Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 404
contained in commercial diets in order for reducing costs. Mostly, the animals get this compound in the form of polysaccharide, as starch.
This complex compound should be digested to be sugars as energy sources through a hydrolysis reaction Bhilave et al., 2013 by breaking
1,4’-a-glycocidic bound Steffens, 1989 Hepher 1990 in Handayani 2006. Krogdahl et al., 2005 explained that polysaccharides
are broken down by a- and b-endoglucosidases whereas di- and oligo-saccharides are hydrolyzed by various brush border enzymes into their constituent monosaccharides.
At the first step of starch digestion, amylase plays an important role to breaks starch down into some maltose and glucose Steffens 1989 Hepher 1990 in Handayani 2006. Some
studies identified the enzyme in the form of alpha-amylases Hidalgo et al. 1999 Al-
Tameemi, et al., 2010. Enzyme secretion is influenced by many factors, such as kinds of food and time Stevens
Hume, 2004; Xu et al., 2003; Bhilave et al., 2013. Feeding habit shows relate to the capability to digest
a wide range of food Fernández et al., 2001 in Castro et al. 2012. Some studies show that temperature storage influence the activity level of enzymes
Thongprajukaew et al., 2010. Based on the circumstance and explanation above, it is important to observe amylase
activities of intestine of some fish species to get some alternatives of enzyme sources.
2. Materials and Methods
The data gained were the amylase activities of 5 species of fish examined from their intestine extracts that were stored at 15 C, 25
C and 35
o
C during 4, 7 and 10 days. The intestines were got from 5 species of fish with 160
– 200 ounces of body weight. The intestines used were got by cutting digestive tract just after ventriculus. After being cut longitudinally and washed in
aquadest, they were crushed in 20 ml of 50 of glycerin. Then, 5 drops of toluene were added into the solution. The extracts resulted were kept in a closed dark bottle and stored at 15
o
C, 25
o
C, and 35
o
C for 4, 7 and 10 days in dark chambers. The amylase activities were examined after 0, 4, 7 and 10 days of storage. In each period, after filtering the extract, the amylase
activities were examined using benedict reagen for getting the level the enzyme qualitatively using color resulted, i.e. blue, greenish blue, light green, green, dark green and very dark
green, representing the level of amylase contents from zero until the highest identified. The data gained were analyzed qualitatively and descriptivelly.
3. Result and Discussion
Amylase activities of 5 species for 4 storage periods 0, 4, 7, and 10 days, and at 4 temperatures 15, 25 and 35
o
C can be seen in Table 1. Based on the data, the amylase activities can be shown from the intestines of 5 fish species
Oreochromis mossambicus
,
Oreochromis niloticus
,
Cyprinus carpio
,
Clarias batrachus and Pangasius pangasius
by greenish blue, light green, green, dark green and very dark green colours. The greens appears
in pathces, however it can be seen that there some tendency of the spreading colours, so that it seem varies among species, and temperatures and period of storages. The appearance of
these colour means that amylase can be got form the intestine of the 5 species. Some authors also identified amylase in some fish and aquatic animals Sabapathy Theo, 1993; German
et al., 2014; Hidalgo, 1999; Handayani, 2006, Thongprajuaew et al., 2010, and Xu et al., 2003.
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 405
The table shows that amylase activities are different among 5 fish species indicated by different colors. Chakrabart et al. 1995 suggest that the production of amylase is family-
specific. It may relate to anatomical and functional differences of the gastrointestinal tract and associated organs Krogdahl et al., 2005. The development of digestive organ structure is
followed by the development of digestive enzyme and the change of feeding habit Handayani, 2006. Reddy and Benarjee 2015 identified groups of fish based on types of
food, carnivorous and omnivorous. The fish have different structure of histology and histochemistry.
Table 1. Amylase Activities in Various Temperatures and Period of Storages of 5 Fish Species
Notes: 1= blue
2 = greenish blue 3 = light green
4 = green 5 = dark green
6 = very dark green
Based on some suggestions, the five fish studied can be divided into 3 groups, i.e. herbivorous, omnivorous and carnivorous. Some authors determine that
Oreochromis mosambicus
Doupe et al., 2010 and
Oreochromis niloticus
Getachew, 2006 are herbivorous. Therefore Al-Tameemi 2010 wrote that
Cyprinus carpio
is an omnivorous or plant oriented omnivorous Smith, 1980. In addition, catfish or
Clarias batrachus
is an animal oriented omnivorous Smith, 1980 or omnivorous
Reddy Benarjee 2015 ,
however, Devi Mishra 2013 mention that the animals are carnivorous. The last
Pangasius pangasius
is grouped as carnivorous Dey et al., 2015. Table 1 shows that the dark green and very dark green colours tend to appear in many samples
of
Oreochromis mossambicus
and
Oreochromis niloticus
extract, followed by
Cyprinus carpio
’s,
Clarias batracus
’s, then Pangasius pangasius’s. It means that the amylase activities tend to dominate in the intestine of the first two species. As discussed that the two animals
are herbivorous, so that it makes a sense that the amylase activities are higher than others’.
Day Species of fish
Control Storage temperature
o
C 15
25 35
1. O. mossambicus 1
- 1
- 2. O. niloticus
1 -
1 -
3. C. carpio 1
- 1
- 4. C. batrachus
1 -
1 -
5. P. pangasius 1
- 1
-
4 1. O. mossambicus
1 5
2 5
2. O. niloticus 1
6 5
5 3. C. carpio
1 2
2 2
4. C. batrachus 1
2 4
4 5. P. pangasius
1 2
2 2
7
1. O. mossambicus 1
1 1
1 2. O. niloticus
1 3
1 4
3. C. carpio 1
3 4
1 4. C. batrachus
1 1
1 1
5. P. pangasius 1
1 1
1
10 1. O. mossambicus
1 2
2 2
2. O. niloticus 1
2 3
2 3. C. carpio
1 2
1 1
4. C. batrachus 1
2 3
1 5. P. pangasius
1 2
2 4
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
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Biology Page 406
Hoffer Sturmbauer 1λκ5; Sabapathy Theo 1λλ3; Ugolev Kuz’mina 1λλ4; and Hidalgo et al. 1999 supported that the amylase activities are higher in herbivorous than in
carnivorous fish. Hidalgo et al. 1999 added that the activities are very low in some carnivores. The differences are explained by some studies that activities of intestinal amylase
correlate positively with carbohydrate intake and feeding intensity Kawai Ikeda 1972;
Cowey Walton 1λκλ; reviewed by Kuz’mina 1λλ6b in Krogdahl et al. 2005. Moreover, Simon 2009 showed significant differences between feeding groups under natural and
formulated diets concerning the activity of digestive enzymes, including amylase, of cultured juvenile spiny lobster,
Jasus edwardsii
. In addition, Le Moullac et al. 1996 identified differences in the amylase activity in
L.vannamei
fed with different protein sources. The authors explained that a decrease in amylase activity and disappearance of one amylase
isoform appeared, when the ammount of dietary casein was increased. It may because of the changes in expression of several genes that relates to metabolic and physiological adjustments
to assimilate the type of food provided Chavez-Calvillo et al., 2010. Castro et al. 2012 add that the ability to digest different food items is related to enzyme profile of a given species.
The presence of numerous amylase isoforms maybe an ecological advantage and may indicate that species are able to benefit more from carbohydrates in diet.
Storage of intestine extracts during periods of time was done for extracting enzyme from intestine glands cells. Beside relating to species differences, the amylase activities differ
among periods of storage see Table 1. There was no amylase activity before storage or just after intestine extraction shown by blue colour resulted. The activities, shown by greenish
blue, light green, green, dark green and very dark green, started from day 4 until the end of experiment day 10. Generally, the highest activities were after 4 day storage. This
phenomena may due to the work of toluene on the cell membrane of intestine gland cells that contain amylase produced. Toluene is an organic solvent that acts on cell membrane by
solubilizing phospholipids and denaturing proteins. Sarathchandiran 2012 added that lipophilic material can be dissolved in toluene
. Just after extracting intestine, this toluene had
not worked on the cell membrane. It seems to work after 4 day storage periods see Table 1. Specifically, in relation to periods of storage, different optimum activities occur among
species. It happen after 4 days storage in
Oreochomis niloticus
,
Oreochromis mossambicus
, and
Clarias bastracus
, followed by 7 days storage in
Cyprinus carpio
, and 10 days storages in
Pangasius pangasius.
These phenomena may due to different structure of cell membrane of amylase glands, so that it inflence the works of toluene. Karakali 1995 reviewed that
structure of gastrointestinal tract differ among animal species, especially membrane composition of enterocytes, and different quantity and location of proteins. Reddy Benarjee
2015 supported that different animals have different structure of histology and histochemistry.
Table 1 shows that the amylase activities varies among temperature storages of intestine extracts. The activities seem different among species. It may due to various factors. Different
species may have different pH and temperatures in relation to the level of amylase activities. Castro, et al., 2012 showed different amylase specific concentration among different sexes
and species through zymogram. The optimal amylolytic activity of shrimp occured at between 40 and 50°C. None of the enzymes from the species were thermally stable at
temperatures above 55°C. Moreover, Bhilave et al. 2014 added that the temperature profile study revealed that the neutral amylase isoform with optimal pH 7 showed optimal
temperature characteristics at 40
o
C and the alkaline amylase isoform with optimal pH 9 had optimal temperatures of 30
o
C and 50
o
C. Other author wrote that lipid membrane permeability depends on temperature Blicher et al., 2009. Thus, the storage temperature may influence
the permeability of enzyme gland cell membrane. As a result, the levels of enzyme secretion
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 407
are different with temperature storages. It causes different level of amylase activities among storage temperatures. Karakali 1995 reviewed that structure differences of gastrointestinal
tracts, especially membrane composition, can determine the variable of transport of some molecules.
4. Conclusion
