ANALYSIS OF FERMENTATION PRODUCT EXPERIMENT I

21 amylose in the granule swells; the crystalline structure of the amylopectin disintegrates and the granule ruptures. The polysaccharides chains take up a random configuration, causing swelling of the starch and thickening of the surrounding matrix such as, gelatinization- a process that renders the starch easily digestible. On cooling drying, recrystallization retrogradation occurs. This take place very fast for the amylose moiety as the linier structure facilitates cross linkages by means of hydrogen bonds. Crystallization of amylose in retrogradation process made starch become resistant and difficult to access by enzyme and reduced starch digestibility Sajilata, 2006. The rate and extent to which a starch may retrograde after gelatinization essentially depends on the amount of amylose present. Repeated autoclaving of wheat starch may generate up to 10 RS. The level obtained appeared to be strongly related to the amylose content, and the retrogradation of amylose was identified as the main mechanism for the formation of RS that can be generated in larger amounts by repeated autoclaving Berry, 1986. Sukuh starch has high amylose content 29.35 ± 0.67. High amylose content in Sukuh starch influence the formation and content of type-III resistant starch. Compared with Purwani and Suhartono 2009, RS 3 from sago and rice starch were treated with pullulanase, α-amylase, and both of them. Among the three treatment of enzyme applied to the starch, using pullulanase alone resulted least breakdown of the starch. Following pullulanase digestion, the liquid present in the flask appeared clear and odorless. It was contrast with using amylase or enzyme cocktail of amylase and pullulanase. When amylase or its combination with pullulanase was applied, the liquid present in the flask showed brown and sweet smelling. Formation of resistant starch also depends on the water content and autoclaving temperature. Indeed, as the amylose concentration increase, RS yield increases. A minimum of water, however, is necessary for plasticization of the environment and for the incorporation into the crystal structure B- type crystal structure indeed contain about 27 water. The influence of the autoclaving temperature varies with starch type. Autoclaving at 148 C, however, results in crytal melting Eerlingen, 1995. Positive effect of starch debranching enzyme also happened on the formation of resistant starch. The hydrolysis of α-1,6 glycosidic bonds would produce more free linear chains in the hydrolyzate. These linear chains, as similar to amylose, could participate in crystal formation by chain elongation and folding. These newly formed crystals could become more perfect during storage of the hydrolizate. Hydrolisis of α-1,6 glycosidic bonds could disentangle, from aylopectin, the double helices and crystallite, which are formed by the re-association of amylopectin A-chains during retrogradation. These disentangled starch entities have unassociated linier chain segments at both ends. Storage at particular temperature and time would like promote association of these free linier chain segments and their close packing. Consequently, the number of perfect starch crystals would increase. Without disentanglement from the amylopectin molecule, the association of the linier segments starch entities and subsequent crystallite formation may be difficult due to lower flexibility of the starch chain segments owing to their closer proximity to the branching points Leong et al., 2007.

4.4 ANALYSIS OF FERMENTATION PRODUCT EXPERIMENT I

In vitro fermentation of RS3 was divided into 2 experiments. Experiment I was design to evaluate the growth of Eubacterium rectale 17629 include fermentation capability SCFA production pattern and optimum time that produce the highest butyrate concentration. Glucose 5 gl 0.5 and RS3 derived from sweet potato of Sukuh variety starch treated with pullulanase in amount of 20 gl 2 were added into medium. Concentration of 2 Sukuh type-III resistant starch was chosen 22 referred to Evalin 2011 that reported 2 Salossa sweet potato type-III resistant starch give highest concentration of butyric acid. In experiment I, the medium with resistant starch as a substrate was distributed into 20 ml of medium and inoculated with 1 ml of 24 hours pre-culture. Incubation was performed at 37 C for 6, 12, 24, 36, and 48 hours. Interval time was chosen to simulate physiological condition in the digestive system.

4.4.1 Turbidimetry and pH measurement

Growth of Eubacterium rectale 17629, its fermentation capability SCFA production pattern and optimum time that produce the highest butyrate concentration was evaluated in Experiment I. Glucose 5 gl and RS3 derived from sweet potato of Sukuh variety starch treated with pullulanase in amount of 20 gl 2 were added into medium. Sampling was done after 6, 12, 24, 36, and 48 hours of incubation at 37 C.Turbidimetry result and pH change is presented in Figure 6 and Figure 7, respectively. Fermentation medium without bacteria culture is used as a blank. Fermentation product for each sampling was measured using spectrophotometry at 660 nm. Higher result of absorbance showed higher cell turbidity and higher growth of the bacteria cell in the fermentation medium. Result showed that the absorbance cell growth increased after 6 and 24 hours. Fermentation reached the highest absorbance at 48 hours of fermentation. It showed that Eubacterium rectale 17629 could use resistant starch substrate from Sukuh sweet potato. Eubacterium rectale 17629 could produce amylase to degrade the resistant starch into glucose. They used resistant starch as carbon source for its growth. Figure 6. Turbidimetry result of E.rectale 17629 growth over 48 h fermentation at 370C 0,186 0,188 0,188 0,193 0,198 0,184 0,186 0,188 0,19 0,192 0,194 0,196 0,198 0,2 10 20 30 40 50 60 Absorb anc e 660nm Fermentation time hours 23 Figure 7. Change of pH fermentation by E.rectale 17629 over 48 h at 370C Decreasing of pH was observed over the time course of fermentation. The lowest pH value 4.16 was achieved at 48 h fermentation. Reduction of pH value occurred as the bacteria produce short chain fatty acid during fermentation process. SCFA production made the fermentation suspension become acid and decreasing pH value of medium. Lower pH value are believed to prevent the overgrowth of pH-sensitive pathogenic bacteria and lower the production of potentially harmful toxic or carcinogenic products in the colon, including secondary bile acids and protein fermentation products ammonia and phenols Topping et al, 2001.

4.4.2 Production of Short Chain Fatty Acid

Bacteria of Eubacterium rectale is reported to be one of the most abundant bacterial species in human feces both from anaerobic cultivation Finegold et al., 1983; Moore Holdeman, 1986 and culture-independent analysis of 16S rRNA sequences Aminov et al., 2006. The genus Eubacterium includes many species of obligately anaerobic bacteria, with Eubacterium limosum designated as the type species. Species of the genus Eubacterium produce mixture of organic acids as fermentation product from carbohydrate, including butyric, acetic, lactic or formic acids, but not propionic and succinic acids, as major products Krumholz Bryant, 1986. Most also produce hydrogen gas. The profile and and proportion of SCFA produced by Eubacterium rectale 17629 is presented in Figure 8 and Table 8. Gas chromatography result showed that acetic, propionic, and butyric acid were produced during 48 hours of fermentation. Chromatogram result is explained in Appendix. Based on chromatogram, peak of acetic acid, propionic acid, and butyric acid is appeared approximately at 8.6; 10.08; and 11.79 minute. Peak of acetic acid firstly appeared at chromatogram because it has higher volatility than propionic and butyric acid. Volatility is influenced by differentiation of fatty acid’s boiled point. Chromatogram result based on fatty acid peak was quantified by fatty acid standard chromatogram. The highest production of butyrate was achieved at 36 hours 50.97 mmolL while propionate was achieved at 36 hours 25.17 mmolL. The value zero was applied for zero or negative concentration based on standard curve calculation. 7,21 5,07 4,79 4,37 4,26 4,16 1 2 3 4 5 6 7 8 10 20 30 40 50 60 pH Fermentation time hours t 3 4 4 i R o r 0,00 10,00 20,00 30,00 40,00 50,00 60,00 Cocentr ati o n mM Figure 8 Table 8. Prop 37 C Fermentation time hours 6 12 24 36 48 Comp 17629. Based the highest SC 36 h, where th 0:1:2.03, but

4.5 ANA