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CHARACTERIZATION OF PROTEASE CRUDE EXTRACT FROM INDIGENOUS LACTIC ACID BACTERIA AND THE PROTEIN DEGRADATION CAPACITY IN LOCAL TUBER AND CEREAL PASTE FLOUR The name and affiliation of authors have removed to support double blind peer review. Abstract Protease hidrolyzed protein in flour in order to more digest by human ulcer. Lactobacillus plantarum B110 and Lactobacillus satsumensis are indigenous lactic acid bacteria that produce protease. The objective of this research is to characterization of protease crude extract from indigenous lactic acid bacteria and the protein degradation capacity in local tuber and cereal paste flour.

Tuber and cereal flour used were purple sweet potato (Dioscorea alata), cassava (Manihot esculenta), rice (Oryza sativa), corn (Zea mays) and wheat (Triticum) as comparison. Protease activity was tested by Horikoshi method (1971) and protein degradation was by formol titration. Research results showed that optimum activities and stabilities of Lactobacillus plantarum B110 were at pH: 7.5, 45oC and pH:5.0-8.0, 35-50oC, while that L. satsumensis EN 38-32 were at pH: 7.0, 40oC and pH:6.0-8.0, 20-45oC.

Increases in protein degradation capacity of the paste flour additional proteases crude extract from L. plantarum B110 were 0.0838% (purple sweat potato), 1.3299% (cassava), 0.5834% (corn), 0.7499% (rice) and 1.5551% (wheat as comparison); while that L. satsumensis EN 38-32 were 0.20% (purple sweet potato), 0.32% (cassava), 0.87% (corn), 1.17% (rice). Based on increases in protein degradation capacity, protease crude extract from L. plantarum B110 and L. satsumensis EN 38-32 were sequently better to hidrolyze protein of cassava and rice paste flour than that other tuber and cereal.

Key words: degradation protein, protease, L. plantarum B110, L. satsumensis EN 38-32, paste flour of local tuber and cereal This paper had been presented in The 6 th JASTIP Symposium of Biodiversity, Genetic Resources and Innovative Bioresources Technology. ICE, BSD City-Tangerang, INDONESIA. November 1, 2018/ 1. INTRODUCTION Protease can be used to hidrolyze protein in tuber and cereal flour to peptide in order to more digest by human ulcer.

Lactic acid bacteria, such as: Lactobacillus plantarum S31 and Lactobacillus delbrueckii subsp. lactis CRL 581produced protease (Budiarto et al., 2016; Villegas and Brown, 2014) The protease activities in hidrolyzing protein in tuber and cereal flour were affected by the type of the bacteria producing protease (Sawant and Nagendran, 2014; Thiele et al., 2002). Furthermore, the different type of tuber and cereal flour caused the different concentration of protein hidrolyzed by microbial proteases in those two flour (Li et al., 2012; Adeniji, 2013).

The protein in tuber and cereal flour were hidrolyzed by the microbial protease activities to simple compounds of peptides (Endo and Okada, 2005; Ganzle et al., 2008). The peptides produced depend on the type of tuber and cereal flour used. Some species of Lactic acid bacteria (LAB) had ability to produce protease, such as: Lactococcus lactis, Lactococcus cremoris and Lactococcus garviae (Adi and Guessas, 2016), Lactococcus lactis 1598, Streptococcus thermophillus t3D1, Lactobacillus lactis 1043 and L. delbrueckii subsp.

bulgaricus b38, b122 and b24 (Atanasova et al., 2014). However, LAB species of Lactobacillus plantarum B110 and Lactobacillus satsumensis EN38-32 producing protease which have potency to degrade protein in local tuber and cereal flour haven’t been known yet. The objective of this research was to characterization of protease crude extract from indigenous lactic acid bacteria and the protein degradation capacity in local tuber and cereal paste flour 2. MATERIALS AND METHODS Production of protease (Tennalli et al.,

2012 modified) The 2 % inoculum culture was poured into 50 mL media of nutrient broth with addition of 1% casein and it was incubated at temperature 37°C for 48 hours. The production media was then centrifuged at 3500 rpm for 15 minutes. Supernatant found was protease crude extract. Optimization of protease activity in various pH (Tennalli et al., 2012 modified) Optimization of protease activity was conducted in pH 4.5. 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. The activity test was conducted at temperature: 37oC and incubation time for 10 minutes.

The highest protease activity in certain pH was stated as optimum protease activity. Optimization of protease activity in various temperatures (Tennalli et al., 2012 modified) Optimization of protease activity was conducted in various temperatures: 20, 25, 30, 35, 40, 45, and 50oC. The activity test was conducted in optimum pH, with incubation time for 10 minutes. The highest protease activity in certain temperature was stated as optimum protease activity. Stability of protease in various pH (Eijsink et al.,

2005; Moran et al., 2012) Stability of protease in various pH were conducted by measuring protease relative activities in pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The activity test was conducted in optimum temperature with incubation time for 60 minutes. Protease relative activities with values: = 50% showed that those activities were in stable condition. Stability of protease in various temperatures (Eijsink et al., 2005; Moran et al.,

2012) Stability of protease in various temperatures was conducted by measuring protease relative activities in temperatures: 20, 25, 30, 35, 40, 45 and 50oC. The activity test was conducted in optimum pH with incubation time for 60 minutes. Protease relative activities with value: = 50% showed that those activities were in stable condition. Production of tuber and cereal paste flour additional protease Tuber and cereal flour used were purple sweet potato (Dioscorea alata), cassava (Manihot esculenta), rice (Oryza sativa), corn (Zea mays) and wheat (Triticum) as comparison.

One (1) gram for each flour from wheat, purple sweet potato, cassava, rice, and corn was soluted in 10 mL aquadest, heated at temperature 70oC with agitation. Heating was conducted up to paste flour formed. Each supernatant (1 unit crude protease) from L. plantarum B110 and L. satsumensis EN 38-32) was added into 10 mL paste flour and incubated in shaker incubator with 100 rpm, at temperature 40oC, for 24 hours. Paste flour treated was then analyzed its protein degradation.

Protease activity (Horikoshi modified, 1971) Protease activity test was conducted by method of Horikoshi (1971) modified. The 0.2 mL protease was poured into reaction tube, added 0.4 mL 2% casein and 0.4 mL buffer phosphate 0,05 M pH 8. The mix was incubated at temperature 37 oC for 10 minutes, added 1 mL 20% TCA and homogenized. Incubation was continued at temperature 37oC for 10 minutes, and solution was centrifuged by rotation 3500 rpm for 5 minutes. Controle was made.

One unit protease was defined as the amount of mL protease needed to produce 1 µmol tirosin every minute with casein as substrate. Protein degradation (U.S.P., 1989 in Nilsang et al., 2005). Protein degradation was tested by formol titration. Sample of 10 mL treated paste flour was added phenolphthalein, and neutralized by NaOH 0,1 N solution. The 10 mL of 37 % Formaldehyde was added into the solution, titrated by standard solution of NaOH 0,1 N up to the colur change to pink.

The protein degradation was then calculated as % Nitrogen x Fc (Conversion Factor). 3. RESULTS AND DISCUSSION Research results showed that the values of protease activities of Lactobacillus plantarum B110 in various pH 4.5-8.0 were in the range 0.4488 – 0.8995 U/mL (Table 1) and in various temperatures: 20-50oC were 0.4415 - 1.0357 U/mL) (Table 2) with relative activities at pH 4.5-8.0 were in the range 47.56 – 100% (Table 3) and at temperatures: 20-50oC were 40.57 - 100% (Table 4). The optimum protease activity of Lactobacillus plantarum B110 was reached at pH: 7.5

(0,8995 U/mL)(Table 1) and temperature: 45oC (1.0357 U/mL) (Table 2) with stabilities at relative activity = 50% were at pH 5.0-8.0 (Table 3) and temperatures: 35-50oC (Table 4). pH Protease Activity (U/mL)  4.5 0.4488 a  5.0 0.4966 a  5.5 0.5408 b  6.0 0.7432 c  6.5 0.7542 c  7.0  0.8848 d  7.5  0.8995 d*  8.0  0.6567 bc  Table 1. Protease Activity from L. plantarum B110 in Various pH Temperature Protease Activity (U/mL)  20 0.4415 a  25 0.4893 a  30 0.5545 a  35 0.5868 a  40 0.6070 a  45  1.0357 c*  50 0.7910 b   Table 2. Protease Activity from L.plantarum B110 in Various Temperatures pH Protease Activity (U/mL) Protease Relative Activity (%)  4.5 0,4230 47.56  5.0 0,4893 55.01  5.5 0,5390 60.60  6.0

0,7082 79.62  6.5 0,7229 81.27  7.0 0,8701 97.82  7.5 0,8895 100  8.0 0,6457 72.59   Table 3. Protease Relative Activity from L. plantarum B110 in Various pH Table 4. Protease Relative Activity from L. plantarum B110 in Various Temperature Temperature Protease Activity (U/mL) Protease Relative Activity (%)  20 0.4157 40.57  25 0.4304 42.01  30 0.4525 44.16  35 0.6052 59.07  40 0.6144 59.97  45 1.0246 100  50 0.7744 75.58   The values of protease activities of L.

satsumensis EN 38-32 in various of pH 4.5-8.0 were in the range 0.2032 – 0.4574 U/mL (Table 5) and in various temperatures of 20-50oC were 0.2385 – 0.7214 U/mL) (Table 6) with relative activities at pH 4.5-8.0 were in the range 34.10 - 100% (Table 7) and at temperatures: 20-50oC were 42.92 - 100% (Table 8). The optimum protease activity of L. satsumensis EN 38-32 was reached at pH: 7.0 (0.4574 U/mL) (Table 5) and temperature: 40oC (0.7214 U/mL)(Table 6), with stabilities at relative activities = 50% were at pH 6.0 - 8.0 (Table 7) and temperatures: 20 - 45 oC (Table 8). Table 5.

Protease Activity from L. satsumensis EN 38-32 in Various pH pH Protease Activity (U/mL)  4.5 5.0 0.2032 a 0.2482 a  5.5 0.3383 b  6.0 0.3834 d  6.5  0.3737 cd  7.0  0.4574 e*  7.5 0.3480 bc  8.0 0.3222 b   Table 6. Protease Activity from L. satsumensis EN 38-32 in Various Temperatures Temperature Protease Activity (U/mL)  20 0.2385 a  25 0.3222 b  30 0.4896 c  35 0.5637 c  40  0.7214 d*  45 0.3866 b  50 0.3576 b   Table 7. Protease Relative from L.

satsumensis EN 38-32 in Various pH at 60 minutes incubation time pH Protease Activity (U/mL) Protease Relative Activity (%)  4.5 5.0 0.0430 0.0478 34.10 37.90  5.5 0.0575 45.60  6.0 0.0773 61.30  6.5 0.0870 68.99  7.0 0.1261 100.00  7.5 0.0795 63.05  8.0 0.0816 64.71   Table 8. Protease Relative Activity from L. satsumensis EN 38-32 in Various Temperature at 60 Minutes Incubation Time Temperature (oC) Protease Activity (U/mL) Protease Relative Activity (%)  20 0.0510 54.31  25 0.0596 63.47  30 0.0666 70.93  35 0.0778 82.85  40 0.0939 100.00  45 0.0687 73.16  50 0.0403 42.92

  The different optimum activities and stabilities between protease from Lactobacillus plantarum B110 and L. satsumensis EN 38-32 were due to the different species of both lactic acid bacteria producing protease. It has been reported that the different species of bacteria may have resulted in the different characteristics of protease produced (Eijsink, 2005; Hayek and Ibrahim, 2013, Naidu, 2011, Sulthoniyah et al., 2015). The optimum temperature and pH of protease B.

subtilis were in the range from 40oC to 50oC and pH 8, respectively (Naidu, 2011), while that Lactococcus species were 37oC and pH 7.2 (Addi and Guessas, 2016). The protein degradation concentration of the tuber and cereal paste flour with addition of L. plantarum B110 protease crude extract were 1.2570% (purple sweat potato), 1.8077 (cassava), 1.9305% (rice), 1.7506 (corn), and 1.5551% (wheat as comparison)(Table 9). The increases of protein degradation concentration of the tuber and cereal paste flour additional L. plantarum B110 crude protease were 0.0838% (purple sweat potato), 1.3299% (cassava), 0.7499% (rice), 0.5834% (corn), and 1.5551% (wheat as comparison)(Table 9).

The increasing of protein degradation concentration from the treated cassava paste flour was highest than that the other local tuber and cereal paste flour (Table 9). Table 9. Protein Degradation Concentrations of Local Tuber and Cereal Paste Flour With and Without L. plantarum B110 EN 38-32 Protease Tuber Paste Flour Protein Degradation Concentration (%) Increase of Protein Degradation (%)   Control* Sample**   Wheat 1.2771 2.8322 1.5551  Purple Sweet Potato 1.1732 1.2570 0.0838  Cassava Rice Corn 0.4778 1.1806 1.1672 1.8077 1.9305 1.7506 1.3299* 0.7499 0.5834  Notes : * Paste flour without addition of L. plantarum B110 protease ** Paste flour with addition of L.

plantarum B110 protease The protein degradation concentration of the tuber and cereal paste flour additional L. satsumensis EN 38-32 crude protease were 0.7300 (purple sweat potato), 0.7900 (cassava), 2.1900% (rice), 1.9600 (corn), and 1.5551% (wheat as comparison)(Table 9). The increases of protein degradation concentration of the tuber and cereal paste flour additional L. satsumensis EN 38-32 crude protease were 0.20% (purple sweet potato), 0.32% (cassava), 0.87% (corn), 1.17% (rice) and 1.83% (wheat as comparison)(Table 10) The concentration increase of protein degradation of the treated rice paste flour was highest than that the other local tuber and cereal paste flour (Table 10) Table 10. Protein Degradation Concentrations of Local Tuber and Cereal Paste Flour With and Without L.

satsumensis EN 38-32 Protease Tuber Paste Flour Protein Degradation Concentration (%) Increase of Protein Degradation (%)   Control* Sample**   Wheat 1.2771 3.8322 1.5551  Purple Sweet Potato 0.5300 0.7300 0.2000  Cassava Rice Corn 0.4700 1.0200 1.0900 0.7900 2.1900 1.9600 0.3200 1.1700* 0.8700  Notes : * Paste flour without addition of L. satsumensis EN 38-32 protease ** Paste flour with addition of L. satsumensis EN 38-32 protease1.17001.1700. The different increases of protein degradation concentration between the tuber (purple sweet potato and cassava) and cereal (corn and rice) paste flour with wheat paste flour (as comparison) additional crude proteases from L. plantarum B110 and L.

satsumensis EN 38-32 were due to the the different protease activities from those two lactic acid bacteria in protein hidrolysis of those two flour. It has been reported that the protease activities from the different bacteria species may have resulted in the different hidrolysis of flour protein (Adeniji, 2013; Gupta et al., 2002; Sawant and Nagendran, 2014). The LAB of Lactococcus lactis 1598, Streptococcus thermophilus t3D1, Lactobacillus lactis 1043 and L. delbrueckii subsp.

bulgaricus b38, b122 and b 24 had high proteolytic activity in the formation of peptides with molecular weight between 5 and 10 kDa (Atanasova et al., 2014) 4. CONCLUSIONS The characterization of protease crude extract from indigenous lactic acid bacteria and its protein degradation capacity in local tuber and cereal paste flour showed that the optimum activity and stability of proteases from Lactobacillus plantarum B110 were at pH: 7.5, 45oC and pH:5.0-8.0, 35-50oC, while that L. satsumensis EN 38-32 were at pH: 7.0, 40oC and pH:6.0-8.0, 20-45oC.

The maximum increase of protein degradation concentration of the tuber and cereal paste flour (with wheat paste flour as comparison) additional proteases crude extract from L. plantarum B110 was at cassava with value of 1.3299%, while that L. satsumensis EN 38-32 was at rice with 1.1700%. Based on the increases of protein degradation concentration, proteases crude extract from L. plantarum B110 and L. satsumensis EN 38-32 were sequently better to hidrolyze protein of cassava and rice paste flour than that the other tuber and cereal paste flour. 5.

ACKNOWLEDGEMENT The authors would like to acknowledge to DIPA Priority Project of Research Center for Biology, Deputy of Natural Sciences, Indonesian Institute of Sciences for giving this research funding, and Desy Septiani S.Si. with Khairrunnisa S.Si for their research assistances in Laboratory 6.