in the food industry , sucrose is the most commonly used sweetener as a result of its physical and chemical and sensory characteristics . however , because of its high caloric value and cariogenic properties , alternative sweeteners are being studied . in the past two decades , there has been an increasingly high interest in the production of isomaltulose ( also known as palatinose or lylose ) . isomaltulose is found naturally in small amounts in both honey and sugarcane and has been considered a promising sucrose replacement . isomaltulose is a reducing disaccharide , a sucrose isomer obtained by microbial enzymatic conversion of sucrose [ 1 , 2 ] . isomaltulose has low cariogenic potential and reduced rates of hydrolysis and monosaccharide formation in the body , and it is therefore recommended for use in beverages and foods intended for diabetics and athletes [ 1 , 3 ] . such items as intermediate disaccharides , polymers such as biodegradable detergents , and surfactants for industrial use may be obtained [ 4 , 5 ] . isomaltulose can also be applied to produce isomaltulose oligomers , which act as prebiotics , stimulating proliferation of intestinal microbiota bifidobacteria . the main isomaltulose derivative is isomalt , a sugar alcohol obtained by hydrogenation resulting in an equimolar mixture of [ 6-o-(-d - glucopyranosido)-d - sorbitol ] and [ 1-o-(-d - glucopyranosido)-d - mannitol ] , a noncariogenic compound with low caloric value . microbial conversion of sucrose into isomaltulose has attracted great commercial interest due to its complexity of chemical synthesis . isomaltulose is currently produced in large scale by using stable continuous columns of immobilized cells . alginate is one of the most commonly used supports for immobilizing whole microbial cells , because its use is simple and cheap ; it is also a reproducible technique and gentle during the immobilization process [ 9 , 10 ] . some papers have recently reported the use of transglutaminase to immobilize cells and enzymes [ 1115 ] . transglutaminase is a microbial enzyme that catalyzes the formation of protein cross - links by creating a link between the carboxyl group of glutamine and the -amino group of lysine . in this paper , experimental design technique and response surface analysis were used to assess both immobilization parameters and effects of gelatin and transglutaminase addition to a calcium alginate immobilization process on the stability and conversion of sucrose into isomaltulose . d12 strain that produces the intracellular enzyme sucrose isomerase , capable of converting sucrose into isomaltulose . the culture was grown in slant test tubes containing 6.0% sucrose ( w / v ) , 4.0% peptone ( w / v ) , 0.4% meat extract ( w / v ) , and 2.0% agar ( w / v ) , for 15 hours , at 30c . after incubation , sterile vaseline was added to the test tubes and the culture was kept at 5c , with transfers every 2 months . d12 strain cell mass was obtained by fermenting the microorganism in a culture medium containing 150 g / l of sugarcane molasses , 20  g / l of corn steep liquor , and 15  g / l of yeast hydrolysate ( prodex lac sd ) , a commercially available yeast extract ; ph was adjusted to 7.5 . the 15-hour culture of that microorganism , as described previously , was inoculated into 250  ml erlenmeyer flasks , containing 50  ml of the above - mentioned culture medium . these flasks were incubated in a shaker ( model series 25 , new brunswick scientific , edison , nj , usa ) at 200  rpm and kept at 30c , for 15 hours . a 300  ml aliquot of preinoculum , prepared following the above description , and 3  ml of dow corning fg-10 ( d'altomare qumica , so paulo , sp , brazil ) , an antifoam agent , were aseptically added to a new brunswick bioflo iic 6.6-liter fermenter ( new brunswick scientific , edison , nj , usa ) containing 2700  ml of that culture medium . after 8 hours of fermentation at 27c , that cell mass was retrieved by centrifuging at 9600  g for 15 minutes ( centrifuge model j2 - 21 , beckman coulter , inc , fullerton , calif , usa ) , at 5c , and washing twice under aseptic conditions , using presterilized distilled water . the experimental design technique was used to assess the effect of immobilization parameters on converting sucrose into isomaltulose . the parameters assessed were sodium alginate concentration , wet cell mass concentration , and cacl2 concentration . the granules containing immobilized cells , following the conditions described in tables 1 and 4 , were transferred to erlenmeyer flasks containing a sucrose solution ( granulated sugar ) and maintained in a new brunswick scientific series 25 shaker incubator ( new brunswick scientific , edison , nj , usa ) . the sugar solutions in each flask were replaced with new sucrose solution samples after every 24 hours of reaction time . the erwinia sp . d12 wet cell mass was obtained as described previously . for immobilization , a cell suspension of wet cells in sterilized distilled water was mixed with sterilized solution ( autoclaved at 121c for 15 minutes ) of sigma sodium alginate ( sigma chemical co. , st . louis , mo , usa ) at a 1  :  2 ratio ( v  :  v ) . the mixture was then dripped with a masterflex l / s peristaltic pump ( cole - parmer instruments co. , vernon hills , ill , usa ) into a previously sterilized cacl2 solution in order to form small granules , which were kept immersed in the same cacl2 solution , at 5c , for 12 hours . the reducing sugars formed were determined by the somogyi   method , using glucose as a standard . blank  was used to adjust the spectrophotometer at 540  nm ( du-70 , beckman coulter , inc . , fullerton , calif , usa ) , replacing 50  l of the sample with 50  l of distilled water in the reaction mixture . carbohydrate analysis was performed using a dionex dx-600 chromatograph ( dionex corporation , 1228 titan way sunnyvale , calif , usa ) equipped with an ip25 isocratic pump and an ed50 gold electrochemical detector . sugars were separated using a carbopac pa 1 column ( 4  mm  250  mm ) , a carbopac pa 1 guard column ( 4  mm  50  mm ) , and 250  mm sodium hydroxide solution as the mobile phase , with 1  ml / min flow , at 20c . carbohydrates were analyzed for retention time , by comparison with fructose , glucose , sucrose , and isomaltulose standards ( sigma ultra , sigma chemical co. , st . gelatin and transglutaminase concentrations were tested in cell immobilization , using experimental design technique and response surface analysis . this solution was autoclaved at 121c for 15 minutes and cooled to room temperature . after cooling , the activa tg transglutaminase enzyme ( ajinomoto interamericana indstria e comrcio ltda ) was added to that gelatin and alginate solution to increase reticulation of the alginate gel . the cell suspension and gelatin+alginate+transglutaminase solution mixture , at a 1  :  2 ( v  :  v ) ratio , was dripped in cacl2 with a masterflex l / s peristaltic pump , in order to form small granules , which were kept immersed in the same solution at 5c , for 12 hours . the conversion of sucrose into isomaltulose in a batch process by erwinia sp . d12 immobilized cells was assessed using experimental design technique . this study showed that the independent parameters assessed had a significant impact on isomaltulose stability and conversion . the parameters assessed in both immobilization and conversion of sucrose into isomaltulose were sodium alginate concentration , wet cell mass concentration , and cacl2 concentration . these tests were performed in 250  ml erlenmeyer flasks and kept in an incubator , shaken at 100  rpm and at 30c . each flask contained 50  ml of 35% sucrose solution ( w / v ) and 10  g of granules containing immobilized cells . the sucrose solution in the flasks was replaced with a new 35% sucrose solution ( w / v ) every 24 hours . samples containing such reducing sugars as isomaltulose , trehalose , glucose , and fructose were analyzed using the somogyi - nelson method . a 2 central composite rotatable design ( ccrd-2 ) was used to assess the following independent parameters : sodium alginate concentration , wet cell mass concentration and cacl2 concentration . table 1 presents the ccrd-2 matrix of 18 tests , including 8 tests for the complete factorial design 2 ( 1 ) , 8 tests for the axial points (  = 1.68 ) , the central point quadruplicate ( 0 ) , and the reducing sugars ( isomaltulose , trehalose , glucose and fructose ) as the dependent parameter as well as the levels studied with their decoded values . table 1 shows the total of sucrose conversion into isomaltulose in each of the 18 tests , reusing those immobilized cells in 7 batches . it was found that most tests presented high conversion in the batches . those granules containing immobilized cells in tests 7 , 8 , 12 , and 14 were more stable in comparison with the other tests and 647.02  mg / ml , 688.42  mg / ml , 812.93  mg / ml , and 582.20  mg / ml were obtained , respectively . 
 table 2 presents effects of independent parameters on converting sucrose into reducing sugars . regarding the main effects , it was observed that the independent parameters , sodium alginate ( l ) , cell mass ( l ; q ) , and cacl2 ( l ; q ) had positive and significant effects within the range studied , with a 95% ( p < 0.05 ) confidence level . only the interaction between sodium alginate and cell mass did not present any significant effect in the levels studied . these results indicate that increasing the concentrations of those independent parameters within the studied range would result in an increased conversion of sucrose into reducing sugars . it was observed that the interactions between sodium alginate and cacl2 and also between cell mass and cacl2 had positive effects , indicating some synergism between those parameters . table 2 also presents the t and p values used to create the quadratic polynomial equation of a sucrose conversion into reducing sugars based on those studied parameters . the p values were used to check the significance of each coefficient and also to indicate how important each independent parameter or interaction between parameters in the equation result is . the greater the t value and the lower the p value , the greater the significance of the coefficient , as observed in table 2 , where only sodium alginate ( q ) and interactions between sodium alginate and cell mass and also between cell mass and cacl2 were not significant at a 95% confidence level ( p < 0.05 ) . cell mass and cacl2 presented the strongest effects and regression coefficient values , indicating a greater influence of such parameters on the conversion of sucrose into reducing sugars . the correlation measurements used to estimate that equation were the correlation coefficient ( r ) and the coefficient of determination ( r ) . the closer r is to one , the stronger the correlation between the equation - predicted conversion values and the observed values . a determination coefficient of 0.92 was obtained , indicating that only 8% of the total variation in responses obtained is not explained by the equation . the f value obtained from anova was 17.63 ( 5.61 times greater than the value of ftabulated = f0;95;7;10 = 3.14 ) , indicating that the equation to convert sucrose into reducing sugars may be considered statistically significant at a 95% confidence level . the quadratic polynomial equation ( 1 ) was obtained after these studied parameters were validated . the statistically non significant parameters were eliminated from the equation and added to lack of fit 
								 ( 1)y=439.14 + 15.66x12 + 49.99x22  + 89.75x2 + 11.74x32 + 68.07x3  + 17.33x1x3 + 40.92x2x3 , 
							 where x1 , x2 , and x3 are sodium alginate concentration , cell mass concentration , and cacl2 concentration , respectively , and y refers to the dependent parameter , reducing sugars ( % ) . figures 1(a ) and 1(b ) were created based on that equation . after analyzing these figures , it can be verified that the concentration of sodium alginate in which the greatest conversion of sucrose into isomaltulose would be obtained was above 2.5% ( w / v ) . regarding the cacl2 variable , a concentration above 0.50  m would be beneficial for cell immobilization , resulting in greater conversion into reducing sugars . figure 1(b ) shows that a cell mass concentration above 40% ( w / v ) would increase conversion by erwinia sp . d12 cells immobilized in calcium alginate in a batch process . to confirm those results , a second test was performed with parameters previously used from tests 7 , 8 , 12 , and 14 , which achieved the best results in converting sucrose into reducing sugars from table 1 ( 2 central composite rotatable design matrix ) . a test designated as a ( alginate 4.7% ( w / v ) , cell mass 47.0% ( w / v ) , and cacl2 ( w / v ) solution 0.54  moll ) was included to represent the immobilization parameters obtained by ( 1 ) and figures 1(a ) and 1(b ) , by which the best cell immobilization and isomaltulose conversion results would be obtained in a batch process . these tests were performed in duplicate 250  ml erlenmeyer flasks and kept in a shaker incubator , at 100  rpm and 30c , as described above . parameters for test 7 ( 3.0% sodium alginate concentration ( w / v ) , 47.0% cell mass concentration ( w / v ) , and 0.3  moll cacl2 concentration ) were chosen for cell immobilization and for continuous testing , as a result of the need for lower sodium alginate and cacl2 concentrations and , consequently , a lower immobilization process cost . effects of adding gelatin and transglutaminase were assessed when converting sucrose into isomaltulose by erwinia sp . the erwinia sp . d12 cell mass used in these tests was obtained as described previously . the immobilization process for a mixture containing both cell mass suspension and alginate solution plus gelatin and transglutaminase was performed as described previously . a 2 central composite rotatable design ( ccrd-2 ) was used to assess effects of a gelatin and transglutaminase concentration on increased reticulation of the granules of cells immobilized in calcium alginate and on conversion of sucrose into isomaltulose . table 4 presents the ccrd-2 matrix of 12 tests , including 4 tests for a complete factorial 2 ( 1 ) , 4 tests for axial points (  = 1.41 ) , the central point quadruplicate ( 0 ) , and the dependent parameter , isomaltulose ( % ) , as well as the studied levels with their decoded values . these tests were performed in 250  ml erlenmeyer flasks and kept in a shaker incubator at 75  rpm and 25c . five grams of granules containing immobilized cells in 25  ml of 35% sucrose solution ( w / v ) were added to each flask . this sucrose solution in those flasks was replaced with a new 35% sucrose solution ( w / v ) every 24 hours . gelatin ( q ) and transglutaminase ( q ) significantly affected ( p < 0.05 ) the conversion of sucrose into isomaltulose ; effects were positive , suggesting that their increased concentration within the range studied would have resulted in increased isomaltulose conversion . table 6 shows the analysis of variance ( anova ) to convert sucrose into isomaltulose . the equation obtained can be considered predictable and significant at a 95% confidence level , as shown by the f - test , in which the f value obtained in our tests ( fexperimental = 10.30 ) was greater than ftabulated = 4.07 . fit of the equation was verified by the correlation coefficient ( r = 0.83 ) . pure error was 1.02 , a low value , indicating good reproducibility of the obtained values . these results were sufficient and satisfactory to obtain an equation representing the actual relationship between the parameters studied and the conversion of sucrose into isomaltulose . after anova and validation of the studied parameters , the quadratic polynomial equation representing isomaltulose production by cells immobilized in calcium alginate plus gelatin and transglutaminase was used to create a response surface and isocurve ( figure 3 ) . equation ( 2 ) predicts the conversion of sucrose into isomaltulose within the studied parameter range : 
 ( 2)y=57.23 + 1.48x12 + 2.03x220.79x2 , 
							 where x1 and x2 are gelatin and transglutaminase concentrations , respectively , and y is the dependent parameter , isomaltulose ( % ) . both response surface and contour curve are represented in figure 3 . note that an increased rate of sucrose conversion into isomaltulose by immobilized cells would take place at the ( maximum and minimum ) extremities of gelatin and transglutaminase concentration , within the studied range . three duplicate tests were performed , with concentrations used in tests 1 , 2 , and 7 of ccrd-2 , in order to confirm the best gelatin and transglutaminase concentrations for immobilization of erwinia sp . the chosen parameter values were based on responses presented in table 4 and figure 3 , in which greater isomaltulose conversion was obtained when lower transglutaminase concentrations were used . new tests with high transglutaminase concentration were not performed due to the high cost of this enzyme . tests were performed as described above , and cells were reused in six batches . d12 cells immobilized in calcium alginate plus gelatin and transglutaminase , in a batch process at 25c . the sucrose conversions into isomaltulose obtained in all three tests were similar , around 5060% . however , from the sixth batch on there was a large decrease in conversion of tests 1 and 7 , in which 28.07% and 14.59% of isomaltulose were obtained , respectively . granules containing immobilized cells in test 2 were more stable , maintaining a constant isomaltulose conversion of about 50% . comparative studies have shown that erwinia rhapontici ncppb 1578 cells immobilized in 5% sodium alginate ( w / v ) and 0.1  moll cacl2 solution , kept in stable continuous columns at 30c , and fed with sucrose solution 1.6 m were 350 times more stable than free cells , reaching a half - life of around 8,600 hours . it was found that activity of immobilized cells decreased with increasing cell concentration , something that normally occurs in diffusionally limited systems , resulting also in less mechanical resistance . the maximum immobilized cell activity , at a ph of 5.5 , was observed after 3 hours and was proportional to the amount of cells , yielding conversions of 18% , 30% , and 44% for 10 , 20 , and 40  mg of cells / ml of solution , respectively . the bacterium klebsiella planticola mx 10 was used by tsuyuki et al .   to convert sucrose into isomaltulose . the cell mass was mixed with a 4% sodium alginate solution ( w / v ) at a 1  :  1 ratio ( v  :  v ) , and cell suspension was dripped into a shaken 0.25  moll cacl2 solution , in order to form immobilized cell granules . these granules were kept in the solution for 1 hour and were later washed with distilled water and placed in a 2% polyethyleneimine solution ( w / v ) , at ph 5.6 . after 5 minutes , these granules were separated and mixed with a 0.5% glutaraldehyde solution ( v / v ) at 5c , for 20 minutes . the polyethyleneimine- and glutaraldehyde - treated immobilized cells completely converted the 25% sucrose solution , yielding 65.4% of isomaltulose and 29.7% of trehalose . the experimental design technique and response surface analysis have been used and were considered useful tools to study the conversion of sucrose into isomaltulose by immobilized cells . ( w / v ) in 1% sodium alginate ( w / v ) and 2% cacl2 solution ( w / v ) . using stable continuous columns , they achieved a yield of about 50% of isomaltulose from sucrose solutions at 2030% concentration ( w / v ) and 35c . studied the effect of various immobilization parameters on converting sucrose into isomaltulose by immobilized cells of erwinia rhapontici ncppb 1578 . using a 30% sucrose solution ( w / v ) , the maximum production of 140  mg / ml was obtained in a batch process when cells were suspended at 5 the experimental design technique was used to assess the effect of parameters when immobilizing erwinia sp . the following independent parameters : sodium alginate concentration , cell mass concentration and cacl2 concentration , had significant effect on the stability and amounts of sucrose converted into isomaltulose . cells immobilized in 3.0% sodium alginate ( w / v ) , 47.0% cell mass ( w / v ) , 0.3  moll cacl2 , 1.7% gelatin ( w / v ) , and 0.15% transglutaminase ( w / v ) presented greater rates of isomaltulose conversion , achieving 5060% in seven consecutive batches . new studies must be performed to ascertain the influence of the parameters studied in this paper on stability and conversion of sucrose into isomaltulose in a continuous process .