PULLULANASE ENZYME ACTIVITY PRO

18 Starch-protein interaction and formation of amylose-lipid complexes reduced yield of resistant starch. Amylose-lipid complexes are enzyme-degradable,however retrogradation of amylose is dificcult to form crystalline structure and reduced yield of resistant starch Sajilata, 2006. In 1987, at the Loisiana State University Agricultural Center LSU AgCenter Larry Ralston developed the Beaurgard, a new variety of high quality, high yield sweet potato and The Evangeline sweet potato released by the LSU AgCenter in 2007 is a new variety of sweet potato developed by Dr Don LaBonte. Futch, 2009. The Beauregard sweet potato yielded a starch with 23.6 amylose, while Evangeline, Jago, and Sukuh sweet potato starch contained 27.1, 25.83, and 29.35 amylose, respectively.Moorthy 2002 found that sweet potatoes have an amylose content around 20. Most of chemical content of two variety is similar with Sukuh sweet potato. The quality of chemical content sweet potato starch is affected mainly by the biological and environmental factors. These factors include genotype, soil types, and climatic conditions, which are very different from one crop to another Katayama et al, 1999. Differentiation result in proximate analysis appeared in the moisture content between Beauregard and Evangeline with Jago and Sukuh. Both of Beauregard and Evangeline have low content of moisture content compared with Jago and Sukuh. Differentiation in method of starch extraction influence the differentiation of moisture content. In extraction of Beauregard and Evangeline, pulp of sweet potato passed through a 150 µm sieve then it was centrifuge at 3000 g. The precipitate was removed and freeze dried Futch, 2009. Compared with method of Jago and Sukuh starch extraction, pulp of sweet potato was not sieved through a 150 µm. Pulp of Jago and Sukuh sweet potato was sieved by adding distilled water with the ratio sweet potato : distilled water was 1 : 3 bv, then sieved by pore material. Starch was precipitated and washed by distilled water, then it was dried by oven. Water removal by freeze drier resulted lower moisture content on Beauregard and Evangeline starch compared with Jago and Sukuh which were dried by dry oven.

4.2 PULLULANASE ENZYME ACTIVITY

Debranching using pullulanase has been applied to produce a sample with linier, low- molecular-weight and recrystallizable polymer chains. Debranching enzymes such as pullulanase rapidly hydrolize only α-1.6-glucosidic bonds, releasing a mixture of long and shorter unit chains from the parent amylopectin molecule. These fragments are linier polymers containing about 10 to 65 anhydroglucose units linked by α,1-4-glucosidic bonds. The debranched starch was then subjected to temperature cycling and incubation to induce retrogradation and yield the RS Leong, 2007. Industrial pullulanase Promozyme®, rather than highly purified pullulanase was used in this study to establish the feasibility of ultimately developing a process for commercial application. Pullulanase enzyme activity assay measured the amount of reducing sugar formed by the hydrolitic action of the enzyme on starch. The original unit IU was defined as the amount of enzyme which produces reducing substances, expressed as glucose, during incubation time 10 minutes under stated condition; although this is not a true representation of the actual course of the reaction. Based on this study, pullulanase activity reached 177, 222.11 IUml. It was defined as the amount of enzyme ml enzyme which produced µmol glucose as released by debranching enzyme Anonim, 1963. While compared with standard activity and density of Promozyme® D2 were 1350 NPUNg and 1.20 gml, respectively. This starch degrading enzyme was obtained from Novo Nordisk through the PT Halim Sakti Pratama, Jakarta. 4 s a b s a s i c g r g o T

4.3 PRO

Gener stage is heatin absorption dur become strong starch granule. amylopectin c starch gel is no in the insoluble Polym critical nuclei growth and pe rate whereas th generally proc of starch, at ab Produ Table 7. Figur Production 1 2 3 4 5 6 7 Yi el d ODUCTION rally, formatio ng process wh ring heating pr ger than starch . The second st hain. Gelatiniz ot stable and fo e short chain p mer recrystalli , propagation erfection. The he maturation eeds rapidly w bout 5 o C Gray uction of type- re 5 explains co Tab Starch Weigh g 20.01 20.00 20.00 20.01 20.03 20.07 20.02 Figure 10,6 10,8 11 11,2 11,4 11,6 11,8 1 11,76 N OF TYPE on of type III r ich will trigge ocess. Water a h molecule affi tage is starch r zed starch is e orm crystal wh polymer and res sation is a th crystal growth e nucleation an rate is more t when the incuba and Bemiller, -III resistant st omparison star ble 7. Yield of t g Resist Produc e 5. Yield of Su 2 6 11,75 1 E-III RESIST resistant starch er gelatinizatio absorption is ca inity inside the retrogradation easier to be di hen cooled retr sistant to diges hree-stage proc h from the nucl nd propagation temperature de ation temperatu 2003. tarch using me rch weigh and r type-III resista tant Starch ct Weigh g 2.35 2.35 2.34 2.21 2.22 2.22 2.22 ukuh type-III r 3 4 11,71 11,05 Producti TANT STA h could be div on. The starch aused by kinet e granule such which cause re igested than th rogradation. S stive enzymes cess that invo lei formed and n rates determi ependent Eerli ure is close to t ethod of Vatan result of type-I ant starch produ Yield 11 11 11 11 11 11 11 resistant starch 5 11,08 1 on ARCH vided into two granule swell tic energy from h that water wi ecrystallization he raw starch Starch retrograd Wasserman et olves nucleatio d maturation c ine the overall ingen et al., 19 the glass transi nasuchart 201 III RS product uction d .76 .75 .71 .05 .08 .05 .09 product 6 7 1,05 11,09 Sukuhs RS o stages, the fi led due to wat m water molecu ill come into t n of amylose a even though t dation will resu t al, 2007 on formation continued crys recrystallisati 995. Nucleati ition temperatu 10 is showed . Mean 11.36 ± 0.36 S 19 rst ter ule the and the ult of tal on on ure in 20 Yield of Sukuh resistant starch product using method of Vatanasuchart 2010 was 11.36 ± 0.36. Sukuh resistant starch has higher yield of RS III product compared with Jago 10.91 ± 0.63 Devega, 2011 but lower than Salossa sweet potato 20 Evalin, 2011, sago RS III 18 Purwani and Suhartono, 2009, cassava RS III dried by extrusion 30, hot air 21.5, and spray drier 14.3. Lower yield of the RS III product caused lost of starch material in each followed step of RS III production. In gelatinization step, starch was moved into plastic then it was moved again into erlenmeyer for hydrolyze sample using pullulanase treatment in shaking waterbath. There was some material which was left in the previous medium when the starch was moved. Beside that, drying using spray drier resulted lower yield of RS III product than using extrusion, hot air, or drum drier Vatanasuchart, 2010. High temperature of spray drier made high amount of starch was evaporated and resulted in lower yield of RS III product. Debranching pullulanase enzyme resulted starch consist of debranched amylopectin unit chains and some hydrolyzed amylose fraction which has lower molecular weigh fraction. RS also contained a starch fraction with structural properties similar with amylose which could be soluble in liquid starch mix. Soluble fraction of the starch could be lost along centrifugation step. Centrifugation separated starch fraction and amylose fraction could be soluble in liquid fraction and separated from precipitate of RS III and resulted in lower yield of RS III product. RS yields in gelatinized starch depend strongly on process condition like gelatinization temperature, enzyme hydrolysis condition pH, temperature, time, enzyme concentration, retrogradation condition, and drying. Retrogradation time and temperature influence strongly with yield of resistant starch product. Indeed, it was concluded that the formation of highly resistant starch fractions in gelatinized starch can be considered as crystallization of amylose in a partially crystalline polymer system Eerlingen and Delcour, 1995. Formation of B-type crystals is needed to make type- III resistant starch while at lower temperature 0-5 C it was observed formed. Crystallinity of the resistant starch fraction was comparable with storage time of the starch gel Eerlingen and Delcour, 1995. Specific method to determine RS in foods is classified as direct method and indirect method. Direct method quantifies RS in the residues obtained after removing digestible starch Berry, 1986. Indirect method determines RS as the difference between total starch and digestible starch Tovar et al., 1990; Englyst et al., 1992. Goni 1996 has been developed a direct method to quantify RS in food and food product. It was derived from Berry 1986 method with essential modifications. The main features of the analytical procedure are: removal 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 Goni, 1996. Resistant starch product derived from Sukuh sweet potato contained higher RS content 30.83 ± 2.44 Table 9 than those from rice starch 21-26, sago starch 31-38 Purwani and Suhartono, 2009, Jago sweet potato starch 28.15 but lower than Salossa sweet potato starch 38.22 Evalin, 2011. The difference could have been caused by several factors such as starch and enzyme used and also heating and cooling condition. Purwani 2009 reported that resistant starch content of Sukuh sweet potato was 13.77. Increasing of resistant starch content happened caused by heating, pullulanase treatment, and retrogradation process of the starch to form crystalline structure of amylose. Amylose content, RS yield and resistant starch content are positively correlated Berry, 1986. Increasing of amylose content influence with low starch degradability and increasing the yield of resistant starch Escarpa et al., 1996. When heated to about 50 C, in the presence of water, the 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