CARBOHYDRATE DEGRADATION OF TUBER PASTE FLOUR BY THE ADDITION OF α-AMYLASE FROM TWO Lactobacillus SPECIES [Penguraian Karbohidrat Tepung Pasta Umbi dengan Penambahan α-Amilase dari Dua Spesies Lactobacillus]

The quality of Indonesia tuber flour can be improved by α-amylases which hydrolyzes the flour amylose to glucose and maltose. These monosaccharides causes the flour to have better homogeniety similar to wheat flour and easier to digest. This research aimed at investigating carbohydrate degradation of tuber paste flour by the addition of α-amylase from two Lactobacillus species. Lactobacillus species used were Lactobacillus bulgaricus and L. plantarum B110, while the flour types were made of local taro (Colocasia esculenta), gadung (Dioscorea hispida) and sweet potato (Ipomoea batatas), as well as wheat (Triticum) as a reference. Crude α-amylase activity and reducing sugars were detected by the Dinitrosalycylic acid (DNS) method. Data were statistically analyzed with ANOVA. Research results indicated that α-amylase from L. bulgaricus and L. plantarum B110 have been characterized for their optimum activity and stabilitiy. The reducing sugar content in taro, gadung, sweet potato paste flour and wheat paste flour added with α-amylase of L. bulgaricus increased by 0.008, 0.006, 0.004 and 0.001%, respectively. Meanwhile, the reducing sugars of the above flours added with amylase from L. plantarum B110, increased by 0.008, 0.008, 0.008, and 0.003%, respectively. Increase in reducing sugar contents in carbohydrate degradation of local tuber paste flour added with L. bulgaricus α-amylases was higher than that in wheat paste flour with a 0.001% increase. Similarly, the 0.008% increase of sugar content in tuber paste added with L. plantarum B110 α-amylase was also higher than that in wheat flour with 0.003% increase. Therefore, local tuber paste flour can be used as an alternative to wheat paste flour.


INTRODUCTION
Indonesian local tuber flour in both powder and paste improves in homogenecity almost like wheat flour by using α-amylase to make the flour able to be digested more easily by human intestine. The quality of tuber flour can be improved by the addition of αamylase. Tuber paste flour were made by using local tubers of taro or Colocasia esculenta, gadung or Dioscorea hispida and sweet potato or Ipomoea batatas, and those pasta flour can be used as basic material for producing baby food and many types of tuber cakes.
The contents of glucose and maltose in the tuber paste flour by the addition of α-amylase from Lactobacillus bulgaricus and Lactobacillus plantarum B110 were not known yet. To make tuber flour increase in homogenecity, the tuber flour could be added with α-amylase from lactic acid bacteria as safe bacteria, with the wheat flour used as a comparison. This research aimed at investigating the carbohydrate degradation of tuber paste flour by the addition of α-amylases from two Lactobacillus species namely Lactobacillus bulgaricus and Lactobacillus plantarum B110.

Materials
Materials used were flour of taro or Colocasia esculenta from farmers at Ratu-Sukabumi harbour,

Tube paste flour
The three local tuber flour of taro or Colocasia esculenta, gadung or Dioscorea hispida and sweet potato or Ipomoea batatas were used as materials in production of tube paste flour, and as a comparison, wheat or Triticum was used. The tube flour was heated at 70°C, 10 minutes to form paste flour.

Carbohydrate degradation of wheat and local tube paste flour by the addition of α-amylase
The 5 g of each tube flour (wheat, taro, gadung and sweat potato) was soluted in 50 mL of aquades, heated, homogenised by thermomagnetic stirrer (Sibata MGH-320, Japan) at 70°C for 10 minutes to form paste flour, added with 1U/mL for each L. bulgaricus and L. plantarum B110 crude amylase, and incubated in a rotary shaker (V-Tech VTRS-1, Model: Platform Size CM, India) at 37°C for 24 hours.

α-Amylase isolation (Sharma and Satyanarayana, 2013)
Each of L. plantarum B110 and L. bulgaricus suspension was subcultured into 50 mL of MRSB media and incubated at 37°C for 24 hours in an incubator. L plantarum B110 or L. bulgaricus crude α-amylase were isolated by subculturing 2% of lactic acid bacteria in 25 mL of sterilised glucose MRSB media (Merck, Germany) was modified with 2% of soluble starch (Merck, Germany) 6 pH, and the incubation was carried out for 24 hours at 37°C by using an incubator, and centrifuged in 9000 rpm for 10 minutes at 4°C (Kubota 5910, Japan). Each crude α-amylase was then tested to investigate its αamylase activity.

Activity of α-amylase (Bernfeld, 1955)
The activity of α-amylase was measured by using a DNS method. The 50 µl crude α-amylase was mixed with 50 µl of 1% of soluble starch with 5.0-8.0 pH, homogenised by vortex (Sibata MGH-320), incubated in waterbath (Memmert, Japan) at 35-65°C for 10 minutes, added with 100 µl DNS reagen (Sigma D0550-100G, United States), homogenised, heated at 100°C for 5 minutes, poured with 800 µl of aquades and revortexed. After the solution cooled, the absorbance was read at λ540 by using spectrophotometer UV-Vis (Shimadzu UV-1700 Pharmaspec, Japan). The α-amylase activity unit was measured as the amount of enzyme in which the reaction produced the same product with 1 µmol glucose per minute at the condition measured.

α-Amylase activity optimisation in various pH and temperatures (Wang et al., 2018)
Optimisation of α-amylase from L. bulgaricus and L. plantarum B110 in various pH detected by pH meter (Horiba pH 1100 Scientific, Japan), at incubation time for 10 minutes was conducted at various pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. The highest α-amylase activity at certain pH indicated the αamylase optimum activity. Optimisation of those αamylase activities in various temperatures in 10 minutes incubation was conducted at various temperatures of 35, 40, 45, 50, 55, 60 and 65°C. The highest α-amylase activity at certain temperature indicated the α-amylase optimum activity.

Stability of α-amylase in various pH and temperatures (Sharma and Satyanarayana, 2013 modified)
The α-amylase stability from L. bulgaricus and L. plantarum B110 was detected by measuring the relative activity of α-amylase in various pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 in 60 minute incubation time, at the temperatures of 50°C. The ≥ 50% relative activity of α-amylase was defined as stability of the α-amylase at a certain pH range. Stability of the two α-amylases was also investigated by measuring α-amylase relative activity at various temperatures of 35, 40, 45, 50, 55, 60 and 65°C, with 5.5 pH. The ≥ 50% α-amylase relative activity was defined as stability of α-amylase at a certain temperature range.
Reducing sugar (Miller, 1959) Reducing sugar was measured by using a DNS method. Reducing sugar (%) was measured by using the standard curve equation of glucose solution. Carbohydrate degradation in tube flour of wheat, taro, gadung, and sweet potato (with or without the addition of L. bulgaricus or L. plantarum B110 crude α-amylase) was centrifuged for 10 minutes at 9000 rpm at 4°C. The 100 µl of tube flour treated was poured into 100 µl DNS reagent, homogenised, and the mixture was heated at 100°C for 5 minutes, poured into 800 µl aquadest, and revortexed. The mixture was then left in a minute, untill the absorbance was read at λ: 540 by using spectrophotometer UV-Vis.

SD %
glucose concentration mg m sample weight mg x Volume of reaction total m x 00% .

Statistical analysis
Data were statistically analysed using analysis of variance (ANOVA) with completely randomised design (CRD), and further analysis was done using the Duncan test to compare the effects of each treatment. Data analysis was conducted by using SPSS 16.0.

RESULTS AND DISCUSSION α-Amylase activity optimisation
The results of this research show that L. bulgaricus α-amylase activity at pH 5.0-8.0 had a value of 0.243-0.539 U/mL and the L. bulgaricus α-amylase optimum activity was reached at 50°C with a value of 0.539 U/mL, with 6 pH, 0.539 U/mL (P<0.05), while the L. plantarum B110 α-amylase had a value of 0.403-0.641 U/mL and the L. plantarum B110 αamylase optimum activity was 50°C with a value of 0.533 U/mL, with 7.0 pH, 0.641 U/mL (P<0.05) ( Table 1). The difference in optimum α-amylase activity at different pH and temperature levels between L. bulgaricus and L. plantarum B110 α-amylases was caused by different species producing α-amylase of the two lactic acid bacteria. It was reported that the different amylolytic lactic acid bacteria species might have resulted in different α-amylase optimum activities of the two bacteria (Santoyo et al., 2003;Tallapragada et al., 2018).

Stability of α-amylase
The activity of L. bulgaricus α-amylase at 60 minute incubation time with pH in the range of 5.0-8.0 had a value of 0.044-0.123 U/mL and the relative activity of α-amylases was in the range of 35.684-100% (Table 2); while at the temperature ranging between 35-65°C, it was 0.05-0.08 U m with the αamylase relative activity was in the range of 54.57-100% (Table 3). The stability of L. bulgaricus αamylase with ≥50% α-amylase relative activity in 60 minute incubation time was reached at pH ranging between 5.0-7.0 (0.061-0.123 U/mL) with the relative activities were 50.019-100% (Table 2), while that at temperature in the range of 35-65°C (0.046-0.084 U/mL) with the relative activity between 54.571-100% (Table 3).
The different α-amylase stabilities measured based on their relative activity at the range of certain pH and temperatures of α-amylase from L. bulgaricus and L. plantarum B110 were caused by the different optimum activity of α-amylase from the two lactic acid bacteria species. It was reported that the different species of lactic acid bacteria producing αamylase might have resulted in the different optimum α-amylase activities from the two lactic acid bacteria species (Kanpiengjai et al., 2015;Santoyo et al., 2003;Shongre-Quottara et al., 2009).

Reducing sugar of wheat and local tube paste flour by the addition of α-amylase
The reducing sugar content of the paste flour of sweet potato, gadung, and taro by the addition of αamylase from L. bulgaricus increased by 0.008, 0.006 and 0.004%, respectively (Table 4), while that of the three paste flours by the addition of α-amylase from L. plantarum B110 increased by 0.008% (Table  4). The reducing sugar content of wheat paste flour by the addition of α-amylase from L. bulgaricus increased 0.001% (Table 4), while the reducing sugar content of wheat paste flour by the addition of α-amylase from L. plantarum B110 increased by 0.003% (Table 4).
The reducing sugar content of paste flour from sweet potato, gadung and taro by the addition of αamylase from L. bulgaricus which increase by 0.008, 0.006, and 0.004%, respectively was higher than that of wheat paste flour which increase by 0.01% (Table 4), and the reducing sugar content of the paste flour of sweet potato, gadung and taro by the addition of α-amylase from L. plantarum B110 which increased 0.008% for each was higher than that of wheat paste flour which showed a 0.003% increase.
The tube paste flour by the addition of each αamylase from L. bulgaricus or α-amylase from L. plantarum B110 increased the homogenecity of the flour, because of the higher reducing sugar level increases in the tube paste flour by the addition of αamylase than that of wheat paste flour, The higher reducing sugar level increases in the paste flour of sweet potato, gadung and taro by the addition of α-amylase from L. bulgaricus or αamylase from L. plantarum B110 than that in wheat paste flour was because the content of carbohydrate in sweet potato, gadung and taro flour was higher than that in wheat flour. It was reported that the reducing sugar level of flour resulting from the lactic acid bacteria amylase activity in carbohydrate degradation was affected by the carbohydrate contents of the flour (do Esperito-Santo et al., 2014;Kanpiengjai et al., 2015;Santoyo et al., 2003).

CONCLUSION
Carbohydrate degradation shown by the increases of reducing sugar contents which was about 0.004-0.008% in local tuber paste flour by the addition of the characterized α-amylase from L. bulgaricus was higher than that of wheat which was 0.001% sugar content, while the increase of reducing sugar contents in the flour by the addition of α-amylase from L. plantarum B110 which was 0.008% was higher than that in wheat which was 0.003%, so that local tuber paste flour can be used as an alternative of wheat paste flour.