15
B. RESISTANT STARCH PRODUCTION
This research used sweet potato starch from Jago variety which is obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor. Sweet pootato
starch then modified into type 3 resistant starch using pullulanase enzyme according to Vatanasuchart et al. 2010.
. According to Wasserman et al 2007, making type 3 resistant starch RS3 can be divided
into two stages, the first stage is heating process which will trigger gelatinitation. The starch granule will swell due to water absorption during heating process. Water absorption is caused by
kinetic energy from water molecule become stronger than starch molecule affinity inside the granule such that water will come into the starch granule. The second stage is starch retrogradation
which cause recrystallization of amylose and amylopectin chain. Gelatinized starch is easier to be digested than the raw starch even though the starch gel is not stable and form crystal when cooled
retrogradation. Starch retrogradation will result in the insoluble short chain polymer and resistant to digestive enzymes.
Polymer recrystallisation is a three-stage process that involves nucleation formation of critical nuclei, propagation crystal growth from the nuclei formed and maturation continued
crystal growth and perfection. The nucleation and propagation rates determine the overall recrystallisation rate whereas the maturation rate is more temperature dependent Eerlingen et al.,
1993. Nucleation generally proceeds rapidly when the incubation temperature is close to the glass transition temperature of starch, at about 5
o
C Gray Bemiller, 2003. Formation of RS3 involves recrystallisation of amylose in a partially crystalline system in a
process that is influenced by the incubation temperature and time Eerlingen et al., 1993a; Haralampu, 2000. In this research, pullulanase enzyme 5 was used at 55
o
C, 24 h incubation hours in pH 5.0. The pullulanase enzyme preferably reacts with pH from 4.5 to 5.5 at a
temperature of 40°C to 60°C Vatanasuchart et al. 2010. Incubation time used was 24 h which refer to Onyago et al 2006 who reported that maximum yields of RS3 of cassava were obtained
after 24 h incubation. Pullulanase is debranching enzyme for hydrolysing α-1,6 glucosidic bond of
the starch. In the present study, debranching using pullulanase was applied to produce linear, low molecular weight and recrystallizable polymer chains. Debranching enzymes such as pullulanase
rapidly hydrolyze only a-1,6-glucosidic bonds, releasing a mixture of long and shorter unit chains from the parent amylopectin molecule. These fragments are linear polymers containing about 10 to
65 anhydroglucose units linked by a-1,4-glucosidic bonds. The debranched starch was then subjected to temperature cycling and incubation at a series of temperature and time to induce
retrogradation and formation of the RS Leong et al. 2007. After incubating at 55
o
C 24 h, the starch slurry was heated at 90
o
C to inactivate the pullulanase enzyme. Starch slurry was then stored in 4
o
C during 24 h to trigger retrogradation. Onyago et al 2006 reported that retrogradation temperature 4
o
C will favour a high nucleation rate and therefore high yields of RS3 in this study : cassava. The resulting retrograded starch or
RS3 was dried by spray dryer. Spray drying method was choosen because of the high efficiency result. Production of RS3 was conducted seven times in this research. Each production used 20
gram sweet potato starch as raw ingredient and resulted in 2.00±0.16 gram RS3 or 10 yield. Yield of RS3 production is presented in Figure 3.
16 Figure 3. Resistant Starch Yield
Resistant starch analysis was conducted according to Goni et al. 1996. The main features of the analytical procedure are removal of protein, removal of digestible starch, solubilization and
enzymatic hydrolysis of RS, and quantification of RS as glucose released x 0.9 where stomach and intestine physiological conditions pH, transit time are approximately simulated. The removal of
protein was introduced to enhance amylase accessibility avoiding starch-protein associations. Moreover, this step is advisable for a better simulation of physiological conditions proteolytic
digestive enzymes, acidic pH. The removal of digestible starch is done to avoid positive mistake when glucose is released.
Purwani et al 2009 reported that resistant starch content of jago sweet potato was 13.77. The analysis showed that resistant starch content of jago sweet potato treated with pullulanase was
28.15, which was lower than Salosa sweet potato starch treated with pullulanase enzyme that contained 38.22 RS Evalin, 2011. Treatment with pullulanase and retrogradation process were
proved could increase resistant starch content of jago sweet potato starch. Purwani et al 2009 reported that sago resistant starch treated with pullulanase contained 31-38 RS and rice resistant
starch treated with pullulanase contained 21-26 RS, whereas Zhao 2009 reported that maize resistant starch treated with pullulanase contained 24.5-32.4 RS. It showed that Jago sweet
potato starch treated with pullulanase contained higher RS content than rice starch treated with pullulanase enzyme, but lower than sago resistant starch and maize resistant starch which also
treated with similar enzyme. The difference could have been caused by several factors such as starch and enzyme used, amylose content, heating and cooling condition, drying method, etc.
C. FERMENTATION OF RS3 by Clostridium butyricum
