Keratinase from newly isolated strain of thermophilic Bacillus for chicken feed modification Ditya Larasati, Nur Tsurayya, Maharani Pertiwi Koentjoro, and Endry Nugroho Prasetyo
Citation: AIP Conference Proceedings 1854, 020022 (2017); doi: 10.1063/1.4985413 View online: https://doi.org/10.1063/1.4985413 View Table of Contents: http://aip.scitation.org/toc/apc/1854/1 Published by the American Institute of Physics
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Keratinase from Newly Isolated Strain of Thermophilic Bacillus For Chicken Feed Modification Ditya Larasati1, Nur Tsurayya1, Maharani Pertiwi Koentjoro2 , Endry Nugroho Prasetyo1,a) 1
Biotechnology Laboratory, Department of Biology, H Building, Sepuluh November Institute of Technology, Surabaya 60111, Indonesia 2 Laboratory of Environmental Microbiology, Department of Biological and Environmental Science, Faculty of Agriculture - Shizuoka University & Structural Biology Research Center, Inter-University Research Institute Corporation - High Energy Accelerator Research Organization (KEK), Tsukuba-Ibaraki, Japan a)
corresponding author: Dr.techn. Endry Nugroho Prasetyo, M.Eng (
[email protected])
Abstract. Keratinase producing bacteria were isolated from Dieng crater and Mojokerto chicken farm. The screening was done by clear zone method. The strains were selected as they produced clear zones suggesting the presence of keratinolytic activity. The clear zone on FM media depended on both the source and activity of keratinase produced by keratinolytic bacteria. Based on keratinase production and activity, Bacillus sp. SLII-1 was selected for further studies. Keratinase produced by Bacillus sp. SLII-1 capable of producing crude keratinase with 2.08 (mg/second)/ml enzyme activity which able to increase digestibility of feather meal until 22.06% based on soluble protein level. Broiler chicken (Gallus domesticus) that consumed feed containing 5% feather meal indicated production performance of 1194.8 gram/head of feed consumption, 567 gram/head of addition of weight, and 2.1 of feed conversion ratio. An enzymatic engineered chicken feathers waste showed the performance of broiler chicken that is better than soybean meal as conventional sources of protein but could not yet substitute the use of conventional protein sources of fishmeal. Keywords: Bacillus sp. SLII-1, Keratinase, Keratin, Feather waste, Feed, Broiler chicken
INTRODUCTION Chicken feather is organic waste that accumulated in bulk quantities as a by-product in poultry industry. In general, each bird has up to 125 gram of feather (Lakshmi et al., 2013) that represent 5-7% of the total weight of mature chickens (Matikevičienė et al., 2009). Meanwhile, more than 400 million chickens being processed every week worldwide (Lakshmi et al., 2013) hence the accumulation of feather waste reaches five million tons (Han et al., 2012). Most feather waste is land filled or burned that cause global environmental issue such as pollution of both air and underground water resources (Cai et al., 2008; Matikevičienė et al., 2009) and protein discharge (Cai et al., 2008). Chicken feathers are high protein resource consist of 90% keratin (Matikevičienė et al., 2009; Cai et al., 2008). Keratin proteins that have α-helix (α-keratin) or β-sheet (β-keratin) structure are linked by disulfide and hydrogen bonds (Riffel and Brandelli, 2006; Mazotto et al., 2011). The structures fold and form complex structures (Kreplak et al., 2004). Structures and linkages of keratin make keratin have high mechanical stability (Mazotto et al., 2011) and resistance to degradation by common proteolytic enzymes such as trypsin, papain, and pepsin (Mousavi et al., 2013). Feathers waste is poorly recycled in nature and has limited utility due to the chemically unreactive nature of keratin (Lakshmi et al., 2013). Despite the rigid structure of keratin, it can be degraded by mechanical, chemical, and biological methods (Mousavi et al., 2013). The major drawback of mechanical and chemical degradation methods are they are required high-energy input and caused environmental problems. These methods are also
Proceeding of International Biology Conference 2016 AIP Conf. Proc. 1854, 020022-1–020022-10; doi: 10.1063/1.4985413 Published by AIP Publishing. 978-0-7354-1528-7/$30.00
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destructive to certain amino acids such as methionine, lysine and tryptophan and in the formation of non-nutritive amino acids such as lysinoalanine and lanthionine (Marcondes et al., 2008) that leads to low protein quality and digestibility (Zerdani et al., 2004). Hence, the feathers waste that converted into feed supplement conventionally resulting in poor quality feed which is unprofitable (Acda, 2010). The alternative method to overcome abundant of feathers waste is by the utilization of keratinolytic microorganism able to keratinase for feather degradation. Bacillus spp. have capability to produce extracellular keratinase (Cai and Zheng, 2009; Macedo et al., 2005; Pillai and Archana, 2008). Research showed that Bacillus subtilis isolated from Suwon Farm, Korea (Kim et al., 2001), Bacillus licheniformis YJ4 isolated from Taiwan farm (Lin and Yin, 2010), and Bacillus licheniformis KA-08 isolated from Muara Labuh springs, West Sumatera, are keratinolytic bacteria that showed keratinase activity. Keratinase belongs to hydrolyses group that capable of hydrolyze keratin more efficient compared to other protease (Vigneshwaran et al., 2010; Kanmani et al., 2011). Keratinase attack disulfide bonds to degrade keratin (Agrahari, 2013). Biodegradation of keratin using keratinase produce peptide and rare amino acids such as serine, cysteine and proline (Mousavi et al., 2013) and essential amino acids such as threonine, valine, methionine, isoleucine, leucine, lysine, histidine and tyrosine (Ali et al., 2011). Chicken feathers waste containing high protein has the potential to be used as an alternative sources of protein and can be applied in the manufacture of animal feed (Sastry et al., 1986) that are cheap and rich in nutrients (Balaji et al., 2008; Khardenavis et al., 2009). Hence, this study was conducted to investigate keratinase production and activity of newly isolated thermophilic bacteria from Dieng crater and Mojokerto chicken farm and also to utilize chicken feather waste that are modified enzymatically by keratinase that are produced by selected strain and then converted it into alternative protein source in broiler chicken (G. domesticus) livestock feed.
MATERIALS AND METHODS Isolation and Purification of Keratinase Producing Bacteria
Soil from Dieng crater and feathers waste from Mojokerto chicken farm were collected in sterilized sampling bags. The sample were brought to laboratory and processed for analysis. Each 10 g of soil and feather waste sample were suspended in 90 ml phosphate buffer by Vortex. These suspensions are diluted by phosphate buffer in serials of dilution till 10-10 dilution factor. These were used as inoculums. Then, 100 μl of inoculums of each series of dilution were inoculated on feather meal agar media by spread plate method. The medium contained 0.5 g NaCl, 0.1 g MgCl2.6H2O, 0.06 g CaCl2, 1.4 g K2HPO4, 0.7 g KH2PO4, 20 g agar and 10 g feather meal in 1 liter distilled water. The cultures were incubated in room temperature for 1-5 days. The colonies which exhibited the largest clear zones were selected and inoculated about to nutrient agar media by streak plate method until pure colonies of keratinase producing bacteria is obtained.
Morphological Studies and Characterization of Keratinase Producing Bacteria Bacterial identification was conducted on morphological, physiological, and biochemical tests. The results were compared using Bergeys’s Manual of Determinative Bacteriology. Morphological studies were done by simple staining method using methylene blue to identify the cell morphology and observation of cultural characteristic. Biochemical tests that were done includes gram staining, spore staining, fermentation test, methyl red test, voges proskauer test, citrate test, oxygen need test, catalase test, urease test, indol test, and H2S test.
Feather Meal Powder Preparation Feather meal powder was prepared based on modified method of Agrahari and Wadha (2010). Raw chicken feathers was washed extensively and boiled for 2-3 h. Then feathers were dried under the sun. The dried feathers were pulverized and the powder was used as feather meal.
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Medium and Culture Conditions for Keratinase Production A modified nutrient medium based on Agrahari and Wadha (2010), was used to cultivate selected keratinolytic bacteria strains to be used as an inoculums and to produce keratinase. The medium contained 0.5 g NaCl, 0.3 g K2HPO4, 0.4 g KH2PO4 and 10 g feather meal in 1 liter of distilled water with adjusted pH to 7. The medium (99 ml in 250 ml Erlenmeyer flask) was autoclaved at 121°C for 15 minutes. The flask was inoculated using 1 ml of selected strain inoculums. The bacteria strain cultured at room temperature on rotary shaker (110 rpm). The keratinase isolated when the bacteria culture in early stationer stage. The culture medium is first filtered to remove remaining feather meal and then centrifuged at 3500 rpm for 30 min. The supernatant was collected for keratinase activity assay and protein determination and further hydrolysis experiment on feather meal.
Keratinase Activity Assay Keratin substrate used for keratinase activity assay were prepared from raw feather becomes feather meal based on modified method of Agrahari and Wadha (2010). The keratinase activity was assayed as follows: 1.0 g keratin that diluted in 160 ml phosphate buffet (50mM, pH 7) was incubated in 0.16 ml keratinase enzyme at 50 °C at water bath for 2 hours. The reaction was stopped by cooling the solution in 0°C by putting ice blocks. Then, the solution was filtered through Whatman No.1. The filtrate that containing water soluble protein is obtained. Water soluble protein was determined based on Bradford method (1967). Keratinase activity in this research was defined as the ability of keratinase hydrolyze keratin into 1 mg water soluble protein every second compared to the control (without enzyme addition) and calculated by the following equation: Keratinase Activity ((mg/second)/ml)= (∆DP⁄T)/V × DF Where: ∆DP T V DF
= Total water soluble protein compared to the control (mg) = Incubation time (second) = Keratinase volume (ml) = Dilution factor
Protein Determination Protein content of keratinase was analyzed using Bradford method (1967) with bovine serum albumin as standard protein. Readings were carried out in a spectrophotometer at 595 nm.
Hydrolysis of feather meal experiments The reaction mixture for hydrolysis experiments contained a gram feather meal properly diluted in 160 ml phosphate buffer (50mM, pH 7) and keratinase enzyme (0.04-0.20 ml to determine the optimum volume of enzyme used to hydrolyze feather meal. The reaction mixture was incubated at at 50 °C in water bath for 2 hours. The reaction is stopped by cooling the solution. Then, solution was filtered through Whatman No.1. Water soluble protein in the obtained filtrate was determined based on Bradford method (1967). The appropriate volume of keratinase that resulted in the highest increase level of water soluble protein was used in conversion of feather meal into chicken livestock feed.
Conversion of feather meal into chicken livestock feed Conversion of feather meal into chicken livestock feed was done based on Poovendran et al. method (2011). The flask containing feather meal properly diluted in phosphate buffer (50 mM, pH 7) and keratinase was incubated at 50 °C in water bath for 2 hours. The flask were taken out and boiled. Simmering continued until all of the liquid was vaporized and a dry powder was left. This hydrolyzed feather meal is used as an alternative sources of protein in livestock feed.
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Experimental diets and management of animals A total of 30 broiler chickens (DOC/ Day Old Chick) were provided by the local broiler hatchery and were used in this research. Broiler chickens were brooded for 6 days on crumbled standard commercial starter that were provided ad libitum with water and food supplement. On the 7th day, chicken were weighed and randomly allocate pens. The chickens were placed in 70×70×60 cm pens with 5 chickens in each pen. The chickens were fed ad libitum experimental diets for 4 weeks during which feed intake and body weight gain were assessed weekly and the feed conversion ratio are calculated. Food supplement such as vitamin and mineral were given via water. The composition of experiment diets were shown in Table 1. TABLE 1. Experiment diets composition for broiler chicken
Ingredients Yellow corn meal (%)1 Rice bran (%)1 Soybean (%)2 Fish meal (%)2 Feather meal (%)2 Protein (%) Carbohydrate (%) Fat (%) Energy (Kkal/kg)
Diet 1 (1st control) 55
Diet 2 (2nd control) 55
55
3 42 0 0 28.92 50.55 3.97 3536.77
3 37 5 0 29.12 49.28 4.02 3497.87
3 37 0 5 30.32 49.26 3.98 3542.52
Diet 3
RESULT AND DISCUSSION Isolation, Screening, and Characterization of Keratinolityc Strains Keratinase producing bacterial isolated from Dieng crater and Mojokerto chicken farm were named SKII-3, SKII-4, SKII-5, SKII-7, SLI-1, SLI-2, SLII-1, SLII-2, SLII-3, and Pt-1. They were selected as they produced clear zones suggesting the presence of keratinolytic activity. The identification of keratinase producing bacteria was conducted on morphological, physiological, and biochemical tests (Table 2). Isolate Pt-1 belong to neisseria genus due to spherical shape, negative gram staining, anaerobe facultative, and fermenting glucose characteristics. SKII-1 that was determined as rod shape, negative gram staining, anaerobe facultative, non motile bacilli, and producing urease, was belong to shigella genus. Isolate SLI-1 that was rod shape, positive gram staining, positive catalase and sporulating, was belong to amphibacillus or sporolactobacillus. Isolates SKII-3, SKII-4, SKII-7, SLI-2, SLII-1, SLII-2, SLII-3 are belong to Bacillus genus due to rod cell morphology, positive gram staining, and sporulating, that were character key of Bacillus (Holt et al., 1994). The hydrolysis activity qualitatively was ability of bacteria isolates to modify keratin that was shown by comparing the diameter of clear zone and the diameter of colony (Pakpahan, 2009). The hydrolysis activity of keratinolytic bacteria was shown in Figure 1. High keratinase activity indicated larger clear zones as an indicator of modified keratin into amino acids and peptides which are dissolved in the medium. Isolate SLII-1 that shown largest clear zone (Figure 2) were selected for further study.
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FIGURE 1. The hydrolysis activity of keratinolytic bacteria
FIGURE 2. Clear zone of Isolate SLII-1 TABLE 2. Morphological, physiological, and biochemical tests of keratinase producing bacteria Source of inoculums Details of experiment SKII-3 SKII-4 Shape of Rod Rod bacteria Endospore + + formation Motility + + Gram + + character Facultative + + anaerobe Colony characteristics Configuration Round Round Elevation Flat Raised Margins Entire Entire Color White White Biochemical characteristics Glucose +/+ +/+ Lactose +/+ +/+ Mannitol -/+/+ Indole + production Methyl red reaction Vogesproskaure reaction Citrate + + utilization Catalase + + Urease production H2S production Genus Bacillus Bacillus
SKII-5 Rod
SKII-7 Rod
Rod
SLI-2 Rod
SLII-1 Rod
SLII-2 Rod
SLII-3 Rod
Mojokerto farm Pt-1 Spherical
-
+
+
+
+
+
+
-
-
+ +
+ +
+ +
+ +
+
+
+ -
+
+
+
+
+
+
+
+
Round Raised Entire White
Round Raised Entire White
Round Raised Entire White
Round Raised Undulate White
Round Raised Entire White
Round Raised Entire White
Round Raised Undulate White
Round Raised Entire White
+/+ +/+ +/+
+/+/+ -/-
+/+ +/+ +/-
+/+ +/+ +/+ -
+/+/+ +/+ -
+/+/+ -/-
+/+ +/+ -/-
+/+ +/+ -/-
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
-
-
+
-
+
+
+
-
-
+ -
+ -
-
+ -
+ -
+ -
+ -
+ -
-
-
-
-
-
-
-
-
Shigella
Bacillus
Amphibacillus or Sporolactobacilllus
Bacillus
Bacillus
Bacillus
Bacillus
Neisseria
Dieng crater SLI-1
Symbol: +: positive; -: negative; +/-: Acid/No gas; -/+: No acid/gas; -/-: No acid/no gas
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Keratinase Production and Isolation Keratinase was produced by cultivation of Bacillus sp. SLII-1 in modified nutrient medium containing feather meal as the only carbon source. Cultivation of Bacillus sp. SLII-1 on feather meal media shows the change visually (Figure 3) on texture of feather meal becomes softer and the color of white feather meal becomes yellowish due to protein hydrolysis (Deliani, 2008; Winarno, 2002). The cultivation is also causing unique smell as a result of protein degradation that can produce components may inflict foul odor like NH 3 and H2S (Deliani, 2008). The keratinase isolated when the bacteria culture in early stationer stage. Figure 4 shows the highest keratinase production happened on 14th hours when culture has entered stationary phase and keratinase accumulating maximally in media (Anitha and Eswari, 2012). Keratinase hydrolyze keratin in order to meet the needs of carbon of Bacillus sp. SLII-1. The culture medium is filtered to remove remaining feather meal and then centrifuged at 3500 rpm for 30 min. The supernatant was collected for keratinase activity assay and protein determination and hydrolysis of feather meal experiment.
FIGURE 3. Cultivation of Bacillus sp SLII-1 in feather meal media. A: 0 h incubation; B: 24 h incubation.
FIGURE 4. Growth curve of Bacillus sp. SLII-1 in feather media
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Keratinase Assay and Protein Determination Keratinase activity and protein determination is necessary to confirm that enzyme produced is keratinase. Keratinase assay and protein determination show that Bacillus sp. SLII-1 is capable of producing enzyme with 2.08 (mg/second)/ml keratinase activity and 6.6 mg/ml protein content. This confirmed that enzyme produced by the Bacillus sp. SLII-1 was keratinase.
The Optimization of Enzyme Used to Hydrolyze Feather Meal Keratin in chicken feathers is water insoluble protein that have low digestibility (Joshie et al., 2007). Keratinase had a role in hydrolyze keratin via termination of hydrogen and disulfide bonds to produce amino acids and peptides (Mousavi et al., 2013) that are indirectly increasing digestibility of chicken feathers waste (Lee et al., 1991). Figure 5 shows the effect on water soluble protein in feather meal due to hydrolysis process. A gram feather meal is optimally hydrolyzed by 0.16 ml keratinase that produce the highest water soluble protein. This is the optimum volume of enzyme where keratinase hydrolyze keratin efficiently. Gaman and Sherrington (1992) said increase level termination of hydrogen and disulfide bond during hydrolysis also increasing the level of protein that can be absorbed by the body and result in growth. The appropriate volume of keratinase that resulted in the highest increase level of water soluble protein was applied in conversion of feather meal into chicken livestock feed.
FIGURE 5. The optimization of enzyme used to hydrolyze feather meal
Performance of Broiler Chicken Table 3 show that difference protein source in diets had a considerable influence (P0.05) with fishmeal. This problem can be resolved with the utilization of soybean meal, fish meal, and feather meal resulting to good quality of protein and balanced amino acids in livestock feed.
CONCLUSIONS Bacillus sp. SLII-1 is capable of producing keratinase with 2.08 (mg/second)/ml keratinase activity and 6.6 mg/ml protein content that can increase water soluble protein level of feathers waste until 22.06%. An enzymatic engineered chicken feathers waste could substitute about 5% of soybean meal protein and showed the performance of broiler chicken that is better than soybean meal conventional sources of protein but could not yet substitute the use of conventional protein sources of fishmeal.
ACKNOWLEDGEMENTS The authors are grateful acknowledge for the grant provided by Biomaterial and Enzyme Technology Research Group for enzyme production and analysis, as well Mr. Dwi Harjono Saputro from PT. Peksi Gunaraharja for isolation of thermophilic bacteria. Special thanks are also given to father Tjahjo Harsojo and mother Theresia Puspita for the motivation, support and prayers; to family of Scylla serrata 2011 and Biomaterial and Enzyme Research Team (2014/2015) for the motivation, support and assistance during the study.
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