Inhibitory effect of Pistia tannin on digestive enzymes of Indian major carps: an in vitro study

Inhibitory effect of Pistia tannin on digestive enzymesof Indian major carps: an in vitro study

Sudipta Mandal • Koushik Ghosh

Received: 26 June 2009 / Accepted: 22 March 2010 / Published online: 6 April 2010

� Springer Science+Business Media B.V. 2010

Abstract Aquatic weeds are one of the major

unconventional feed ingredients tested for aquafeed

formulation. Tannin content in the water lettuce,

Pistia, has been quantified (26.67 mg g-1; dry

weight) and graded levels of which (12.5–200 lg)

have been incorporated in the reaction mixtures to

evaluate any change in the in vitro activity of the

principal digestive enzymes from the three Indian

major carps (IMC), namely rohu (Labeo rohita), catla

(Catla catla) and mrigala (Cirrhinus mrigala). Result

of the experiment revealed that the Pistia tannin (PT)

significantly inhibit/lower the activities of the diges-

tive enzymes from three IMCs in a dose-dependant

manner, even at very low concentration. Significant

variation in the reduction of the enzyme activities

was noticed between the three fish species, as well as

between the three enzymes studied. Among the three

species studied, digestive enzymes from L. rohita

were found to be the most sensitive to the PT,

whereas enzymes from C. catla were found to be

comparatively least affected. On the other hand,

protease and lipase activities were comparatively

more affected than the amylase activity. The results

of the study suggest that more stress should be given

on the elimination of tannin while incorporating feed

ingredients of plant origin in fish diets.

Keywords Indian major carps � Tannin �Protease � Amylase � Lipase � Pistia

Introduction

The growing demand to substitute fish meal in

aquafeed has compelled to search for alternative less

expensive and protein-rich sources. Plant proteins are

considered to be the most viable alternative in this

respect for economic fish production in most of the

developing countries (Wee and Wang 1987; Mukho-

padhyay and Ray 1996; Becker and Makkar 1999).

The use of plant-derived materials (e.g., aquatic

weeds, legume seeds, different types of oilseed cake,

leaf meals, leaf protein concentrates and root tuber

meals) as fish feed ingredients is limited by the

presence of a wide variety of antinutritional sub-

stances (Francis et al. 2001). Even though possibilities

for uses of plant ingredients have been investigated

and achieved experimental success (Hasan et al. 1990;

Patra et al. 2002; Kalita et al. 2007). The water lettuce,

Pistia, is an aquatic weed; infestation of which causes

great problem in the aquaculture ponds. In compar-

ison with the terrestrial conventional vegetal meals,

the use of aquatic weeds like Pistia involves minimum

S. Mandal � K. Ghosh (&)

Aquaculture Laboratory, Department of Zoology,

The University of Burdwan, Golapbag, Burdwan 713104,

West Bengal, India

e-mail: [email protected]; [email protected]

123

Fish Physiol Biochem (2010) 36:1171–1180

DOI 10.1007/s10695-010-9395-6

expenditure as it requires only the cost of labor for

collection. Ray and Das (1995) have shown that Pistia

has the potential to serve as one of such alternate

ingredient in fish feed. However, higher levels of

incorporation of Pistia meal in aquafeeds were

associated with poor growth, feed conversion and

protein utilization, which may be due to the interfer-

ence caused by the antinutrients therein. Tannins are

one such naturally occurring plant polyphenols that

may interfere with the digestive processes (Liener

1989). These are secondary compounds of various

chemical structures which are generally divided into

hydrolysable and condensed tannins (Francis et al.

2001). Experimental evidence has shown to induce

feed rejection by hydrolysable tannin when included

in the feed of the common carp, Cyprinus carpio

(Becker and Makkar 1999). Poor growth performance

has also been recorded in the same species as a result

of feeding tannic acid incorporated diet (Hossain and

Jauncey 1989). However, studies on the interaction of

tannin with fish intestinal enzymes are scanty. Maitra

and Ray (2003) demonstrated the inhibition of the

intestinal enzymes in Labeo rohita fingerlings to a

significant level by the tannin extracted from Acacia,

though the effect of the hydrolysable tannin on the

digestive enzymes of the other carp species has not

been worked out. Indian major carps might not share

same pattern of inhibition with regard to the digestive

enzymes as they vary in feeding regime specificity

and also in the enzyme activities (Jhingran 1997).

Therefore, the present study was undertaken to

demonstrate the comparative effect of the hydrolysa-

ble tannin extracted from Pistia on the digestive

enzymes of three Indian major carps, rohu, Labeo

rohita; catla, Catla catla and mrigal, Cirrhinus

mrigala.

Materials and methods

Extraction of crude tannin

Tannin was extracted from Pistia leaves following

the method described by Schanderi (1970). Pistia

leaves were collected from a local water body, oven

dried (at 55 ± 5�C) for 48 h and finely powdered in a

mixer grinder (Remi Laboratory Blender). The pow-

dered material (5 g) was mixed with distilled water

(200 ml) and kept at room temperature for overnight.

After soaking, the mixture was boiled for 30 min,

cooled and centrifuged at 2,000 rpm for 20 min. The

supernatant was collected and used as tannin extract.

The extracted tannin was not purified further.

Estimation of extracted tannin

Concentration of tannin in the extract was measured

by Folin–Denis method (Schanderi 1970) with minor

modifications. The crude extract (0.2 ml) was diluted

with 8.3 ml of distilled water and then mixed with

0.5 ml of Folin–Denis reagent. The reaction mixture

was alkalinized by the addition of 1 ml of 15% (w/v)

sodium carbonate solution and kept in dark for

30 min at room temperature. The absorbance of the

solution was read at 700 nm using spectrophotometer

(Shimadzu UV/VIS-1700), and the concentration of

tannin in the extract was determined using pure

tannic acid (MERCK, India) as standard.

Experimental fish

Fingerlings of Labeo rohita, Catla catla and Cirrhi-

nus mrigala were collected from three local compos-

ite carp culture farms and acclimatized separately

according to their source in glass aquaria (75 L) for

10 days, during which fish were fed ad libitum with a

diet containing approximately 40% crude protein

having fish meal as the chief protein source. Average

weight of the fishes examined and their feeding habits

are presented in Table 1.

Preparation of crude enzyme

The experimental fish were weighed to the nearest

gram on a single-pan top-loading balance, and the

anterior and middle intestinal parts were dissected out

on cooled plates and placed in prechilled Petri dishes.

Blood and other debris were washed out with chilled

phosphate buffer (0.1 mol L-1, pH 7.4) containing

0.89% sodium chloride (phosphate buffered saline,

PBS). The cleaned intestinal parts were minced with

scissors, and a 10% homogenate was prepared in the

same buffer using tissue homogenizer (REMI, Model

RQ-127A2). The homogenate was centrifuged

(REMI, Model No.C24) at 10,000 rpm for 30 min

at 4�C. The supernatant was separated and used as

enzyme extract. Preparation of enzyme extract and

inhibition assay was done on the same day, and the

1172 Fish Physiol Biochem (2010) 36:1171–1180

123

extract was kept at 4�C in between. Protein content of

the extract was determined following the method

described by Lowry et al. (1951) using BSA as

standard. Three specimens of each fish species from

each collection site were used for preparation of

enzyme extract for every replicate, and there were

three replicates for each fish species.

Inhibition assay

Inhibitory effect of tannin extracted from Pistia leaf

meal on the activity of principal digestive enzymes

were studied in vitro by adding graded levels of

extracted tannin (12.5–200 lg of tannin) to the test

tubes containing enzyme extracts. Then, substrate and

PBS were added to the tubes and incubated at 37�C

for the optimal time period. Any minor change in pH

and volume of the reaction mixture due to the

addition of tannin was corrected by adding PBS. A

concurrent control set without any tannin was main-

tained for each experimental set. Decrease in the

enzyme activity due to the addition of tannin was

expressed as percent reduction when compared to the

control set (100% activity). There were three repli-

cates for each of the experimental set.

Protease assay

Protease activity was measured according to Anson

(1938) using bovine serum albumin (BSA) as

substrate. Graded levels of extracted tannin were

added to 0.1 ml of enzyme extract followed by

0.1 ml of substrate (10 mg ml-1 BSA solution),

0.5 ml of phosphate buffer (pH 7.4, 0.1 M) and

0.3 ml of distilled water. The reaction mixture was

incubated for 1 h at 37�C. One milliliter of 10%

trichloroacetic acid (TCA) was then added to stop the

reaction and precipitate any remaining substrate. It

was then centrifuged at 3,000 rpm for 10 min. To

2 ml of the supernatant, 1 ml of distilled water and

1 ml of ninhydrin reagent were added in a test tube

and heated in a boiling water bath for 20 min, placing

a marble on top of each tube. After cooling to room

temperature, the developed color was read at 570 nm

in a spectrophotometer (Shimadzu UV/VIS-1700).

Blanks were obtained by adding TCA to the substrate

prior to incubation. The amino acid liberated was

measured following Moore and Stein (1948). Prote-

ase activity was expressed as lg of glycine liberated

h-1 mg protein-1.

a-Amylase assay

a-Amylase activity was determined following the

method described by Bernfeld (1955). Graded levels

of extracted tannin were added to 1 ml of enzyme

extract followed by 1 ml of substrate (1% soluble

starch). The mixture was incubated at 37�C for

20 min. Then, 2 ml of dinitrosalicylic acid reagent

(DNSA reagent, containing 1% dinitrosalicylic acid,

30% sodium potassium tartrate in 0.4 N NaOH

solution) was added to each tube and kept in a

boiling water bath for 5 min. Then, the tubes were

cooled and intensity of the color developed was read

at 540 nm. Blanks were obtained by adding DNSA

reagent prior to incubation. Amylase activity was

expressed as mg maltose liberated h-1 mg protein-1.

Lipase assay

Lipase activity was measured following the method

described by Colowick and Kaplan (1955) using olive

oil as substrate. Briefly, graded levels of extracted

tannin (12.5–200 lg of tannin) were added to 0.5 ml

of enzyme extract followed by 2.5 ml of substrate

(olive oil emulsion in 2% polyvinyl alcohol) and

0.5 ml of calcium chloride solution (110 mM). The

reaction mixture was taken in 100-ml conical flask

and incubated at 37�C for 1 h in a shaker incubator

(Lab. companion, SL-300R) with continuous shaking

Table 1 Food habit and average weight of the fishes examined

Fish species Food habit Average weight (g) (±SD)

Catla catla Zooplanktophagous 17.67 ± 1.8

Labeo rohita Omnivorous, mostly plant matter 12.44 ± 1.67

Cirrhinus mrigala Detritivorous 10.11 ± 1.45

Values are mean ± standard deviation (SD) of nine specimens

Fish Physiol Biochem (2010) 36:1171–1180 1173

123

(120 rpm). Then, 10 ml of acetone–ethanol mixture

(1:1) was added to stop the reaction. Few drops of

phenolphthalein indicator were added, and fatty acid

liberated as a result of enzymatic action was titrated

with 0.02 N NaOH solutions till the appearance of

faint pink color. One milliliter of 0.02 N NaOH is

equivalent to 100 lM of free fatty acid. Blanks were

obtained using boiled enzyme. Lipase activity was

expressed as l mole of fatty acid liberated h-1 mg

protein-1.

Statistical analysis

The data obtained on the enzyme activity in presence

of the tannin were subjected to regression analysis,

using the concentration of the inhibitor as the

independent variable. Further, the data on the prote-

ase, amylase and lipase enzymes were applied for

three-way ANOVA followed by Tukey’s test to infer

about the differences in the enzyme inhibition

between the three fish species. All the statistical

analyses were carried out following Zar (1999) using

SPSS Ver10 (Kinnear and Gray 2000) software.

Results

The tannin content in powdered Pistia was

26.67 mg g-1 of leaf meal. The concentration of

tannin in the tannin extract was 0.89 mg mL-1. The

results of the experiment revealed that the Pistia

tannin (PT) significantly inhibit/lower the activities

of the digestive enzymes from three IMCs in a dose

dependant manner (Fig. 1) as evident from the

regression equations (Table 2). In all the instances

irrespective of the carp species, the enzyme activity

was noted to decrease with the increase in the tannin

concentrations. However, variation in the reduction

of enzyme activities was noticed between the three

fish species, as well as the enzymes studied. Among

the fish species, digestive enzymes from L. rohita

were found to be the most sensitive to the PT

followed by C. mrigala, whereas enzymes from

C. catla were comparatively least affected. The

activities of protease and lipase were more affected

compared to the amylase.

Percent inhibition of the enzyme activities are

presented in Table 3. Protease activities in the

control sets (without tannin) in L. rohita, C. catla

and C. mrigala were 14.85 (±0.15), 11.52 (±0.07)

and 10.32 (±0.13) lg of glycine liberated h-1 mg

protein-1, respectively. There was a reduction of 9.65

(±0.13)% to 88.28 (±0.68)% enzyme activity in

L. rohita when 12.5–200 lg of tannin was added.

With similar doses of tannin, reduction in the

protease activities ranged between 4.37 (±0.06)%

to 43.96 (±0.09)% and 6.43 (±0.17)% to 82.53

(±0.99)% in C. catla and C. mrigala, respectively.

Amylase activities in the control sets were 7.34

(±0.04), 8.43 (±0.06) and 6.73 (±0.07) mg maltose

liberated h-1 mg protein-1 in L. rohita, C. catla and

C. mrigala, respectively. Reduction in the enzyme

activity due to the addition of tannin was reduced as

3.22 (±0.07)% to 37.88 (±0.77)% in L. rohita, 3.12

(±0.03)% to 21.63 (±0.33)% in C. catla and 3.67

(±0.14)% to 32.74 (±0.39)% in C. mrigala.

Activities of lipase in the control sets in L. rohita,

C. catla and C. mrigala were recorded as 12.27

(±0.11), 9.17 (±0.05) and 10.82 (±0.06) lmol of

fatty acid liberated h-1 mg protein-1, respectively.

Reduction in the enzyme activities varied between

7.93 (±0.2)% to 87.07 (±0.59)% in L. rohita, 4.43

(±0.04)% to 41.35 (±0.4)% in C. catla and 6.41

(±0.03)% to 75.35 (±0.62)% in C. mrigala with the

addition of tannin.

The results of the three-way ANOVA (Table 4)

revealed significant differences in enzyme activity

due to tannin in terms of dose, type of enzyme and

the species. The post hoc Tukey test revealed

significant differences in protease, amylase and lipase

activity between the three fish species (Between

L. rohita and C. catla: 1.09; between L. rohita and

C. mrigala: 0.51; between C. catla and C. mrigala:

1.61; for all values P \ 0.001).

Discussions

Chemical compounds that render plant tissues unpal-

atable are known to be widely distributed in the plant

kingdom (Liener 1989; Makkar 1993; Hagerman et al.

1997). Tannins are one such group of phenolic

compounds which have received a lot of attention

with respect to their possible nutritional and physio-

logical interference. The nutritional value and possi-

bilities of utilization of aquatic weeds have been

evaluated in some of the literatures in the last decade

(Edwards et al. 1985; Ray and Das 1992, 1994, 1995;


123

Hasan et al. 1990; Patra and Ray 1988; Patra et al.

1999, 2002). In view of accessing effectiveness of

aquatic weeds in carp diet formulation, quantification

of tannin in the dried Pistia leaf meal has been

performed in the present investigation. Previous study

by Mandal and Ghosh (2009a) depicted tannin

concentration in some natural and potential fish food

items/ingredients of plant origin that ranged between

5.38 (coconut oil cake) to 34.3 (phytoplankton)

mg g-1 dry weight. Result of the present study

revealed tannin concentration as high as

26.67 mg g-1 in the Pistia leaf meal. Such com-

pounds or antinutritional factors are believed to be the

defense weapon of plants against the herbivores

(Becker and Makkar 1999). While studying the

enzyme inhibitory effect of the Pistia tannin in the

present study, it has been revealed that the digestive

enzymes from L. rohita were most sensitive to the

Pistia tannin (PT) whereas enzyme from C. catla were

comparatively least affected. Herbivory is more

Fig. 1 The activity of the intestinal protease (lg glycine liberated mg-1 protein h-1), amylase (mg maltose liberated mg-1 protein

h-1) and lipase (lmol fatty acid liberated mg-1 protein h-1) from the Indian major carps under different doses of tannins (lg)


123

common in the L. rohita than C. mrigala; on the other

hand, C. catla feeds on zooplanktons (Jhingran 1997;

Mohanty 2003). Most of the previous studies reported

L. rohita to be herbivore (Alikunhi 1958; Khan and

Siddiqui 1973); however, some of the recent inves-

tigations have focused on omnivorous feeding apti-

tude of this species by analyzing feed intake/

preference (Mohanty 2003; Rahman et al. 2006) or

symbiotic gut microbial community (Ghosh et al.

2002). Therefore, maximum inhibition of digestive

enzymes from L. rohita by plant tannin and appear-

ance of omnivorous feeding aptitude may be viewed

as co-evolution where tannin in the leaf meal appears

to play as a defense compound. To reduce damage

caused by herbivory, plants have evolved a broad

array of defenses, majority of which are chemicals.

Herbivores prefer to avoid ingesting tannin if alternate

foods are available (Wynne-Edwards 2001). Thus,

defenses and counter defenses lay down the step for

the co-evolution of plants and herbivores.

Hydrolysable tannins are easily degraded in bio-

logical systems by non-specific esterase, and the

hydrolyzed products entering into the blood may

cause organ toxicity (particularly in liver and kidney)

once the level in blood rises beyond the detoxification

capability of these organs (Garg et al. 1992; Muller-

Harvey and McAllan Mueller-Harvey and McAllan

1992). In contrast, the condensed tannins are complex

and larger molecules which are not hydrolyzed in the

biological system, indicating their least effect in fish

(Makkar et al. 1995). Condensed tannin did not

appear to bind to proteins and other nutrients and

decrease their bioavailability in the carp intestine

(Becker and Makkar 1999). In the present study,

inhibition of digestive enzymes in carps must be from

their exposure to the hydrolysable tannin (or, tannic

acid), indicating its potential to interfere with the

digestive process.

Commonly used ingredients in formulated fish

feed like mustard oil cake and sun flower oil cake

contain tannin as high as 21.25 mg and

26.25 mg g-1 dry weight, respectively (Mandal and

Ghosh 2009a). It may be assumed that if fish of 100 g

biomass is fed at the rate of 3% of the body weight

daily with a formulated diet having only 10% of these

ingredients, the fish will be exposed to more than

6 mg of tannin daily. Natural feeding on phytoplank-

ton tends to increase this value. In the present study,

concentration of tannin used (12.5–200 lg) are much

lower than this presumptive value. The results of the

present study clearly indicated that tannin, even in

very low concentration, can inhibit or lower the

activities of protease, amylase and lipase in all the

three carp species studied, which is in agreement with

the study made by Maitra and Ray (2003) in L. rohita

fingerlings. In a study based on growth and hemato-

immunological parameters, Prusty et al. (2007)

opined that tannic acid in feed (up to 2%) may not

be detrimental for Labeo rohita fingerlings. However,

they did not address enzyme activities in the short-

term feeding trial of 60 days. Similarly, Becker and

Makkar (1999) assumed that tannic acid may not

affect nutrient availability and enzyme activity in

gastrointestinal tract of carp. In contradiction to these

observations, in vitro studies made in the present

experiment clearly indicated inhibition of the diges-

tive enzyme activities in carps by the tannic acid

extracted from the Pistia. Although experiment

Table 2 Regression equations of the activities of enzymes from the Indian major carps against graded levels of tannin

Enzymes Fish species Regression equation

(y = enzyme activity, x = dose of tannin)

Protease L. rohita y = 13.36 - 0.064x; F = 181.98, df = 1,19; p \ 0.001; R2 = 0.905

C. catla y = 11.37 - 0.023x; F = 834.63, df = 1,19; p \ 0.001; R2 = 0.978

C. mrigala y = 10.12 - 0.04x; F = 848.1, df = 1,19; p \ 0.001; R2 = 0.978

Amylase L. rohita y = 7.08 - 0.013x; F = 254.35, df = 1,19; p \ 0.001; R2 = 0.93

C. catla y = 8.3 - 0.009x; F = 614.45, df = 1,19; p \ 0.001; R2 = 0.97


Lipase L. rohita y = 11.98 - 0.05x; F = 2687.66, df = 1,19; p \ 0.001; R2 = 0.993

C. catla y = 8.84 - 0.02x; F = 348.86, df = 1,19; p \ 0.001; R2 = 0.948



123

conducted with commercial tannin may not corre-

spond with the tannins of the feed components

(Becker and Makkar 1999). Tannins may reduce

macro nutrient utilization by forming tannin–protein

complexes with various digestive enzymes preclud-

ing the formation of the product absorbable by the

small intestine (Carmona et al. 1996). In addition, it

may inhibit transport systems concerning carbohy-

drate assimilation such as glucosidase/maltase

(Bjorck and Nyman 1987), sucrase (Welsch et al.

1989) and the intestinal sodium-dependent glucose

uptake system (Karasov et al. 1992). Bean tannins

were shown to strongly inhibit pancreatic trypsin,

chymotrypsin and a-amylase (Sing 1984; Carmona

et al. 1991).

Tannins are considered as plant secondary metab-

olites which are distinguished from other polypheno-

lic compounds by their ability to precipitate proteins

(Silanikove et al. 2001). It has been postulated that

tannins interfere with protein and dry matter digest-

ibility by inhibiting protease and also forming

indigestible complexes with dietary protein (Krog-

dahl 1989). However, little is known about the effect

of tannin on fish (Makkar and Becker 1998). Vohra

et al. (1966) reported that tannins caused growth

depression in chickens at levels as low as 0.5% of the

diet. Similarly, Hossain and Jauncey (1989) observed

poor growth response in common carp (Cyprinus

carpio) fed diets containing 0.57 and 1.14% tannins.

In addition, high levels of tannins in feed have been

shown to have adverse effect on herbivorous and

omnivorous fish (Al-Owafeir 1999; Becker and

Makkar 1999; Olvera et al. 1988). The extents of

these growth inhibitory effects varied in different fishTa

ble

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3.2

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3.6

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6.4

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6.7

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0.6

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3.6

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85

.75

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12

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6

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8.5

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24

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17

.62

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25

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10

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7.3

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62

0.7

5±

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13

8.6

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82

2.3

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71

2.6

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31

7.2

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0.1

54

9.2

8±

0.6

72

9.8

7±

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3.1

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15

07

1.4

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0.6

27

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±0

.27

54

.12

±0

.42

26

.52

±0

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16

.81

±0

.32

1.8

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36

2.8

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13

7.2

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0.7

55

9.0

9±

0.1

5

20

08

8.2

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0.6

84

3.9

6±

0.0

98

2.5

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0.9

93

7.8

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0.7

72

1.6

3±

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33

2.7

4±

0.3

98

7.0

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0.5

94

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±0

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Table 4 Results of the three-way factorial ANOVA for the

effect of tannin on the enzyme activities in the Indian major

carps (F-values are significant at P \ 0.001)

Source Sum of squares df Mean square F-value

DOSE (D) 631.45 5 126.29 9,222.37

ENZYME (E) 51.07 2 25.54 1,864.87

FISH SP. (F) 72.76 2 36.38 2,656.61

D 9 E 142.28 10 14.23 1,038.99

D 9 F 81.24 10 8.12 593.26

E 9 F 36.78 4 9.19 671.55

D 9 E 9 F 43.79 20 2.19 159.91

Error 1.48 108 0.01

Total 1,060.86 161


123

species studied and also plant material used in the fish

feed formulation. Present study also revealed varied

degrees of tannin-induced inhibition of digestive

enzymes from different carp species studied. In

agreement with the reports discussed above, it may

be assumed from the results of the present study that

tannin may affect feed utilization efficiency and

digestibility in the major carps if used in feed

formulation. Although results obtained from such in

vitro studies should not be extrapolated directly to in

vivo conditions as several other factors, such as

presence of food, pH, microbiota, intestinal secre-

tions, etc. may influence the adversity of dietary

tannin. However, result of the present study empha-

sizes the need for eliminating tannin from aquatic

macrophytes or other feed stuffs of plant origin to

replace fish meal successfully. Low-cost physical

methods like heat inactivation and water soaking

(Egounlety and Aworh 2003; Ramachandran and Ray

2008) or bioinactivation processes using tannase

producing microbiota may be practiced as indicated

in some of the recent literatures (for review see

Ghosh and Mukhopadhyay 2006; Mandal and Ghosh

2009b).

Although decrease in enzyme activity has been

assumed as the result of formation of insoluble

complexes with the dietary protein in most of the

observations (Makkar 1993), in the present study,

tannin at such low concentration in the in vitro

reaction mixture may form no or negligible amount

of complex which may rule out the possibility of non-

availability of substrate for enzymatic degradation

(Maitra and Ray 2003). Moreover, tannin not only

affected the protease activity, but also inhibited

comparatively lipase activities to a greater extent

and amylase activities to a lesser extent. Lee and Pan

(2003) indicated a non-competitive mixed-type inhi-

bition of in vitro trypsin activity from grey mullet

(Mugil cephalus) by tannin which may throw some

light on possible inhibitory mechanism of such

compound. Limited literature on this particular issue

suggests that more effort should be given to gain a

better insight to find out the mechanism of tannin-

mediated inhibition of digestive enzymes of fish and

its probable effect at the physiological level.

Acknowledgments The authors are grateful to the Head,

Department of Zoology, The University of Burdwan, West

Bengal, India and The Department of Science and Technology

(FIST programme), New Delhi, India for providing research

facilities. The authors are obliged to Dr. G. Aditya, Department

of Zoology, The University of Burdwan for rendering help in

statistical analyses of data. The first author is grateful to The

University of Burdwan, Burdwan for awarding the university

fellowship.

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Inhibitory effect of Pistia tannin on digestive enzymes of Indian major carps: an in vitro study

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