72
inasua fermentaion. In addiion, the sugar contained in coconut sap can be also converted into lacic acid
by lacic acid bacteria Atputharajah et al., 1987. Fermented coconut sap is a combinaion of alcoholic,
lacic acid, and aceic acid fermentaion Njoki et al., 2015.
Shelf life of inasua can last for a long ime. Good processing can extend the shelf life of this
fermentaion product to more than a year. Long shelf life of a product fermented ish very inluenced the
diversity of microbes, especially lacic acid bacteria. The diversity of microbes in the inasua fermentaion
using coconut sap has not been done. Inasua processing that uilizes various demersal ish species
may allow the ideniicaion of lacic acid bacteria involved in the fermentaion of ish. Based on the
descripion above, it is necessary to isolaion and characterizaion of lacic acid bacteria in inasua.
2. Materials and methods
2.1. Sample Collecion Inasua samples were processed using
three diferent types of ish and has undergone fermentaion for 5 months is taken from the people at
TNS-Waipia District , Ceram Island. The three sample is Samandar inasuaberonang Siganatus gutatus ,
Gala - gala inasua Lutjanus sp , and inasua sikuda lencam Lethrinus ornatus. Each of samples was
taken 250 g in an Tupperware and then transported to the Laboratory of Microbiology IPB to measure
pH, calculaions of total plate count, isolaion and
characterizaion of lacic acid bacteria in inasua. 2.2. Calculaion of Microbes Number in Inasua
A total of 25 g sample was mixed with 225 ml of sterile peptone 0.1 w v peptone is
then homogenized using a stomacher at 10 x g for 1 minute. A total of 100 mL of sample inasua
homogenized further diluted serially and spreaded linearly on the de Man, Rogosa and Sharp Agar
MRSA medium, which has been added 1 w v of calcium carbonate CaCO
3
and Nutrient Agar NA medium. Incubaion was performed at room
temperature for 48 hours. Diluions used are 10
-2
, 10
- 3
, and 10
-4
. Colonies that grew on NA media is used to determine the total plate count TPC while colonies
grown on media MRSA+CaCO
3
is used to determine the amount of lacic acid bacteria
2.3. Isolaion and Characterizaion of Microbial in Inasua
Colonies that have diferent morphological characterisics were scratched at MRSA+ CaCO
3
to obtain pure cultures which are considered as isolates.
Further isolates characterized macroscopically. All isolates were stained with Gram stain and catalase
test using hydrogen peroxide 3 vv. Formaion of bubbles indicates isolates tested are posiive catalase.
Isolates are Gram posiive and negaive catalase subsequently observed under a light microscope
1000x magniicaion to determine the microscopic characterisics of lacic acid bacteria Fan et al. 2013;
Kusmarwai et al .2013. One selected bacteria were tested for physiological characterisics to hydrolyze
the carbohydrates using API 50 CHB kit. Incubaion was performed for 24 hours and 48 hours and then
analyzed using sotwere API bioMérieux.
3. Results
3.1. The Number of Microbes in Inasua The measurement of pH from inasua samples
were 5.9, 5.0 and 5.8, respecively. The measurement of total plate count of bacterial isolates from inasua
by using Nutrient Agar NA medium, and for lacic acid bacteria by using de Man, Rogosa dan Sharp
Agar MRSA + CaCO
3
medium. The highest number of lacic acid bacteria was found from Gala – gala
inasua was 2,5x10
7
cfug sample Table 1.
Mahulete et al Journal of Tropical Biodiversity and Biotechnology, Volume 1, Issue 2, December 2016, 71-76
Table 1. Total plate count of the third inasua that grow on NA and MRSA+CaCO3 medium
Sources Total plate count cfug
NA Media MRSA+CaCO
3
Media
Samandar inasua 1.4 x 10
6
± 2.68 1.2 x 10
6
± 1.64 Gala-gala inasua
2.8 x 10
7
± 0.10 2.5 x 10
7
± 0.70 Sikuda inasua
3.5 x 10
5
± 1,94 6.2 x 10
4
± 3.10 3.2. Morphological Characterisics of Microbes in Inasua
Isolaion of all isolates bacteria from inasua showed that a total of 7 isolates of bacteria was obtained from Samadar inasua, 9 isolates from Gala-gala inasua, and 7 isolates from Sikuda inasua. From a total of 23
isolates, only 6 isolates had characterisic as lacic acid bacteria that were Gram posiive, negaive catalase, and cocci shape Table 2.
The colony characterisics of the six isolates on MRSA-CaCO
3
medium were round with smooth edges, convex elevaion, and creamy white. The microscopic characterisics of the isolates are coccid in pairs or
uniforms which combine to form tetrads Figure 1
73
Table 2. Microscopic characterisics and catalase test of microbes in inasua
Isolate Resources
Cell shape Gram stain Catalase test
Sm 01
Samandar inasua rod
negaive posiive
Sm 02
Samandar inasua cocci
posiive negaive
Sm 03
Samandar inasua cocci
posiive negaive
Sm 04
Samandar inasua rod
negaive posiive
Sm 05
Samandar inasua rod
negaive posiive
Sm 06
Samandar inasua cocci
posiive posiive
Sm 07
Samandar inasua rod
negaive posiive
Gg 01
Gala-gala inasua cocci
posiive negaive
Gg 02
Gala-gala inasua rod
negaive posiive
Gg 03
Gala-gala inasua rod
posiive posiive
Gg 04
Gala-gala inasua rod
negaive posiive
Gg 05
Gala-gala inasua cocci
posiive posiive
Gg 06
Gala-gala inasua rod
negaive posiive
Gg 07
Gala-gala inasua rod
negaive posiive
Gg 08
Gala-gala inasua cocci
posiive negaive
Gg 09
Gala-gala inasua cocci
posiive posiive
Si 01
Sikuda inasua cocci
posiive negaive
Si 02
Sikuda inasua rod
negaive posiive
Si 03
Sikuda inasua rod
posiive posiive
Si 04
Sikuda inasua rod
posiive posiive
Si 05
Sikuda inasua rod
negaive posiive
Si 06
Sikuda inasua cocci
posiive negaive
Si 07
Sikuda inasua batang
posiive posiive
Isolates that hav
e characterisics such as LAB
a b
c
Figure 1. Microscopic characterisic of lacic acid bacteria in Samandar inasua a, Gala-gala inasua b, and Sikuda Inasua c.
3.3 Characterisics of Microbial Physiology in Inasua Catalase test using 3 hydrogen peroxide showed that six isolates were proposed as lacic acid bacteria.
All isolates did not form bubbles of gas on catalase test, so it can be concluded that these isolates are negaive catalase. Carbohydrate hydrolysis test using API 50 CHB kit showed that 13 kinds of carbohydrates are fermented
by selected isolate ater incubaion for 48 hours Table 3. The results of bacterial ideniicaion kit API 50 CHB can not show a species of lacic acid bacteria.
Table 3 The test results of carbohydrate uilizaion of selected isolates using API 50 CHB kit ater incubaion for 48 hours
Types of carbohydrate Test results
Types of carbohydrate Test results
Control -
Esculin -
Glycerol +
Salicin -
Erythritol -
Cellobiose -
Mahulete et al Journal of Tropical Biodiversity and Biotechnology, Volume 1, Issue 2, December 2016, 71-76
74
D-Arabinose -
Maltose +
L-Arabinose -
Lactose +
Ribose -
Melibiose -
D-Xylose -
Sucrose +
L-Xylose -
Trehalose +
Adonitol -
Inuline -
Methyl xyloside -
Melezitose -
Galactose +
Rainose -
Glucose +
Starch -
Fructose +
Glycogen -
Mannose +
Xylitol -
Sorbose -
Gentobiose +
Rhamnose -
D-Turanose +
Dulcitol -
D-Lyxosa -
Inositol -
D-Tagatose -
Mannitol +
D-Fucose -
Sorbitol -
L-Fucose -
Met-D-Mannoside -
D-Arabitol -
Met-D-Glucoside -
L-Arabitol -
NAc-Glukosamine +
Gluconate -
Amygdalin -
2-Kt-gluconate -
Arbuin -
5-Kt-gluconate -
4. Discussion
