Food and Agricultural Immunology

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Effect of Natural Fermentation on the Lectin of

Lentils Measured by Immunological Methods

Carmen Cuadrado , Gyongyi Hajos , Carmen Burbano , Mercedes M.

Pedrosa , Gema Ayet , Mercedes Muzquiz , Arpad Pusztai & Eva Gelencser

To cite this article: Carmen Cuadrado , Gyongyi Hajos , Carmen Burbano , Mercedes M.
Pedrosa , Gema Ayet , Mercedes Muzquiz , Arpad Pusztai & Eva Gelencser (2002) Effect of
Natural Fermentation on the Lectin of Lentils Measured by Immunological Methods, Food and
Agricultural Immunology, 14:1, 41-49, DOI: 10.1080/09540100220137655

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Food and Agricultural Immunology ( 2002) 14, 41– 49

Effect of Natural Fermentation on the Lectin of Lentils

Measured by Immunological Methods

CARMEN CUADRADO,1 GYONGYI HAJOS,2 CARMEN BURBANO,1

MERCEDES M. PEDROSA,1 GEMA AYET1 MERCEDES MUZQUIZ,1
ARPAD PUSZTAI5 AND EVA GELENCSER2
1
Area de Tecnologia de Alimentos, SGIT-INIA, Aptdo. 8111, 28080 Madrid, Spain;
2
Central Food Research Institute, Budapest, Herman O. u.15, Hungary; 3The
Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, Scotland, UK

( Original manuscript received 1 August 2000; revised manuscript accepted 24 August 2001)

The effects of natural fermentation upon the lectin in the seeds of Lens culinaris cultivar
Magda 20 were investigated. Suspensions of lentil flour were allowed to ferment naturally at
different lentil flour concentrations ( 79, 150 and 221 g l–1) and temperatures ( 28, 35 and
42°C). During fermentation, samples were taken at daily intervals (0, 24, 48, 72, and 96 h)
and lentil lectin activity was measured by haemagglutination test. With the progress of
fermentation there was a rapid decline in haemagglutination activity in all the batches. The
largest decrease occurred between 0 h and 24 h of fermentation in all the conditions. The
lectin concentration showed the maximum reduction at 72 and 96 h, under the fermentation
conditions of 79 g l–1 and 42°C, when the initial lectin content measured by ELISA was
reduced by 98 and 97.8%, respectively. The changes in lentil lectin were also followed by
SDS-polyacrylamide gel electrophoresis and immunoblotting. The results confirmed those
obtained by ELISA and indicated that the lectin almost disappeared after 72 h of natural
fermentation under the optimum conditions of flour concentration and temperature.

Keywords: Lentil, natural fermentation, Lectin, ELISA, SDS-PAGE

INTRODUCTION
Fermentation is one of the oldest and most economical methods of processing and preserving
food. Foods are fermented for many reasons, including the enhacenment of nutritive value
and improvement of sensory characteristics such as odour and taste. In some parts of the
world large amounts of fermented food are produced to serve as an essential part of the diet.
Legume-based fermented foods originated centuries ago in these areas and are often referred
to as indigenous fermented foods. They are popular because changes in their flavour, aroma
and appearance are regarded as desirable and this is reinforced by improvements in their

Correspondence to: C. Cuadrado., Fax: + 34 913572293; E-mail: cuadrado@inia.e s

ISSN 0954-0105 (print )/ISSN 1465-3443 ( online )/02/010041-0 9 © 2002 Taylor & Francis Ltd
DOI: 10.1080/09540100220137655
42 C. CUADRADO ET AL.

nutritive value, due to the partial or complete elimination of antinutritional factors ( Reddy &
Salunke, 1989).
Lentils ( Lens culinaris ) are a rich source of easily available and cheap protein both for
human and animal nutrition, and due to their essential amino acid profile, they can
complement cereal proteins in the diet. Unfortunately, as other legumes, lentils contain non-
nutritive factors which limit their acceptability as staple food. However, some of them, such
as phytic acid ( Cuadrado et al., 1996 ) and phenolic compounds and trypsin inhibitors ( Tabera
et al., 1995) can be greatly reduced in amounts by natural fermentation.
Lectins are natural carbohydrate reactive proteins which occur widely in plants. Most
plant-based food/feedstuffs contain appreciable amounts of lectins, some with striking
biological activities on the gut (Pusztai, 1991). As lectins react with the surface epithelium
of the digestive tract, they can cause antinutritional, mild allergic or other subclinical effects
in higher animals and humans (Pusztai et al., 1997 ). Although lentils are utilised by animals
with moderate efficiency, when some isolated lectins are incorporated in diets for rats, there
is a growth retardation and the activities of intestinal proteolitic enzymes, intestinal
disaccharidases and hepatic dehydrogenases are considerably reduced ( Jindal et al., 1982 ).
Also, the transport of sugars and amino acids by brush border membrane vesicles is
considerably impaired in vitro by the lentil lectins (Utal et al., 1990 ). Thus, to make these
foods safe, either the lectins themselves or their activity need to be eliminated or their
amounts reduced before consumption ( Pusztai, 1989). Although in general lectins are more
resistant to heat-denaturation than other plant proteins, legume lectins can be inactivated by
moderately prolonged cooking. However, as heat-processing is expensive and potentially
damaging, it is usually kept to a minimum even with legumes, particularly when the product
is to be used in animal nutrition ( Pusztai & Bardocz, 1996). Unfortunately, there is very little
published information on the effects of fermentation on lectin content. Reddy and Pierson
( 1994 ) have reported that the lectin content of beans can be reduced up to 95% in Tempeh
and other fermented foods which also involved additional heat-treatment as cooking or
steaming. To date there is no information about the effects of fermentation on lentil lectin.
The aim of the present work was to study the changes produced by different conditions of
natural fermentation ( time, temperature and flour concentration) in the amounts and properties
of lentil lectin to obtain a flour with higher nutritive value than the raw legume.

MATERIALS AND METHODS

Fermentation
Lentil var. Magda 20 seeds from Albacete ( Spain) were finely ground in a ball mill, sieved, and
the 0.05– 0.25 mm fraction was collected. Suspensions of lentil flour in sterilized tap water
were aseptically prepared at concentrations and temperatures set up by the experimental design
and were allowed to ferment naturally ( with the only microorganisms on the seeds) for 4 days
without aeration in a stirred fermentor (Infors ISF-100, Infors AG, Switzerland) according to
Cuadrado et al., ( 1996 ). The time zero means the time requiered for preparing the lentil
suspension ( always less than 40 min). The pH, partial pressure of O2 ( PO2 ), titrable acidity and
lactic acid values of the broth were measured and registered every 24 h ( Cuadrado et al., 1996 ).
Samples were collected and daily freeze-dried before analysis.
A 22 complete factorial design with three replicate centrepoints (Bayne & Rubin, 1986)
was used to study the effects of the temperature and the suspension concentration on the
fermentation performance. Experimental conditions for the different fermentation batches are
shown in Table 1.

Microbial Analyses
Total viable counts ( TVC) were determined on PCA ( plate count agar) ( Adsa-Micro) and
lactobacilli were enumerated on double-layer MRS agar ( Adsa-Micro) both incubated at
 EFFECT OF NATURAL FERMENTATION ON THE LECTIN OF LENTILS 43

TABLE 1. Complete 22 factorial design with three

added contrepoints for temperature and
concentration factors

Temperature Concentration
Batches ( °C) ( gl–1)

F1 28 79
F2 42 79
F3 28 221
F4 42 221
F5 35 150
F6 35 150
F7 35 150

32°C for 2 days. Identification of microorganisms was carried out according Harrigan and
McCance (1979).

Preparation of the Samples

Raw and fermented samples were extracted in 0.1 M-phosphate-buffered saline ( PBS), pH
7.4, containing 0.1 M-D-glucose, at concentration 200 mg ml–1 using an Ultraturrax homoge-
nizer ( 2 min). After centrifugation ( 4300 ´ g, 20 min, 4°C) the supernatants diluted 4 or 100
times were used for the haemagglutination tests and ELISA assays.

Haemagglutination Activity
Haemagglutination activity was estimated as a measure of the lectin content of the samples
using the method described by Grant ( 1991 ). Trypsin treated rat erythrocytes were used in the
haemagglutination test. One unit of haemagglutinating ( lectin) activity ( HU) was defined as the
amount ( g) of sample in the last dilution which caused agglutination of 50% of the blood cells.
For comparison purposes the activity of the samples was calculated as HU kg–1 of dry matter.

Purification of Lentil Lectin

This was performed by affinity chromatography on Sephadex G-100 as described before
( Cuadrado et al., 1997). Lentil meal was sequentially extracted with 20 mM-sodium acetate-
acetic acid buffer pH 5.0 and 0.05 M-Tris-HCI buffer, pH 7.6. The freeze-dried extracts
containing haemagglutination activity were dissolved in 0.05 M-Tris-HCI buffer. The lentil
lectin contained in such extracts was purified by affinity chromatography on Sephadex
G-100. The column was equilibrated and run with 0.05 M-Tris-HCI buffer, pH 7.6. Elution of
adsorbed lectin from the column was with the same buffer also containing 0.1 M-D-glucose.
Non-adsorbed as well as adsorbed fractions were collected, extensively dialysed against
distilled water and recovered by freeze-drying. All fractions recovered were tested by
haemagglutination and SDS-PAGE. The lectin preparations from adsorbed fractions were
shown by SDS gel electrophoresis to be of high purity and showing two bands of 8 kDa
( a-chain) and 18 kDa (b-chain) ( Fliegerová et al., 1974; Foriers et al., 1981 ). The pure lentil
lectin ( Lens culinaris agglutinin, LCA) obtained was used to obtain rabbit anti-LCA
antibodies as well as standard in the ELISA and immunoblotting assays.

Development of Antisera to Lentil Lectin

Antibodies to lentil lectin ( LCA) were developed in rabbits according to the method of
Harboe and Inglid ( 1973 ).
44 C. CUADRADO ET AL.

Competitive Indirect ELISA

Plates coated overnight at 4°C with 0.5 mg ml–1 of the pure lentil lectin previously obtained
( LCA) (in 0.05 M-sodium carbonate-bicarbonate buffer, pH 9.8) were washed four times with
PBST ( 0.01 M-PBS containing 0.01% (v/v) Tween 20, pH 7.4). Then, 0.2 ml of PBSG
( 0.01 M-PBS containing 0.5% ( w/v) gelatin, pH 7.4) was added and after incubation for 1 h at
37°C the plates were washed four times with PBST. After this, 0.05 ml of pure standard LCA
diluted in PBS containing 0.1 M-D-glucose or lentil samples (also diluted in PBS containing
D-glucose ) with unknown content of lectin were added, followed by 0.05 ml of rabbit anti-LCA
IgG antibody ( diluted 1:100 in PBS). Determinations were performed in triplicate for each data
point. After incubation for 1 h at 37°C, the plates were washed four times with PBST and 0.1 ml
of horseradish peroxidase ( HRP)-conjugate goat anti-rabbit IgG ( HUMAN, Hungary) diluted
with PBS ( 1:10 000, v/v) was added to each well and incubated for a further 1 h at 37°C. After
washing ( four times) with PBST, 0.1 ml of substrate solution ( 0.34 mg ml–1 o-phenylene-
diamine in 0.05 M-phosphatecitrate buffer, pH 5.0 containing 0.03% ( v/v) hydrogen peroxide)
was added to each well. After 5 min, the reaction was stopped by adding 0.05 ml of 3 M-H2SO4
and the optical density measured at 492 nm using a DYNATECH plate reader. The lectin
content of the samples was estimated from the calibration curve ( 0.001–1000 mg ml–1 of pure
standard LCA). Results were expressed in g kg–1 of dry matter.

SDS-Polyacrylamide Gel Electrophoresis and Transblotting

SDS-PAGE electrophoresis was carried out as described by Hajos et al. ( 1995 ). The total
acrylamide content of the running gel was 17.60% with 0.45% cross-linkage and that of the
stacking gel was 3.95% with 1.42% cross-linkage. Samples ( 1 mg 100 ml–1) were incubated
at 100°C for 5 min in 0.01 M-Tris-glycine buffer ( pH 8.3) containing 3% ( w/v) SDS and 0.1%
( v/v) b-mercaptoethanol before electrophoresis. The gels were stained with 0.5% ( w/v)
Coomasie Brilliant Blue R ( Sigma).
Semidry transblotting onto nitrocellulose membranes was performed according Hajos et
al. ( 1995 ) using the same anti-LCA antibody than in the ELISA assay.

Statistical Analysis
Analysis of variance was made to study the effect of fermentation process itself by comparing
control ( raw) and fermented samples on the lectin content and haemagglutination activity. Also
the significance of time of fermentation was studied by analysis of variance and t-test.

RESULTS AND DISCUSSION

Natural fermentation of legumes is a typically lactic acid fermentation characterised by
decreases in pH ( Ragaee et al., 1985; Vidal-Valverde et al., 1993 ). The pH drop during the
fermentation experiments performed in this work was from 6.4 to 4.6–3.8. The titratable
acidity ( g of lactic acid kg–1 of dry matter) increased rapidly for the first 24 h in all batches.
Treatment with the lowest flour concentration and the highest temperature (F2) produced the
highest change in titratable acidity between 0 and 96 h ( 19.0–127.8 g of lactic acid kg–1 d.m.).
At the start of the natural fermentation the dissolved PO2 dropped to values near zero and
remained there during the rest of the process.
The counts of total flora at 0 h were less than 103 Colony forming units ( CFU) g–1 being
Bacillus the predominant microorganism in all batches. At 24 h the counts of total flora and
lactic bacteria reached similar values (108–109 CFU g–1) remaining constant afterwards. The
presence of Lactobacillus and Pediococcus was observed at 24 h of the natural fermentation
of lentils, with Pediococcus becoming the predominant microorganism after 48 h of
fermentation in almost all batches, except at 42°C ( F2 and F4) where only Pediococcus were
found after 24 h of fermentation. Ragaee et al. ( 1985 ) reported that lactic acid bacteria play
a major role in natural fermentation of lentils and pH decreases from 6.9 to 3.9. Similar
 EFFECT OF NATURAL FERMENTATION ON THE LECTIN OF LENTILS 45

changes in pH were observed in the present study. This decline in pH during fermentation is
known to act as a bacteriostatic and preservative factor against bacteria associated with
spoilage and against nondesiderable and pathogenic microorganisms which cannot proliferate
under these conditions ( Wang & Hesseltine, 1981)
Figure 1 shows the effect of time, temperature and lentil flour concentration during natural
fermentation on the lectin activity of lentils measured using haemagglutination tests. The
reproducibility of the fermentation process was high since similar results were obtained in
batches F5, F6 and F7 fermented at identical conditions ( 35°C, 150 g l–1). These data were
combined and presented as mean and standard deviation (Fx ). With the progress of
fermentation there was a rapid decline in haemagglutination activity in all the batches. The
largest decrease occurred between 0 and 24 h of fermentation in all the conditions. After 96 h

FIG. 1. Effect of natural fermentation upon the lectin activity expressed as haemagglutination units ( HU)
per kg of dry matter of lentil flour suspensions F1, F2, F3, F4 , and Fx ( mean and standard deviation
data of F5, F6 and F7).
46 C. CUADRADO ET AL.

of natural fermentation the minimal haemagglutination activity values were obtained under
the fermentation conditions of 79 g l–1 and 42°C ( F2). In our previous work ( Cuadrado et al.,
1996 ) the influence of natural fermentation on the inositol phosphate content was analysed
to establish the effect of different conditions of temperature and broth concentration. From
the results it is concluded that 79 g l–1 and 42°C are the most effective fermentation
conditions to reduce the total inositol phosphate content of the meal ( 63% of reduction at
72 h). Tabera et al. ( 1995 ), using the same lentil variety and fermentation procedure, reported
a slight increase of total protein content after 4 days of fermentation in comparison with the
raw lentils, and a 62.5% reduction of trypsin inhibitor activity after the same fermentation
period at 42°C and 79 g l–1. The same conditions were also found to be the optimum to reduce
lectin activity in the present experiment.
The loss in lectin or haemagglutination activity detected could initially be due to either
changes in the part of the lectin protein structure which is responsible for the sugar binding
capacity and haemagglutination properties of LCA or as a result of the proteolytic degradation
of the lectin protein, the LCA content of the meal was reduced. Therefore, a more detailed study
was carried out with the fermented samples that showed the strongest reduction in
haemagglutination activity ( F2, 79 g l–1 and 42°C) to know the cause of such reduction.
Haemagglutination assays are at best semi-quantitive methods to determine the presence of
lectins. Moreover, some lectins may have their agglutination activity affected by high level of
sugars in the extract, or by the presence of compounds which damage red blood cells before
agglutination occur (Peumans & van Damme, 1996). Taking this into account and that the
measurement of lectins in a natural product require sensitive and specific methods, a competive
indirect ELISA technique has been developed, using specific polyclonal antibodies (PABs), to
quantify the lectin concentration in lentil samples. Indirect ELISA methods are more precise
than direct for assaying low concentrations of antigens ( Hajos et al., 1996).
Table 2 reports the lectin content of raw and F2 fermented samples quantified by
competitive indirect ELISA. The reduction of lectin concentration (LCA) in the fermented
samples is also reported as a percentage of the initial content ( raw seeds). The data in fact
indicated that a significant decrease in the LCA content occurred throught the fermentation
time ( 0–96 h). This decrease was parallel to that observed in haemagglutination units ( HU)
for the same fermented samples ( Figure 1). These results showed that a major reduction in
the lectin concentration occurred in the first 24 h of fermentation and that the greatest
decrease was achieved by 72 and 96 h of fermentation when the initial lectin content was
reduced by 98%, respectively.
Similar to following enzymatic modifications in proteins ( Hajos et al., 1996), changes in
the lentil lectin during fermentation were studied by SDS-polyacrylamide gel electrophoresis

TABLE 2. Changes in lectin concentration ( LCA), measured by

competitive indirect ELISA, during natural fermentatio n
of F2 lentil samples ( 42°C and 79 g l–1 )

Fermentation LCA content Reduction

time ( h) ( g kg–1 dry matter ) rate (%)

Raw lentil 4.0 ± 1.1a

0 6.0 ± 1.2a No reduction
24 1.4 ± 0.5ab 65
48 1.1 ± 0.3b 72
72 0.08 ± 0.02c 98
96 0.09 ± 0.08c 98

Values are the mean of three determinations ± SE. Means in the

same column with different superscripts were shown by t-test to be
significantly different at 5% level.
 EFFECT OF NATURAL FERMENTATION ON THE LECTIN OF LENTILS 47

FIG. 2. Densitometric evaluation of the band corresponding to the b-chain ( 18 kDa) of lentil lectin ( LCA) on
the basis of immunoblotting patterns of raw and fermented lentil samples after SDS-PAGE separation.
The proteins were reacted on nitro-cellulose membrane with anti-LCA rabbit IgG. Raw lentil, 100%
relative LCA content ( Peak 1); fermented lentil samples: 0 h, 77% relative LCA content ( Peak 2); 24 h,
58% relative LCA content ( Peak 3); 48 h, 37% relative LCA content (Peak 4); 72 h, 13% relative LCA
content (Peak 5); 96 h, 3% relative LCA content (Peak 6); lentil lectin standard, LCA ( Peak 7).

and immunoblotting. The pure lentil lectin used as standard showed two spaced bands of 8
and 18 kDa in SDS-PAGE in agreement with evidences that lentil lectin consisted of two light
a-chains and two heavy b-chains ( Fliegerová et al., 1974; Foriers et al., 1981 ). The
immunoblots of the pure lentil lectin showed only one band of 18 kDa which should
correspond to the b-chain. Figure 2 shows the immunoblotting patterns of transblotted raw
and F2 fermented samples after SDS-PAGE separation and the densitometric evaluation of
the band corresponding to the b-chain of lentil lectin ( LCA). From this densitometric
evaluation a peak area for each sample is obtained, which enable to calculate the percentage
of LCA of each sample relative to the raw lentils. These results confirmed those obtained by
ELISA and indicated that the lectin was partially degraded after 24 h (58% of LCA) and
almost disappeared after 96 h ( 3% of LCA) of natural fermentation at 42°C and 79 g 1–1. On
the bases of the SDS-PAGE patterns of F2 fermented samples related to the raw flour there
was evidence of proteolisis during fermentation in some bands between 31–18 kDa. Data
previously published (Tabera et al., 1995 ) indicated that the total protein content estimated
by N content did not decrease under these fermentation conditions. Probably, the different
methodology used can explain these results.
The present study clearly shows that the lectin content ( LCA) of the lentil meal was
essentially completely removed using a natural fermentation procedure and that this occurred
without any additional heat-treatment. The scarce information on the influence of fermentation
on lectin content indicated that fermentation with microorganisms with additional controlled
heat treatment, as cooking or steaming, decreases lectin activity from beans ( Reddy & Pierson,
1994 ). However, based on the present and previous findings for other antinutritive factors
( Tabera et al., 1995; Cuadrado et al., 1996 ) it can be concluded that the natural fermentation
itself, without any additional heat-treatment, is a powerful method capable of improving the
nutritional value of lentils by reducing the level of non-nutritive compounds.
Trugo and von Baer ( 1998 ) indicated that immunological methods are a more specific and
sensitive approach for the quantitative measurement of lectins than erythrocyte agglutination
tests. Our results show that the immunochemical assays used in the present work have adequate
sensitivity to detect residual amounts of lentil lectin in products processed for food use.
48 C. CUADRADO ET AL.

ACKNOWLEDGEMENTS
This work was supported by the INIA ( Project SC97–057) and by OMFB and FM of
Hungary and it is a part of COST-98 Action ( EU). The authors wish to thank the INIA for
awarding postdoctoral grants to CC and MMP and EU/COST 98 for Short Term Scientific
Missions to CC. The highly skilled assistance of Mrs Kissne-Valentin Eva, Betty Zarai and
Mr Istvan Makk is gratefully acknowledged.

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