Phytate degrading activities of lactic acid bacteria

Phytate degrading activities of lactic acid bacteria isolated from traditional fermented food Ema Damayanti, Febiyani Ndaru Ratisiwi, Lusty Istiqomah, Langkah Sembiring, and Andi Febrisiantosa

Citation: AIP Conference Proceedings 1823, 020053 (2017); doi: 10.1063/1.4978126 View online: https://doi.org/10.1063/1.4978126 View Table of Contents: http://aip.scitation.org/toc/apc/1823/1 Published by the American Institute of Physics

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Phytate Degrading Activities of Lactic Acid Bacteria Isolated from Traditional Fermented Food Ema Damayanti1, a), Febiyani Ndaru Ratisiwi2, b), Lusty Istiqomah1, Langkah Sembiring2 and Andi Febrisiantosa1 1

Research Unit for Natural Product Technology, Indonesian Institute of Sciences (LIPI) Jl. Jogja-Wonosari, km. 31,5, Gading, Kec. Playen, Kab. Gunungkidul, D.I. Yogyakarta 2 Faculty of Biology, UniversitasGadjahMada, Yogyakarta, Indonesia Jl. Teknika Selatan, Sekip Utara, Yogyakarta, Indonesia a)

Corresponding author: [email protected] b) [email protected]

Abstract.The objective of this study was to determine the potential of LAB with phytate degrading activity from fermented traditional food grain-based and legume-based. Lactic acid bacteria were isolated from different sources of traditional fermented food from Gunungkidul Yogyakarta Indonesia such as gembus tempeh (tofu waste), soybean tempeh, lamtoro tempeh (Leucaena bean) and kara tempeh. Isolation of LAB was performed using Total Plate Count (TPC) on de Man Rogosa Sharpe Agar (MRSA) medium supplemented with CaCO3. They were screened for their ability to degrade myo-inositol hexaphosphate or IP6 by using qualitative streak platemethod with modified de Man RogosaMorpholinoPropanesulfonic Acid Sharpe (MRS-MOPS) medium contained sodium salt of phytic acid as substrate and cobalt chloride staining (plate assay) method. The selected isolates were further assayed for phytase activities using quantitative method with spectrophotometer and the two selected isolates growth were optimized. Furthermore, thhe isolates that shown the highest phytase activity was characterized and identified using API 50 CH kitand 16S rRNA gene sequencing. The results showed that there were 18 LAB isolates obtained from samplesand 13 isolates were able to degrade sodium phytate based on qualitative screening. According to quantitative assay, the highest phytate degrading activities were found in TG-2(23.562 U/mL) and TG-1 (19.641 U/mL) isolated from gembus tempeh. The phytate activity of TG-2 was optimum at 37 °C with agitation, while the phytate activity of TG-1 was optimum at 45 °C without agitation. Characterization and identification of TG-2 isolate with the highest phytate degrading activity using API 50 CH and 16S rRNA showed that TG-2had homology with Lactobacillus fermentum. It could be concluded that LAB from from fermented traditional food grain-based and legume-based produced the extracellular phytase. Keywords: lactic acid bacteria, tempeh, phytatedegrading activity

INTRODUCTION Phytate the major storage form of phosphorus in the plant seed, food or feed of plant origin [1]. Phytic acid also in the predominant form of phosphorus in cereals, oil-seeds, and seeds of leguminous plants and as such is the major natural phosphorus source in animal feed [2]. Phytic acid (myo-inositol hexaphosphate) is an important plant phosphorus storage form and accounts for 50 – 80 % of total phosphorus present in cereal grains and legumes commonly used in livestock animal feeds [3]. Phytic acid has often been considered as an antinutrient due to its ability to bind minerals and proteins, either directly or indirectly, and thus change their solubility, functionality, absorption, and digestibility [2]. It forms insoluble complexes with numerous divalent and trivalent metal cations, particularly at slightly alkaline pH values, as prevailing in the small intestine, the major site of mineral absorption in the human gastrointestinal tract [4]. The formation of insoluble metal cation-phytate complexes at physiological pH values is regarded as the major reason for poor mineral availability, because these complexes are essentially nonabsorbable from the GIT. Phytate in could

International Conference on Chemistry, Chemical Process and Engineering (IC3PE) 2017 AIP Conf. Proc. 1823, 020053-1–020053-9; doi: 10.1063/1.4978126 Published by AIP Publishing. 978-0-7354-1491-4/$30.00

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interfere the minerals absorption by chelate the divalent cations such as Zn 2+, Fe2+ and Mg2+ as InsP6 negatively charged thus decreased the minerals absorption [5]and formed an insoluble complex became phytate-mineral in the gastrointestinal tract [6,7]. Phytic acid in plants was always bound to the cation, protein, or carbohydrate. Phytic acid also interacts with several enzymes such as trypsin and pepsin thus decreased its activity [8]. By binding plants protein, phytic acid decreased the solubility and digestibility, thereby reduced the nutritional value of feed. Phytic acid binds protein and made it resistant to proteolytic degradation [9]and binds carbohydrates[10]. However, phytate-phosphorus has low bioavailability and is underutilized due to the lack of endogenous phytate-degrading enzymes in non-ruminant livestock including poultry and swine. Additionally, phytic acid exerts anti-nutritive effects, sequestering essential cations including calcium, magnesium, iron, and zinc and reducing their bioavailability [2]. Phytases, a specific group of phosphatases, arerequired to initiate the release of phosphorus from phytate. In the context of human and animal nutrition, the following two aspects of phytate are critically important monogastric animals have only low levels of phytate-degrading enzymes in their digestive tracts, and since phytic acid itself is not absorbed, feed for pigs and poultry is commonly supplemented with inorganic phosphate to meet the phosphorus requirements of these animals [1]. Phytases [myo-inositol hexakisphosphatephosphohydrolase] have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues [11;12]. In feed administration of microbial phytases to improvedigestibility of phytic acid is widely used in the production of poultry and other livestock [2]. Lactic acid fermentation was reported to significantly reduce the phytate content in plant-based foods with a concomitant improvement of mineral solubility. The dephosphorylation of phytate is initiated by a class of enzymes called phytases and extracellular phytase activity was suggested to be responsible for the observed reduction in phytate content during lactic acid fermentation [4]. In European countries, cereals, like wheat and rye are used for sourdough production, which is traditionally prepared by adding a pre-fermented sourdough to the dough [13].Since lactic acid bacteria (LAB) have been used traditionally in fermented foods, and have also been isolated from natural vegetable fermentations, they may be a source of microbial phytases. If they do produce phytases, they can be used as suitable starter cultures for legume and cereal fermentations. Moreover, LAB has the potential to be used as probiotics, and if they can produce these enzymes in the gut, it could be advantageous. There have been reports that LAB can degradephytate and exhibit phytase activity in the fermentation medium [1]. Many studies revealed that lactic acid bacteria have phytase degrading activity such as LAB for phytate degradation during cereal dough fermentation[4], Lactobacilluscasei DSM 20011 and L. plantarum W42 [13], L.plantarum [1], L. plantarum, L. reuteri [14]. Many traditional food based on grain and legume as raw material in Yogyakarta Indonesia used fermentation process [15]such as gembus tempehfrom processed tofu soy (Glycine max), soybean tempeh(Glycine max), lamtoro tempeh (Leucaenaleucocephala), and karabenguk tempeh (Mucunapruriens).Lactic acid bacteria is one of the dominant microbial fermentation processes involved in traditional grain and legumes based foods [16]. However, the pyhtate degrading activity from LAB in traditional fermented foods grain-based and legume-based was unknown. The objective of this study was to determine the potential of LAB with phytate degrading activityfrom fermented traditional food grain-based and legume-based.

MATERIALS AND METHOD Isolation of Lactic Acid Bacteria from Traditional Food Lactic acid bacteria were isolated from traditional fermented foods derived from grains such as gembus tempeh, soybean tempeh, lamtorotempeh and karatempeh. Samples of food was blend to obtain a suspension of bacteria, further serial dilution was performed to obtain bacterial isolates by inoculated in the de Man ROGOSA Sharpe (MRS) Agar and Broth medium [17]. Each serial dilution was plated in deMannRogosa Sharpe (MRS) Agar media (Oxoid) with pH 6.2 supplemented with 0.2% CaCO3 (Merck) and incubated at 37 °C for 24 h. The LAB colonies were detected by clearing zone appearance. The LAB identification procedures consisted of morphology, catalase, gas production, Gram staining, and motility tests. The pure LAB isolates were maintained on microbank (Pro-lab) containing 15% of glycerol.

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Screening for Phytate Degrading Activities The selected LAB were grown on modified MRS Agar medium (MRS-MOPS), in which inorganic phosphate (KH2PO4) wasreplaced by 0.65 g/l of sodium phytate and 0.1 M 3-[N-Morpholino]propanesulfonic acid (MOPS, SRL, India). The contents of glucose,yeast extract and beef extract were reduced to 10, 2 and 4 g/L,respectively to reduce the final phosphate content and to promote theenzyme synthesis. MRS-MOPS medium was inoculated with 5% (v/v)overnight culture propagated in same conditions for two generationsand incubated until the stationary phase of growth was attained (18–24 h).After the incubation, thecolonies were washed from the agar surface using double distilledwater and petri plates were flooded with 2% (w/v) aqueous cobaltchloride solution [18].After 5 min of incubation at room temperature the cobalt chloride solution was replaced with a freshly prepared solution containing equal volumes of a 6.25% (w/v) aqueous ammonium molybdate solution and 0.42% (w/v) ammonium metavanadate solution. After 5 min incubation, the ammonium molybdate/ammonium vanadate solution was removed and the plates were examined for zone of phytate hydrolysis.

Phytase Activities Phytase activity was assayed by measuring the amount of liberated inorganic phosphate from sodium phytate. The enzyme activity was determined by incubating a mixture of 350 mL of 0.2 M Na-acetate (pH 6), 350 mL of 5 μM Na-phytate, and 20 mL of enzyme at 37 °C for 30 min. The reaction was stopped by adding 0.2 mL of 1 M trichloroacetic (TCA) then 3 mL of freshly prepared solution of acetone: 5 N sulfuric acid: 10 mM ammonium molybdate (2:1:1 v/v) was added to the reaction mixture. Blank was prepared by adding an enzyme sample after TCA solution. The inorganic phosphorus (P) released was determined by the ammonium molybdate method that had been modified and quantified at λ355 nm using a spectrophotometer [19].One unit of phytase (U) was defined as the amount of enzyme that produces 1 nmol of inorganic phosphorous per min at a state of optimal measurement [20,21].To compare the level of activity among different isolates, the data were calculated in units per milligram of protein (specific activity) [13].The standard was prepared by dissolving 0.0068 g of KH2PO4 into 100 mL of 0.2 M Na-acetate buffer pH 6. The concentration of the standard carrier was 500 μM. To liberate the phosphate quantity, the calibration curve was made at a concentration of 50-500 μM KH2PO4 [19].

Specific Enzyme Activity Assay Specific phytase activity is was defined as U per mg protein. The concentration of protein in the enzyme preparation was measured by the Folin-Lowry method [22], using Bovine Serum Albumin (BSA) as a standard solution and distilled water as a blank. A total of 0.5 mL sample plus reagent modifications MRSB Medium-MOPS. Medium-MOPS MRSB modifications made by dissolving 1 g peptone (Oxoid, London), 0.4 g of lab-LEMCO powder (Oxoid, London), 0.2 g of yeast extract (Oxoid, London), 1 g of glucose, 0, 1 ml of sorbitan monooleate (Merck, Germany), 0.5 g of sodium asetat.3H2O, 0.2 g of tri-ammonium citrate, 0.02 g MgSO4.7H2O, 0.005 g MnSO4.4H2O, 2.09 g MOPS and KH2PO4 replaced with 0.065 g Na-phytate into 100 mL of distilled water, continued by sterilization using an autoclave at 121°C for 15 minutes. After coupled with reagent Medium-MOPS MRSB modifications homogenation with vortex, incubation at room temperature for 15 minutes and was coupled with a 1.5 mL reagent glycerol. Glycerol reagent consists of 35.29 mL of glycerol were dissolved in 100 mL of distilled water. Glycerol solution and then sterilized using an autoclave at 121 °C for 15 minutes. After coupled with glycerol reagent, homogenized solution is returned to the vortex and incubated at room temperature for 45 minutes. The absorbance of samples were measured with a spectrophotometer at a wavelength of 750 nm. Standard solution prepared by dissolving 0.015 g BSA into the flask to 50 mL to obtain a standard mains concentration of 0.3 mg/mL.The specific enzyme activity was calculated by using formula according to [23]: Specific enzyme activity (U per mg) =

ா௡௭௬௠௘௔௖௧௜௩௜௧௬ሺ௎௣௘௥௠௅ሻ ௉௥௢௧௘௜௡௖௢௡௧௘௡௧௜௡௦௔௠௣௟௘ሺ௠௚௣௘௥௠௅ሻ

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........................(1)

Optimization Growth of Selected Isolates Medium MRS broth in a test tube was inoculated by 1% (v/v) new culture propagated under the same conditions for two generations, then incubated at 30, 37 and 45°C, anaerobically with the addition of CO2, aerobics without agitation and aerobics with agitation 100 rpm. Control tube containing MRS broth medium without the addition of culture.Absorbance was measured at λ600nm using a spectrophotometer during incubation for 48 hours. Optimization of growth do to obtain optimal growth of LAB isolates.

Identification of the Selected Isolates Selected isolates with the highest phytase activity were further identified using the kit API 50 CH (API System, bioMérieux, France). The LAB cultures in API 50 CH assay wereincubated at 37 °C for 48 hours and observed the color change. Substrate color change was observed after 24 and 48 hours and the results were analyzed by using a web API. This biochemical assay was subsequently confirmed with molecular identification using 16S rDNA sequence analysis.

Data Analysis Specific phytase activity data were analyzed using One-Way ANOVA with LAB as a fixed factor to determine significant differences between strains and statistical analysis followed by Duncan test to find out where the real difference. Determination of LAB isolates growth optimization is done based on the stability of LAB isolates growth phase and highest density.

RESULT AND DISCUSSION Aflatoxin Level in Experimental Diets Eighteen isolates from fermented traditional food hadcharacteristic as lactic acid bacteria such as negative catalase assay, positive motility, Gram-positive, and rod shape of cell. The selected isolates had different activities for phytatedegrading (TABLE 1). Isolate

TG-1 TG-2 TG-3 TG-4 TG-5 TK-1 TK-2 TK-3 TK-4 TK-5 TL-1 TL-2 TB-1 TB-2 TB-3 TB-4 TB-5 TB-6

TABLE 1. Lactic acid bacteria isolated from traditional fermented food Catalase Assay Motility Gram Cell Form Phytase Activity

-

+ + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod

+ + + + + + + + + +s + + +

Note: TG: gembus tempeh, TK: soybean tempeh, TL: lamtoro tempeh, TB: karabenguk tempeh

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In the qualitative method, the positive results were marked by the formation of a clear zone due to LAB ability to degrade phytic compounds in modified MRS medium agar (MRS-MOPS). In this assay,theability of LAB isolates to degrade sodium phytate by the presence of calcium. Calcium ion was needed for phytase activity in L. sanfranciscensis[24]. Calcium is not involved in the reaction, but is required for enzyme activity. Calcium ions in the process of facilitating enzyme substrate binding by electrostatic contributes to creating an environment appropriate[25].

(a)

(b)

FIGURE 1.Sodium phytate degrading activity by LAB isolates. LAB isolates were able to degrade sodium phytate characterized by the formation of a clear zone (a) and LAB isolates that could not degrade sodium phytate (b).The existence of a clear zone is marked with red arrows

Sodium phytate is hydrolyzed by the phytase from LAB to produced inorganic phosphate that will be usedby the bacteria's metabolic processes. A clear zone formed around colonies in modified MRS medium agar (MRS-MOPS contained sodium phytate indicated that the synthesis of phytase from LAB depend on the presence or absence of sodium phytate in the growth medium. It could be statedthat the synthesis of phytase from 13 isolates of LAB induced by phytate acid in the growth medium. Some studies showed that the synthesis of extracellular phytase differ depend on the type of microbes.Phytase on Bacillus[26;27], Candida krusei[28], Aspergillus oryzae [29], and Klebsiella [30]induced by phytic acid in the growth medium. While the E. coliphytase production induced by the presence of phosphorus[31]. Most isolates showed differences on degradation activity based on variations in the clear zone formed. The result showed that from 18 isolates of LAB isolated from samples, there were 5 isolates (TG-5, TK-5, TB-1, TB-2, TB-3) performed highactivity to degrade sodium phytateProduction of phytate degrading enzymes in LAB is inconsistent[11]. Phytase activity assaywas conducted by modifying thequantitative methods using medium MRS Broth (MRSMOPS) for the production of extracellular enzymes. Measurement of soluble proteins aimed to determine the specific activity of enzyme per milligram protein. TABLE2 showed that a protein in the enzyme does not affect the catalytic power of enzymes. TG-2 isolatehad the highest phytase activity (23.56 U/mL)with a protein content of 0.464 mg/mL, otherwise TG-3 isolate had the highest protein content (0.567 mg/mL)and phytase activity of 12.876 U/mL. This could be due LAB isolates had a higher enzyme specificity towards other substrates. In addition to phytase activity, LAB had cellulolytic and amylolytic activity[32]. Specificity phytase of the 13 isolates showed low specific activity of the phytase substrate. Lactobacillus plantarumshowed a very low specificity to phytate, with a higher hydrolysis rate against monofosforilasi compounds such as acetyl phosphate[1].There are exceptions to the phytase enzyme produced by Bacillus, this enzyme is very specific to the phytic acid but has a low specific activity[33]. In contrast, phytase with high specificity against phytic acid include phytase from E. coli, A. niger and A. terrushad a specific activity ranging from 103 U/mg to 811 U/mg.

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TABLE 2. Protein level and phytase degrading activities of lactic acid baceteria. Spesific activities of phytase (U/mg) Protein level (mg/ml) Phytase activities (U/ml)

Isolate

TG-2 TG-1 TL-1 TG-3 TB-4 TB-6 TL-2 TK-3 TK-4 TG-4 TK-2 TB-5 TK-1

0.464 0.518 0.409 0.567 0.512 0.494 0.552 0.549 0.509 0.540 0.522 0.516 0.549

50.780c 37.892bc 33.611abc 22.695abc 17.297ab 14.630ab 13.076ab 7.203ab 6.489ab 4.905a 3.383a 3.232a 3.036a

23.562 19.641 13.758 12.876 8.856 7.222 7.222 3.954 3.301 2.647 1.765 1.667 1.667

Note: Number followed by different letters within a column indicate significant difference at the level of 5%, TG: gembus tempeh, TK: soybean tempeh, TL: lamtoro tempeh, TB: karabenguk tempeh

The specific activity of phytase from TG-2, TG-1 and TL-1 include sufficiently high activity when compared with Bacillus subtilisisolated from limestone mountain Holiwood Gresik (HG) Java in the amount of 0,044 U/mg at fraction deposition crude phytase enzyme with (NH4)2SO460-80% [34]but lower than Citrobacter braakii(3457 U/ mg), Candida krusei(1210 U/mg) and Peniophora Lycii(1080 U/mg) [33] . The highest specific activity of phytase revealed by TG-1 and TG-2 isolated from gembus tempeh samples. Determination of the optimum growth was intended to obtain the optimum temperature and conditions.Variations in temperature, oxygen consumption and physical treatments are severalfactors that influence the growth and viability of microbes[35]. TG-1 isolate had the optimum growth at 45 °C (FIGURE 2) while the TG-2 isolatehad the optimum growth conditions at 37 °C with agitation (FIGURE 3). Both isolates could beclassified into mesophyll bacteria, because it grownwell at 25 - 45 °C. 3

OD (Optical Density)

2,5

2 TG-1 (30 ºC) TG-1 (37 ºC) 1,5

TG-1 (45 ºC) TG-1 (aerobic, 37 ºC)

TG-1 (anaerobic, 37 ºC)

1

TG-1 (agitation 100 rpm, 37 ºC)

0,5

0 0

3

6

9

12

18

24

36

48

Incubation time (hour)

FIGURE 2. Growth curve of TG-1 lactic acid bacteria isolated from gembus tempeh

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3


2,5

2 TG-2 (30 ºC) TG-2 (37 ºC)

1,5

TG-2 (45 ºC) TG-2 (aerobic, 37 ºC) 1

TG-2 (anaerobic, 37 ºC)

TG-2 (agitation 100 rpm, 37 ºC) 0,5

0 0

3

6

9

12

18

24

36

48


FIGURE 3. Growth curve of TG-2 lactic acid bacteria isolated from gembus tempeh

Optimization of phytase production from TG-1 and TG-2 isolates wascarried out to determine the optimum enzyme activity during 48 hours of incubation as shown in FIGURE 4. The enzyme activity of two isolates increased in line with the enhancement of incubation time and reached the maximum activity at 12 hours of incubation (TG-2)and 24 hours of incubation (TG-1). This result showed that both isolates had the highest ability to decipher sodium phytate be phosphorus. Phytase activity of TG-2 isolate was higher than TG-1 isolate. This condition affected bytime and temperature of phytase in deciphering the phytic acid into the phosphor varies depending on the type of microorganism phytase[36]. After reached the maximum activity and increased the incubation time, the enzyme activity in both isolates gradually decline. The declinein the phytase activity was likely caused by the presence of inhibitors originated from metabolic waste isolatesresulted in the reduction of enzyme catalytic ability. 40 35


30 25 20

TG-1 TG-2

15 10 5 0 0

4

8

12

24

36

48


FIGURE 4.Growth curve of phytase production TG-1 and TG-2 lactic acid bacteria isolated from gembus tempeh

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The results of biochemical tests using API 50 CH kits resulted thatTG-2 had homology withLactobacillus fermentum with a resemblance to the level of 98.5%. The similarity level of TG-2 isolate with L. fermentumwas based on ability to ferment carbohydrates such asL-Arabinose, D-Ribose, D-galactose, D-glucose, D-fructose, DManosa, D-maltose, D -Laktosa, D-Mellibiosa, D-saccharose and D-Raffinosa. The 16S rRNA gene sequencing revealedthat TG-1 isolate had 99.9% of similarity with Lactobacillus fermentum strain NBRC 15885, while TG-2 isolate had 99,9% of similarity withL. fermentum strain CIP 102980 based on BLAST analysis performed by Gen Bank National Center for Biotechnology Information (NCBI).Lactobacillus fermentumTG-2 isolated from gembus tempeh. In fermented product such as tempeh and other processed foods commonly found lactic acid bacteria strains belong to Lactobacillus genus [37]. Members of Lactobacillus genus such as Lactobacillus paracasei, L. plantarum, L. casei [38], L. acidophilus and L. bulgaricus [39] were commonly inoculated to ferment soybeans processed products.

CONCLUSION Phytate degrading activities revealed by Lactobacillus fermentum TG-2 and L. fermentumTG-1 were 23.56 U/mL and 19.64 U/mL respectively. Both isolates were isolated from fermented traditional food grain-based (gembus tempeh) as waste production from tofu soybean protein processing.The identification results showed that TG-2 isolate was Gram-positive bacteria, rod-shape of cell, negative catalase, and had homology with Lactobacillus fermentum.

ACKNOWLEDGMENTS The authors would like to expressour most sincere thanks to all who have assisted and supported the research in this study particularly BPTBA LIPI for funding the program through DIPA 2014.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

M. Zamudio, A. G. Âlez and J. A. Medina, Letters Appl. Microbiol. 32, 181 – 184 (2001). T. Požrl, M. KoPJar, I. KureNT, J. HrIbar, A. JaNeš and M. SIMčIč, Czech J. Food Sci. 27, 29–38 (2009). T. E. Askelson, A. Campasino, J. T. Lee and T. Duong, Appl. Environ. Microbiol. 80, 3, 943-950 (2014). A. Reale, U. Konietzny, R. Coppola, E. Sorrentino and R. Greiner, J. Agric Food Chem. 55, 8, 2993-2997 (2007). H. Rostami and A. Giri, Int. J. Advanced Biotech. 4, 62-71 (2013). X. G. Lei and J. M. Porres, Biotechnol. Letters 25, 1787-1794 (2003). V. Raboy, Phytochem. 64, 1033–1043 (2003). E. Rodriguez, J. M. Porres, Y. Han and X. G. Lei, Arch. Biochem. Biophys. 365, 262-267 (1999). F. A. Igbasan, O. Simon, G. Milksch and K. Manner, Arch. Anim. Nutr. 53, 353-373 (2000). M. Z. Yao, Y. H. Zhang, W. L. Lu, M. Q. Hu, W. Wang and A. H. Liang, J. Applied Microbiol. 112, 1–14 (2011). U. Konietzny and R. Greiner, Braz. J. Microbiol. 35, 11-18 (2004). E. T. Kornegay, “Digestion of Phosphorus and Other Nutrients: The Role of Phytases and Factors Influencing their Activity,” in Enzymes in Farm Animal Nutrition, edited by M. R. Bedford and G. G. Partridge (CAB International, Canada, 2001), pp. 240. M. Haros, M. Bielecka, J. Honke and Y.Sanz, Poland. J. Food Nutr. Sci. 58, 33-40 (2008). Z. Didar, A. Pourfarzad, M. H. Khodaparast, Inter. J. of Biol., Biomol., Agric., Food and Biotech. Eng. 4 (2010). B. Sarwono, Usaha Membuat Tempe dan Oncom. (PT Niaga Swadaya, Surabaya, Indonesia 2010, pp 45-46). E. Damayanti, Y. R. Indriati, L. Sembiring, H. Julendra and A. A. Sakti, “Antifungal Activities of Lactic Acid Bacteria against Aspergillus flavus, A. parasiticus and Penicillium citrinum as Mycotoxin Producing Fungi,” in Proceedings of the 16th AAAP Animal Science Congress Vol. II (Gadjah Mada University, Yogyakarta, 2014) pp 1742–1745. E. Damayanti, A. E. Suryani, A. Sofyan, M. F. Karimy and H. Julendra, J. Agritech, 35, 164–169 (2015). H. D. Bae, L. J.Yanke, K. J. Cheng and L. B. Selinger, J. Microbiol. Methods 39, 17-22 (1999).

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19. A. S. M. Hussin, A. Farouk, R. Greiner, H. M. Salleh and A. F. Ismail, World J. Microbiol. Biotechnol. 23, 1653-1660 (2007). 20. P. Raghavendra and P. M. Halami., Int. J. Food Microbiol. 133, 129–134 (2009). 21. M. Sumengen, S. Dincer and A. Kaya, Food Biotechnol. 27, 105-118 (2013). 22. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem. 193, 265–275 (1951). 23. R. A. Anatriera, “Aktivitas spesifik katalase jaringan ginjal tikus yang diinduksi hipoksia hipobarik akut berulang”, Undergraduate thesis, Indonesia University, 2009. 24. E. J. Mullaney and A. H. J. Ullah, Biochem and Biophysic. Res. Commun. 312, 179–184 (2003). 25. E. J. Mullaney and A. H. J. Ullah. Biochem and Biophysic. Res. Commun. 312, 179–184 (2003). 26. V. K. Powar and V. Jagannathan, J. Bacteriol. 151, 1102-1108 (1982). 27. Y. M. Choi, H. J. Suh and J. M. Kim, J. Protein Chem. 20, 287-292 (2001). 28. C. Quan, L. Zhang, Y. Wang and Y. Ohta, J. Biosci. Bioeng. 9, 154-160 (2001). 29. J. Fujita, Y. I. Yamane, H. Fukada., Y. Kizaki, S. Wakabayashi, S. Shigata, O. Suzuki and Kono, J. Biosci. Bioeng. 95, 353-384 (2003). 30. V. Shah and L. J. Parekh, Indian J. Biochem. Biophys. 27, 98–102 (1990). 31. R. Greiner, E. Haller, U. Konietzny and K. D. Jany, Arch. Biochem. Biophys. 341, 201–206 (1997). 32. A. Meryandini, V. Melani and T. C. Sunarti, African J. Food Sci. and Technol. 2, 30-31 (2011). 33. A. P. Kusumadjaj, “Penapisan, Karakterisasi Fitase dan Analisis Homologi Gen Penyandi Fitase dari Bakteri Termofilik Kawah Ijen Banyuwangi,” Doctoral thesis, Universitas Airlangga, 2013. 34. L. Yuanita, A. Puspita, S. Surodjo, S. Hidayati, F. A. Amin and A. Budiman, Jurnal Penelitian Berkala Hayati 15, 113 (2010). 35. Jutono, S. Hartadi, S.S. Kabirun, Susanto, S. Judoro and D. Suhadi, Mikrobiologi Untuk Perguruan Tinggi Jilid I (Departemen Mikrobiologi Fakultas Pertanian UGM, Yogyakarta, 1975), pp 130-132. 36. Y. Marlida, Periadnadi and R. Delfita. 2008. Isolasi dan Karakterisasi Enzim Phytase Mikroflora Endofitik Tanaman Kedelai (Glycine max L.). (Artikel Hibah Bersaing. Universitas Andalas Padang. http://repository.unand.ac.id/766/. 11/09/2014) 37. A. B. Ray, Fundamental Food Microbiology (CRC Press LLC, Florida, 2004). 38. B. H. Lee, Y. H. Lo and T. M. Pan, J. Funct Foods 5, 905–913 (2013). 39. W. Shurtleff and A. Aoyagi. History of Fermented Soymilk and Its Products, (http://www.soyinfocenter.com/HSS/fermented_soymilk.php. 19/09/2014)

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