al., 2003). The former have the inherent disadvantage of lack of specificity.

The capacity to haemolyse erythrocytes is one of the important properties of saponins

(Gauthieret al., 2009). Haemolytic property has been used for detection of saponins on thin layer

chromatograms (Muetzel et al., 2003). However, the protocol used did not provide sharp spots of

saponins against a clear background. Hence, the classic methods based on haemolytic activity of

saponins and the precipitation methods are now only of historical interest.

1.1.4.1 Spectrophotometry

Saponins have also been determined by spectrophotometric methods, using oleanolic acid as

reference. The spectrophotometric methods utilize the colour produced by the reaction of

saponins with vanillin or anisaldehyde. These methods are not suitable for estimating saponins in

plant extracts due to the fact that the reactions are not specific, and coloured products can be

produced from other compounds such as flavonoids (Oakenfull, 1981).

1.1.4.2 Chromatographic methods

A number of Chromatographie methods have been used for saponin analysis (thin layer

chromatography, gas chromatography, HPLC, etc.). Special attention has focused on the use of

gas chromatography, but it has its limitations. It can only be used for the separation and

quantification of the aglycon portion or the saponin after hydrolysis and derivatization (which

involves both the loss of structural information about the glycosidic portion of the molecule and

the potential loss of material during hydrolysis and derivatization).

TLC

Thin layer chromatography (TLC) has the advantage of speed of analysis and comparison of

many samples simultaneously, versatility of supports, solvent systems and detection reagents

(Stahl, 1969). These attributes make TLC an ideal classic tool for first stage of phytochemical

analysis as well as for monitoring of column chromatography fractions during purification of

natural plant products.

Sharma et al. (2011) have reported an improved method for thin layer chromatographic analysis

of saponins. In their reported method, the solvent system was n-butanol: water:acetic acid

(84:14:7). Detection of saponins on the TLC plates after development and air-drying was done

by immersion in a suspension of sheep erythrocytes, followed by washing off the excess blood

on the plate surface. Saponins appeared as white spots against a pink background. The protocol

provided specific detection of saponins in the saponins enriched extracts. The method has been

applied to saponin extracts from ten saponin-rich plants and compared with the common

detection method based on acid based spray and heating (Kerem et al., 2005). The protocol is

convenient, inexpensive, does not require any corrosive chemicals and provides specific

detection of saponins.

HPLC

The use of HPLC in the separation of saponins is now widespread. Although the determination

of optimal parameters for the separation of individual components of a mixture is generally time-

consuming, the process becomes indispensable in many cases for its efficient and successful

separation of pure saponins.

A modified HPLC method described by Kesselmeir et al. (1981) was used for the determination

of saponins by Ruales a'b and Nair (1993). The saponins were extracted from 15 g of flour with

150 ml of methanol for 24 h, using a Soxhlet apparatus, after the seeds were defatted with 150 ml

of petroleum ether for 16 h. After the extraction, the methanol was evaporated at 35°C using

vacuum, and the residue was dissolved in 5 ml of methanol. Separa tion of the saponins was

performed by injecting 20 /xl of the sample into a high-performance liquid chromate graph

(Varian Model 5000 Liquid chromatograph, Varian Associates, Sunnyvale, CA, USA) equipped

with a column (4 mm x 250 mm) packed with LiChrospher 100 CH-8/2 (5 /~m) a UV detector

and an integrator (Shimadzu C-R3A Chromatopac, Kyoto, Japan). The detection wavelength

was 200 nm. A gradient elution was performed with 25 to 40% acetonitrile in water during 15

min. The flow rate was 2.0 ml/min. Saponins A and B isolated from quinoa seeds were used as

standards.

The HPLC method is suitable for the analysis of saponins in crude extracts of plant tissues. It has

the advantage over other methods used as it has high accuracy and precision. However, suit able

standards of saponins are necessary.

1.1.4.3 Other methods

The technique of DCCC (droplet counter-current chromatography) has found application in

isolation and purification of saponins (Hostettman et al., 1979). The centrifugal liquid

chromatography has the potential for separating closely related saponins as shown by Kitagawa

et al. (Kitagawa et al., 1983; Kitagawa et al., 1983). Flash chromatography (Still et al., 1979) is

basically an air-pressure driven hybrid of medium pressure and short column chromatography

which is optimized for particularly rapid separations. This technique has great potential for the

large-scale isolation of plant saponins (Price and Fenwick, 1984). Several of these techniques are

necessary for the separation of individual saponins from a complex mixture. Nevertheless, the

integration of silica gel column chromatography, semipreparative HPLC, and repeated

preparative TLC may yield expected separation in most cases.

1.1.5 Lectin

Lectins (agglutinins) are cell-agglutinating proteins or glycoproteins of non-immune origin that

bind carbohydrates without modifying them chemically. Lectins are either soluble or membrane-

bound. They exist in a wide variety of plants, animals, bacteria and viruses.

Isolation, purification and determination

The purification of lectins to homogeneity poses problems not commonly encountered in the

purification of other proteins. Lectins may appear in multiple forms that possess more or less

similar biological activities and differ only slightly in their chemical and physical properties.

Many lectins are composed of subunits, which may undergo different association-dissociation

reactions. The chromatographic behavior and other characteristics of lectins may depend on the

experimental conditions, especially the presence of certain metal ions. The specific affinity for

certain sugar residues can be used for the purification of lectins. Lectins can be obtained in a

single step in relatively pure form and in excellent yield by affinity chromatography. The use of

affinity chromatography has permitted the purification of many lectins. Lectin affinity

chromatography is a form of affinity chromatography where lectins are used to separate

components within the sample. Lectins, such as Concanavalin A are proteins which can bind

specific carbohydrate (sugar) molecules. The most common application is to

separate glycoproteins from non-glycosylated proteins, or one glycoform from another

glycoform.

Formalized erythrocytes can also be used for the isolation of lectins from crude plant extracts.

Advantage may be taken of the fact that most lectins are glycoproteins and can therefore, interact

with some other lectins. Thus, concanavalin A covalently bound to Sepharose can be used for the

removal of different lectins from crude plant extracts.

The detection of lectins in plant extracts is still performed mostly by the simplest assay i.e. some

variation of a basic procedure that involves serial dilution and in which the end-point is

determined by the highest dilution (least amount of lectin) that still gives a clumping of the cells

as perceived by visual inspection. A microdilution technique has found widespread acceptance

because of the minute amounts of sample needed. It can be performed with a single seed or part

of a seed. Commercial microtitration kits are available for this purpose and are suitable for

routine testing of multiple samples. The presence of sodium chloride or some other salt is

required for agglutination. The washed red blood cells should be activated by 0.5 h treatment

with a suitable proteinase, pronase, trypsin or papain, since the sensitivity of the agglutinin

reaction is usually enhanced considerably by this treatment. A control with a plant extract known

to contain a lectin of specificity similar to the one being investigated must be included in the

experiment. A positive control is always required with samples of unknown activity. Human and

rabbit blood are most frequently used but may not be suitable for the detection of some specific

lectins. Lectin activity is most commonly determined by measuring the least amount of test

sample necessary to agglutinate the red blood cells of a given animal. The agglutination-dilution

test is only semi-quantitative and has been critically evaluated by Burger (1974).

One can also ascertain the specificity of the lectin by incorporating various concentrations of a

given sugar fact that one must choose the blood or blood group for which the lectin is specific.

Improper selection of red blood cells may result in low sensitivity or even negative results. In the

specific case of the soybean lectin, trypsinated or Papain-treated human or rabbit erythrocytes

have proven to be the most sensitive blood system.

Kaul et al.(1991) have proposed the substitution of polystyrene latex beads, to which various

glycoproteins have been bound, in place of red blood cells. This technique not only obviates the

need for fresh blood, which may not always be available, but also provides a convenient method

for the determination of the specificity of a given lectin. In the case of the soybean lectin, the

covalent coupling of the latex beads to Af-acetyl-galactosamine or lactosamine proved to be the

most sensitive agglutinating system.

A quantitative method devised by Liener (1955) is based on the photometric measurement of the

density of a suspension of erythrocytes that are not agglutinated by the lectin. Several more

sophisticated methods have been proposed for investigating the lectin-cell surface interaction.

For example, Hwang et al. (1974) and Kaneko et al. (1975) have applied spectrophotometric

measurements to the study of the binding of lectins to cancer cells.

Vargas-albores et al. (1987) assayed hemagglutinating activity in legume sample using microtiter

sets. The lectin solution was diluted successively twofold with 0.15 M phosphate buffered saline

+ 1 mM CaC12, pH 7.2, and a 2% suspension of donkey erythrocytes was added. Agglutinating

activity was measured after standing for one hour at room temperature.

An immunoenzymatic method for the quantitative determination of dietary lectin activities

employing immobilized glycoproteins was studied by Vincenzi et al. (2002). Lectins from wheat

germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with

ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with

lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the

concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA.

Inhibition assays carried out with different saccharides confirmed that the binding of lectins to

immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing

the quantitative determination of lectin activities on raw samples by simple dilution of the

extracts. Examples of application to wheat germ and roasted peanut extracts are reported.

Rizzi et al. (2003) have described modified immunoenzymatic method for the quantitative

determination of biologically active lectins in unknown samples to measure the concentration of

active soybean lectin (SBA) in food stuffs. In their work, a modified immunoenzymatic method

previously developed (Vincenzi et al., 2002) for the quantification of biologically active lectins

in raw samples of wheat germ and roasted peanut has been described and applied to SBA

determination in soybean-derived foodstuffs and soy sprouts.

References

Phytic acid

1. Blatny, P., Frantisek, K. and Kenndler, E. 1995. Determination of phytic acid in cereal

grains, legumes and feeds by capillary isotachophoresis. Journal of Agricultural Food

Chemistry, 43, 129–133

2. Bradbury MG, Egan SV, Bradbury JH (1999). Determination of all forms of cyanogens

in cassava roots and cassava products using picrate paper kits.J. Sci. Food. Agric.79, 593-

601.

3. Brunner JH (1984). Direct spectrophotometric determination of saponin. Anal. Chem.42:

pp 1752-1754.

4. Camire, A. L. and Clydesdale, F. M. 1982. Analyses of phytic acid in foods by HPLC.

Journal of Food Science, 47, 575–578.

5. Cilliers, J. J. L. and Van Niekerk, P. J. LC. 1986. Determination of phytic acid in food

by postcolumn colorimetric detection. Journal of Agricultural a n d Food Chemistry,

34, 680–683.

6. Cosgrove, D. J. 1980a. Inositol Phosphates. Their Chemistry, Biochemistry and

Physiology. Amsterdam: Elsevier.

7. Cosgrove, D. J. 1980b. The determination of myo-inositol hexakisphosphate (phytate). J.

Sci. Food Agric. 31:1253-1256.

8. Dawra RK, Makkar HSP, Singh B (1988). Protein binding capacity of microquantities of

Tannins. Analytical Biochemistry, 170, pp. 50-53.

9. De Boever , J. L., Eeckhout, W. and Boucque, C. V. 1994. The possibilities of near

infrared reflection spectroscopy to predict total-phosphorus, phytate-phosphorus a n d

phytase activity in vegetable f e e d s t u f f s . Netherlands Journal of Agricultural

Science, 42, 357–369.

10. De Boever, J. L., W. Eeckhout, and C. V. Boucque. 1994. The possibilities of near

infrared reflection spectroscopy to predict total-phosphorus, phytate-phosphorus and

phytase activity in vegetable feedstuffs. Neth. J. Agric. Sci. 42:357-369.

11. De Koning, A. J. 1994. Determination of myo-inositol and phytic a c i d b y gas

chromatography using s c y l l i t o l a s internal standard. Analyst, 119, 1319–1323.

12. Ellis, R. and Morris, E. R. 1986. Appropriate resin selection for rapid phytate

a n a l y s e s by ion-exchange chromatography. Cereal Chemistry, 63, 58–59.

13. Ellis, R., Morris, E. R. and Philpot, C. 1977. Quantitative determination of phytate i n

the presence of high inorganic phosphate. Analytical Chemistry, 77, 536–539.

14. Erso Z, A., Akgu n, H. and Aras, N. K. 1990. Determination of phytate in Turkish diet

by phosphorus-31 Fourier trans-form nuclear m a g n e t i c resonance spectroscopy.

Journal of Agricultural and Food Chemistry, 38, 733–735.

15. Fasset DW (1996). Oxalates. In: Toxicants occurring naturally in foods. National

Academy of Science Research Council, Washington D.C, U.S.A. Maga JA (1983).

Phytate: Its chemistry, occurrence, food interaction, nutritional significance and methods

of analysis. J. Agric Food Chem. 30: 1-9.

16. Feil, B. 2001. Phytic Acid. Journal of New Seeds, 3:3, 1-35.

17. Frolich, W., Drakenberg, T. and Asp, N.G. 1986. Enzymic degradation of phytate (myo-

inositol Hexaphosphate) in whole grain flour suspension and dough. A comparison

between 3 1 P NMR spectroscopy and ferric ion method. Journal of Cereal Science,

4, 325–334.

18. Fru Hbeck, G., Alonso, R., Marzo, F. and Santidria´ N, S. A. 1995. Modified method

for the indirect quantitative analysis of phytate in foodstuffs. Analytical

Biochemistry, 225, 206–212.

19. Fujita, Y., Mori, I., Tanaka, T., Koshiyama, Y. and Kawabe, H. 1986. Application of o-

hydroxyhydroquino- nephtalein–iron (III) complex to determination of organic

compounds containing phosphorus. Chemical Pharmaceutical Bulletin, 34, 2236–

2238.

20. Han, O., M. L. Failla, A. D. Hill, E. R. Morris, and J. C. Smith Jr. 1994. Inositol

phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line. J.

Nutr. 124:580-587.

21. Han, O., M. L. Failla, A. D. Hill, E. R. Morris, and J. C. Smith Jr. 1994. Inositol

phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line. J.

Nutr. 124:580-587.

22. Harland, B . F. and Oberleas, D. 1986. Anion-exchange method for determination of

phytate in foods: Collaborative study. Journal of the AOAC, 69, 667–670.

23. Harland, B. F. and Oberleas, D. A 1 9 7 7 . Modified method for phytate analysis

using an ion-exchange procedure: Application to textured vegetable proteins. Cereal

Chemistry, 54, 827–832.

24. Harland, B. F., and G. Narula. 1999. Food phytate and its hydrolysis products. Nutr. Res.

19:947-961.

25. Hatzack, F., and S. K. Rasmussen. 1999. High-performance thin-layer chromatography

method for inositol phosphate analysis. J. Chromatography B 736:221-229.

26. Haug, W., and H.-J. Lantzsch. 1983. Sensitive method for the rapid determination in

cereals and cereal products. J. Sci. Food Agric. 34:1423-1426.

27. Henry TA (1973). Organic Analysis of Alkaloids. 6 : 163-187.

28. Heubner, W. and Standler, H. (1914). Ubereine titrationsmethode zur Bestimmung des

Phytins. Biochemical Zeitschrift, 64, 422–437.

29. Jaffe WG (1979). Haemagglutinin in toxic constituents of plant foodstuff (Liener JE Ed.)

Academy Press. N.Y. p. 71.

30. Kemme, P. A., Lommen, A., de Jonge, L. H., van der Klis, J. D. Jongbloed, A. W.,

Mroz, Z. and Beynen, A.C. 1999. Quantification of inositol phosphates using P-31

nuclear magnetic resonance spectroscopy in animal nutrition. J. Agric. Food Chem.

47:5116-5121.

31. Latta, M. and Eskin, M. A. 1980. Simple and rapid colorimetric method for

p h y t a t e determination. Journal of Agricultural and Food Chemistry, 28, 1315–

1317.

32. Lee, K. and Abendroth, J. A. 1983. High performance liquid chromatographic

determination of phytic acid in foods. Journal of Food Science, 48, 1344–1345, 1351.

33. Lehrfeld, J . 1994. HPLC separation and quantitation o f phytic a c i d and some

inositol phosphates in foods: problems and solutions. Journal of Agricultural and Food

Chemistry, 42, 2726–2731.

34. Lehrfeld, J. and Morris E.R. 1992. Overestimation of Phytic Acid in Foods by the

AOAC Anion-Exchange Method. J. Agric. Food Chem. 1992, 40, 2208–2210.

35. Lehrfeld, J. and Morris, E. R. 1992. Overestimation of phytic acid in foods by the

AOAC anion-exchange method. Journal of Agricultural and Food Chemistry, 40,

2208–2210.

36. Lehrfeld, J. 1994. HPLC Separation and Quantitation of Phytic Acid and Some Inositol

Phosphates in Foods: Problems and Solutions. J. Agric. Food Chem. 42, 2726–2731.

37. Lehrfeld, J.; Morris E.R. 1992. Overestimation of Phytic Acid in Foods by the AOAC

Anion-Exchange Method. J. Agric. Food Chem. 40, 2208–2210.

38. Liener IE (1980). In: Advances in legume science. RJ Summerfield and AH. Bunting

(eds.), Academic Press, New York, London.

39. Lönnerdal, B., A.-S. Sandberg, B. Sandström, and C. Kunz. 1989. Inhibitory effects of

phytic acid and other inositol phosphates on zinc and calcium absorption in suck- ling

rats. J. Nutr. 119:211-214.

40. March, J. G., Villacampa, A. I. and G r a s e s , F. 1995. Enzymatic-spectrophotometric

determination of phytic ac id with phytase from Aspergillus ficuum. Analytica Chimica

Acta, 300, 269–272.

41. Mathews, W. R., Guido, D. M. and Huff, R. M. 1988. Anion- exchange high-

performance liquid chromatographic analysis of inositol phosphates. Analytical

Biochemis t ry , 168, 63–70.

42. McCance, R. A. and Widdowson, E. M. 1935. Phytin in human nutrition. Biochemical

Journal, 29, 2694–2699.

43. Meek, J. L. and Nicoletti, F. 1986. Detection of inositol trisphosphate and other

organic phosphates by high- performance liquid chromatography using an enzyme-

loaded post-column reactor. Journal of Chromatography, 351, 303–311.

44. Meek, J. L. 1986. Inositol bis-, tris-, and tetrakis (phosphate)s: Analysis in tissues by

HPLC. Proceedings of the National Academy of Sciences of the United States of

America, 83, 4162-4166.

45. Oatway, L., Vasanthan, T. and Helm, J.H. 2001. Phytic acid. Food Reviews International.

17 (4): 419-431.

46. Oberleas, D. 1971. The determination of phytate and inositol phosphates. In: Glick, D.

(Ed), Methods of Biochemical Analysis. New York: Wiley, pp. 87–101 (1971)

47. Oberleas, D. 1983. Phytate content in cereals and legumes and methods of determination.

Cereal Foods World 28:352-357.

48. Oberleas, D., and B. F. Harland. 1986. Analytic methods for phytate. In Phytic Acid

Chemistry and Applications, ed. E. Graf, 77-100. Minneapolis: Pilatus Press. p.77.

49. Phillippy, B. Q., Johnston, M. R., Tao, S.-H. and Fox, M. R. S.1988. Inositol

phosphates in processed foods. Journal of Food Science, 53, 496–499.

50. Plaami, S. and Kumpulainen, J. 1991. Determination of phytic acid in cereals using

ICP-AES to determine phosphorus. Journal of the AOAC, 74, 32–36.

51. Plaami, S., and J. Kumpulainen. 1995. Inositol phosphate content of some cereal-based

foods. J. Food Comp. Anal. 8:324-335.

52. Reddy, N. R., Pierson, M. D., Sathe, S. K. and Salukhe, D. K. 1989. Influence of

p r oce s s i ng technologies on phytate. Cooking. In: Phytates in Cereals and

Legumes. Advances in Food Research, Vol. 28, pp. 111–135 (1989).

53. Reddy, N.R.; Pierson, M.D.; Sathe, S.K.; Salunkhe, D.K.1989. Phytates in Cereals and

Legumes; CCRC Press, Inc.: Boca Raton, FL.

54. Rounds, M. A. and Nielsen, S. S. 1993. Anion-exchange high- performance liquid

c h r o m a t o g r a p h y wi th post-column detection for the analysis of phytic acid and

other inositol phosphates. Journal of Chromatography A, 653, 148–152.

55. Sandberg, A.S. and Ahderinne, R.1986. HPLC Method for determination of inositol

tri-, tetra-, penta-, hexaphosphates in foods and intestinal contents. Journal of Food

Science, 51, 547–550.

56. Sandberg, A.S., Carlsson, N.G. and Svanberg, U. 1989. Effects of Inositol tri-, tetra-,

penta-, and hexaphosphates on in vitro estimation of iron availability. Journal of Food

Science, 54, 159–186.

57. Sirkka and Plaami, 1997. Myoinositol Phosphates: Analysis, C o n t e n t in Foods and

Effects i n Nutrition. Lebensm.-Wiss. u.-Technol., 30, 633–647.

58. Soetan, K.O. and Oyewole, O. E. 2009. The need for adequate processing to reduce the

antinutritional factors in plants used as human foods and animal feeds: A review. African

Journal of Food Science Vol. 3 (9), pp. 223-232.

59. Talamond, P., S. Doulbeau, I. Rochette, J.-P. Guyot, and S. Treche. 2000. Anion-

exchange high-performance liquid chromatography with conductivity detection for the

analysis of phytic acid in food. J. Chromatography A 871:7-12.

60. Uppstro M, B. and Svensson, R. 1980. Determination of phytic acid in rapeseed meal.

Journal of the Science of Food and Agriculture, 31, 651–656

61. Wheeler, E. L. and Ferrel, R. E. 1971. A method for phytic acid determination in wheat

and wheat fractions. Cereal Chemistry, 48, 312–320.

62. Xu, P., Price, J. and Agge t t , P. J. 1 9 9 2 a . Recent advances i n methodology for

analyses of phytate and inositol phosphates in foods. Progress in Food and Nutrition

Science, 16, 245–262.

63. Xu, P., J. Price, A. Wise, and P. J. Aggett, 1992b: Interaction of inositol phosphates with

calcium, zinc, and histidine. J. Inorg. Chem. 47:119-130.

64. Young, L. 1936. The determination of phytic acid. Biochemical Journal, 30, 252–257.

Polyphenols

1. Achilli, G., Cellerino, G.P., Gamache, P.H.; Melzi d´eril., J. Chromatogr. A., 1993, 632,

111.

2. Bailey, R. G., McDowell, I.; Nursten, H.E., J. Sci. Food Agric. 1990, 52, 509.

3. Baldi, A., Romani, A., Mulinnaci, N., Vincieri, F.F.; Casetta, B., J. Agric. Food Chem.,

1995, 43, 2104.

4. Bartolomé, B., Bengoechea, M.L., Gálvez, M.C., Pérez-Ilzarbe, J., Hernández, T.,

Estrella, I.; Gómez- Cordovés, C., J. Chromatogr. A. 1993, 655, 119.

5. Bartolomé, B., Hernández, T., Bengoechea, M.L., Quesada, C., Gómez-Cordovés, C.;

Estrella, I., J. Chromatogr. A, 1996, 723, 19.

6. Betés-Saura, C., Andrés-Lacueva, C.; Lamuela- Reventós, J. Agric. Food Chem., 1996,

44, 3040.

7. Bilyk, A., Hicks, K.B., Bills, D.D.; Sapers, G.M., J. Liq. Chromatogr., 1988, 555, 137.

8. Bronner, W.E.; Beecher, G.R., J. Chromatogr. A., 1995, 705, 247.

9. Brune, M., Hallberg, L.; Skanberg, A.B., J. Food Sci., 1991, 56, 128.

10. Buiarelli, F., Cartoni, G.P., Coccioli, F.; Ravazzi, E., Chromatographia, 1991, 31, 489.

11. Cancalon, P. F.; Bryan, C.R., J. Chromatogr. A, 1993, 652, 555.

12. Cancalon, P.F., Food Technol (Chicago), 1995, 49(6), 52.

13. Carmona, A., Seidl, D ; Jaffé, W.G., J. Sci. Food Agric., 1991, 56, 291.

14. Castillo, J., Benavente-García, O.; Del Río, J.A., J. Liq. Chromatogr., 1994, 17(7), 1497.

15. Celesste, M., Tomás, C., Cladera, A., Estela, J.M.; Cerdá, V., Analytica Chimica Acta

1992, 269, 21.

16. Del Río, J.A., Fuster, M.D., Sabater, F., Porrás, I., García-Lindón, A.; Ortuño, A. J.

Agric. Food Chem., 1995, 43, 2030.

17. Delage, E., Bohuon, G., Baron, A.; Drilleau, J.F., J. Chromatogr. A. 1991, 555, 125.

18. Di Stefano, R.; Cravero, M.C., Riv. Vitic. Enol., 1991, 2, 37.

19. Dick, A. J., Redden, P.R., DeMarco, A., Lidster, P.D.;

20. Drawert, F., Leupold, G.; Pivernetz, H., Chem. Mikrobiol. Technol. Lebensm., 1980, 6(6),

189.

21. Drawert, F., Pivernetz, H., Leupold, G.; Ziegler, A., Chem. Mikrobiol. Technol. Lebensm,

1980, 6(5), 131.

22. Escarpa, A., González, M.C., Chromatographia, 2000, 51, 37

23. Escarpa, A., González, M.C., J. Chromatogr. A, 1998, 823, 331

24. Escarpa, A., González, M.C., J. Chromatogr. A, 1999, 830, 301

25. Fernández de Simón, B., Pérez-Ilzarbe, J., Hernández, T., Gómez-Cordovés, C.; Estrella,

I., J. Agric. Food Chem. 1992, 40, 1531.

26. Fernández de Simón, B., Pérez-Ilzarbe, J., Hernández, T., Gómez-Cordovés, C.; Estrella,

I., Chromatographia, 1990, 30, 35.

27. Finger, A., Kurh, S.; Engelhardt., J. Chromatogr. A., 1992, 624, 293.

28. Franke, A.A., Custer, L.J., Cerna, C.M.; Narala, K.K., J. Agric. Food Chem., 1994, 42,

1905.

29. Gao, L.; Mazza, G., J. Agric. Food Chem., 1995, 43, 343.

30. Gao, L.; Mazza, G., J. Food Sci., 1994, 59, 1057.

31. Ghiselli, A., Nardini, M., Baldi, A. Scaccini, C., J. Agric. Food Chem., 1998, 46, 361

32. González-San José, M.L.; Díez, C., Food Chem., 1992, 43, 193.

33. Greenham, J., Williams, C.; Harborne, J., Phytochem. Anal., 1995, 6, 211.

34. Grindley, T.B., J. Agric. Food Chem. 1987, 35, 529.

35. Guyot, S., Doco, T., Souquet, J.M., Moutounet, M.; Drilleau, J.F., Phytochemistry 1997,

44, 351.

36. Heimhuber, B., Galensa, R.; Herrmann, K., J. Chromatogr. A, 1988, 439, 481.

37. Hernández, T., Hernández, A.; Martínez. C., J. Agric. Food Chem., 1991, 39, 1120.

38. Hertog, M.G.L., Hollman, P.C.H.; Katan, M.B., J. Agric. Food Chem., 1992, 40, 2379.

39. Hertog, M.G.L., Hollman, P.C.H.; Venema, D.P.J., J. Agric. Food Chem., 1992, 40,

1591.

40. Hong, V.; Wrolstad, R., J. Agric. Food Chem., 1990, 38, 698.

41. Hong, V.; Wrolstad, R., J. Agric. Food Chem., 1990, 38, 708.

42. Kamiya, S., Esaki, S.; Konishi, F., Agric. Biol. Chem., 1972, 36, 1461.

43. Kanner, J., Frankel, E., Granit, R., German, B.; Kinsella, J.E., J. Agric. Food Chem.

1994, 42, 64.

44. Kanner, J., Frankel, E., Granit, R., German, B.; Kinsella, J.L., J. Agric. Food Chem.,

1994, 42, 64.

45. Keinänen, M., Julkunen-Tiitto, R., J. Chromatogr. A, 1998 , 793, 370

46. Kinosita, E., Sugimoto, T., Ozawa, Y., J. Agric. Food Chem. 1998, 46, 877

47. Kitada, Y., Ueda, Y., Nakazaba, H.; Fujita, M., J. Chromatogr. A. 1886, 366, 403.

48. Lea, A.G.H., J. Sci. Food Agric. 1979, 30, 833.

49. Mangas, J., Suárez, B.; Blanco, D., Z. Lebensm. Unters Forsch., 1993, 197, 424.

50. Mondello, L., Dugo, P., Bartle, K.D., Frere, B.; Dugo, G., Chromatographia, 1994, 39,

529.

51. Morin, Ph., Archambault, J.C., André, P., Dreux, M., Gaydou, E., J. Of Chromatogr.

A,1997, 791, 289

52. Mouly, P., Gaydou, E.M., Auffray, A., J. Chromatogr.A, 1998, 800, 171

53. Munekazu, M., Matsuura, S. Kurogochi, K.; Tanake, T., Chem. Pharm. Bull., 1980, 28,

717.

54. Nishiura, M., Esaki, S.; Kamiya, S., Agric. Biol. Chem., 1969, 33, 1109.

55. Nogata, Y., Ohta, K., Yoza, K.I,, Berhow, M.; Hasegawa, S., J. Chromatogr. A., 1994,

667, 59.

56. Oleszek, W., Amiot, M.J.; Aubert, S., J. Agric. Food Chem. 1994, 42, 1261.

57. Ooeghe, W.C., Ooeghe, S.J., Detavernier, C. M.; Huyghebaert, A., J. Agric. Food Chem.,

1994, 42, 2183.

58. Ooeghe, W.C., Ooeghe, S.J., Detavernier, C. M.; Huyghebaert, A., J. Agric. Food Chem.,

1994, 10.

59. Ortuño, A., García-Puig, D., Fuster, M.D., Sabater, F., Porras, I., García-Lindón, A.; Del

Río, J.A., J. Agric. Food Chem., 1995, 43.

60. Pérez-Ilzarbe, J., Hernández, T.; Estrella, I., Z. Lebensm.-Unters. Forsch. 1991, 192, 551.

61. Perfetti, G., Joe, F., Fazio, T.; Page, S., J. Assoc. Off. Anal. Chem., 1988, 71, 469.

62. Pietta, P., Gardana, C.; Mauri, P., J. High Resolut. Chromatogr., 1992, 15, 136.

63. Pietta, P.G., Mauri, P.L., Zini, L.; Gardana, C,. J. Chromatogr. A, 1994, 680, 175.

64. Poon, G.K., J. Chromatogr. A, 1998, 794, 63

65. Revilla, E., Alonso, E.; Estrella, I., Chromatographia, 1986, 22, 1.

66. Robards, K., Haddad, P.R.; Jackson, P.E. Principles and Practice of Modern

Chromatographic Methods, Academic Press, Londres, 1995.

67. Robards, K.; Antolovich, M. Analyst, 1997, 122, 11R

68. Roggero, J.P. American Laboratory News 1997.

69. Roggero, J.P., Archier, P.; Coen, S., ACS Symposium Series, 1997, 2, 6.

70. Roggero, J.P., BioFactors 1997, 6, 441.

71. Roggero, J.P., Coen, S.; Archier, P., Bull liasions Groupe Polyphenols. 1990, 15, 244.

72. Rommel, A.; Wrolstad, R., J. Agric. Food Chem., 1993, 41, 1941.

73. Rommel, A.; Wrolstad, R., J. Agric. Food Chem., 1993, 41, 1951.

74. Rommel, A.; Wrolstad, R., J. Agric. Food Chem.,1993, 41, 1237.

75. Rouseff, R. L., Seetharaman, K., Naim, M., Nagy, S.; Zehavi, U., J. Agric. Food Chem.,

1992, 40, 1139.

76. Sato, M., Ramarathnam, N., Suzuki, Y., Ohkubo, T., Takeuchi, M.; Ochi, H., J. Agric

Food Chem. 1996, 44, 37.

77. Sendra, J.M., Navarro, J.L.; Izqquierdo, L., J. Chromatogr. Sci., 1988, 26, 443.

78. Spanos, G.A. Wrolstad, R. E.; Heatherbell, D.A., J. Agric. Food Chem. 1990, 38, 1572.

79. Spanos, G.A.; Wrolstad, R.E., J. Agric. Food Chem. 1990, 38, 1565.

80. Strack, D.; Wray, V, In Methods in Plant Biochemistry,Vol. I, Plant Phenolics, ed.

Harborne, J.B., Academic Press, Londres 1989, pp. 325-356.

81. Suarez-Vallés, B., Santamaría-Victorero, J., Mangas.Alonso, J.J.; Blanco-Gomis, D. J.

Agric. Food Chem., 1994, 42, 2732.

82. Swain, T.; Goldtein, J.L., in Methods in Polyphenol Chemistry, ed. Pridham, J.B.,

Pergamon Press, Oxford, 1964, pp. 131-146.

83. Tomás-Barberán, F.A., Phytochem. Anal., 1995, 6, 177.

84. Tomás-Lorente, F., García-Viguera, C., Ferreres, F.;Tomás-Barberán, F.A., J. Agric.

Food Chem. 1992, 40, 1800.

85. Tsuchiya, H., Sato, M., Kato, H., Okubo T., Juneja, LR., Kim, M., J. of Chromatogr.B,

1997, 703, 253

86. Venkataraman, K. In The Chemistry of Flavonoid Compounds, ed. Geissman, T.A.,

Pergamon Press, Oxford, 1962, p.70

87. Watanabe, M., J. Agric. Food Chem. , 1998, 46, 839

88. Weintraub, R. A., Ameer, B., Johnson, J.V.; Yost, R.A., J. Agric. Food Chem., 1995, 43,

1966.

89. Winter, M.; Herrmann, K., J. Agric. Food Chem., 1986, 34, 616.

Trypsin inhibitor

1. Brandon, D. L., Bates, A. H., and Friedman, M.1988. Enzyme-linked immunoassay of

soybean Kunitz trypsin inhibitor, J. Food Sci., 53, 102, 1988.

2. Brandon, D. L., Bates, A. H., and Friedman, M., Monoclonal antibody-based enzyme

immunoassay of the Bowman-Birk protease inhibitor of soybeans, J. Agric. Food Chem.,

37, 1189, 1989.

3. Brandon, D. L., Haque, S., and Friedman, M., Interaction of monoclonal antibodies with

soybean trypsin inhibitors, J. Agric. Food Chem., 35, 195, 1987.

4. Dietz AA, Rubenstein HM, Hodges L (1974) Measurement of alpha-1-antitrypsin in

serum, by immunodiffusion and by enzymatic assay. Clin Chem 20: 396–399.

5. Hammerstrand, G. E., Black, L. T., and Glover, J. D., Trypsin inhibitor in soy products:

modification of the standard analytical procedure, Cereal Chem., 58, 42, 1981.

6. Kakade, M. L., Rackis, J. J., McGhee, J. E., and Puski, G., Determination of trypsin

inhibitor activity of soy products: a collaborative analysis of an improved procedure,

Cereal Chem., 51, 376, 1974.

7. Kakade, M. L., Simons, N., and Liener, I. E., An evaluation of natural vs. synthetic

substrates for measuring the antitryptic activity of soybean samples, Cereal Chem., 46,

518, 1969.

8. Krogdahl, A. and Holm, H., Inhibition of human and rat pancreatic proteinases by crude

and purified soybean proteinase inhibitors, J. Nutr., 109, 551, 1979.

9. Krogdahl, A. and Holm, H., Pancreatic proteinases from man, trout, rat, pig, mink, and

fox. Enzyme activities and inhibition by soybean and lima bean proteinase inhibitors,

Comp. Biochem. Physiol., 74B, 403, 1983.

10. Krogdahl, A. and Holm, H., Soybean proteinase inhibitors and human proteolytic

enzymes: selective inactivation of inhibitors by treatment with human gastric juice, J.

Nutr., 111, 2045, 1981.

11. Rascon, A., Seidl, D. S., Jaffe, W. G., and Aizman, A., Inhibition of trypsins and

chymotrypsins from different animal species: a comparative study, Comp. Biochem.

Physiol., 82B, 375, 1985.

12. Roozen, J. P. and de Groot, J., Analysis of low levels of trypsin inhibitor activity in

food, Lebensm. Wiss. & Technol., 20, 305, 1987.

13. Roozen, J. P. and de Groot, J., Electrophoresis and assay of trypsin inhibitors in

different stages of tempeh production, J. Food Biochem., 9, 37, 1985.

14. Roy, D.N. and Rao, P.S. 1971. Evidence isolation purification and some

properties of a trypsin inhibitors in Lathyrus sativus. J. Agric. Food Chem. 19 :

257-259.

15. Smith, C., Van Megen, W., Twaalfhoven, L., and Hitchcock, C., The determination of

trypsin inhibitor levels in foodstuffs, J. Sci. Food Agric., 31, 341, 1980.

16. Vanderjagt, D.J., Freiberger, C., Vu, T.N., Mounkaila, G., Glew, R.S. and Glew, R.H.

2000. The trypsin inhibitor content of 61 wild edible plant foods of Nige. Plant Foods for

Human Nutr. 55: 335-346.

Saponins

1. Gauthier, C., Legault, J., Girard-Lalancette, K., Mshvildadze, V. and Pichette, A.2009.

2. Hemolytic activity, cytotoxicity and membrane cell permeabilization of semisynthetic

and natural lupane- and oleanane-type saponins. Bioorganic and Medicinal Chemistry.

17: 2002–2008.

3. Hostettman, K., Hostettman-Kaldas M. and Nakanishi, K. 1979. J. Chrom., 170:355.

4. Kerem, Z., German-Shashoua, H. and Yarden, O. 2005. Microwave-assisted extraction of

bioactive saponins from chickpea (Cicer arietinum L.). J. Sci. of Food and Agri. 85:

406–412.

5. Kesselmeir, J. and Stack, D. 1981. High performance liquid chromatographic analysis of

steroidal saponins from Avena sativa L. Z Naturforsch., 36C, 1072-4.

6. Kitagawa, I., Wang, H. K. and Yoshikawa, M. 1983. Chem. Pharm. Bull., 31, 664.

7. Kitagawa, I., Wang, H. K. Saito, M. and Yoshikawa, M. 1983. Chem. Pharm. Bull., 31,

674.

8. Muetzel, S., Hoffmann, E. M. and Becker, K. 2003. Supplementation of barley straw with

9. Oakenfull, D. 1981. Saponins in food--A review. Food Chem., 6, 19-20.

10. Price, K. R. and Fenwick, G. R. 1984. J. Sci. Food Agric., 35, 887.

11. Price, K. R., Johnson, I. T. and Fenwick, G. R. 1987. The chemistry and biological

significance of saponins in food and feedingstuffs. CRC Crit. Rev. Food Sci. Nutr., 26(1):

27- 135.

12. Radomir Lásztity , Máté Hidvégi and Árpád Bata. 1998. Saponins in food. Food

Reviews International, 14:4, 371-390.

13. Ruales a'b, J. and Nair, B. M. 1993. Saponins, phytic acid, tannins and protease inhibitors

in qulnoa (Chenopodium qumoa, Willd) seeds. Food Chemistry 48: 137-143.

14. Sesbania pachycarpa leaves in vitro: Effects on fermentation variables and rumen

microbial population structure quantified by ribosomal RNA-targeted probes. British

Journal of Nutrition. 89: 445–453.

15. Sharma, O.P., Kumar, N., Singh, B. and Bhat, T.K. 2012. An improved method for thin

layer chromatographic analysis of saponins. Food Chem. 132:671–674.

16. Stahl, E. 1969. Thin layer chromatography – a laboratory handbook. Berlin: Springer

Verlag.

17. Still, W. C., Kahn, M. and Mitra, M. 1979. J. Org. Chem., 43, 2933.

Lectins

1. Burger, M.M. 1974. In: „Methods in enzymology‟ (Fleischer, and Packer, eds). Vol. 32,

pp. 615-621. Academic Press, New York.

2. Hwang, K.M., Murphree, S.A. and Sartorelli, A.C. 1974. Cancer Res. 34: 3396-3402.

3. Kaneko, I., Hayatsu, H. and Ukita, T. 1975. Biochem. Biophys. Acta. 392: 131-140.

4. Kaul, R., Read, and Mattiasson, B. 1991.Screening for plant lectins by latex agglutination

test. Phytochem. 30: 4005.

5. Liener, I.E. 1955. Arch. Biochem.Biophys. 54: 223-231.

6. Vargas-albores, F., de la Fuente, G., Agundis, C. and Córdoba, F. 1987. Purification and

Characterization of a Lectin from Phaseolus acutifolius Var. Latifolius. Preparative

Biochem. 17 (4): 379-396.

7. Vincenzi, S., Zoccatelli, G., Perbellin, I. F., Rizzi, C., Chignola, R., Curioni, A. and

Peruffo, A.D. 2002. Quantitative determination of dietary lectin activities by enzyme-

linked immunosorbent assay using specific glycoproteins immobilized on microtiter

plates. J Agric Food Chem. 50(22):6266-70.