Effects of culture conditions on keratin degrading capability of bacillus subtilis k15

  • Số trang: 28 |
  • Loại file: PDF |
  • Lượt xem: 14 |
  • Lượt tải: 0
minhtuan

Đã đăng 15929 tài liệu

Mô tả:

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY EFFECTS OF CULTURE CONDITIONS ON KERATIN DEGRADING CAPABILITY OF BACILLUS SUBTILIS K15 SUPERVISOR Dr. BUI THI MINH DIEU STUDENT TRUONG VAN THO Student’s code: 3082631 Session: 34 (2008 - 2013) Can Tho, 5/2013 APPROVAL SUPERVISOR Dr. BUI THI MINH DIEU STUDENT TRUONG VAN THO Can Tho, May, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract Microorganisms grow at different ranges of environmental conditions. The aim of this study was to investigate the culture conditions affecting keratin degradation by bacillus subtilis k15, a feather-degrading mesophilic bacterium. The bacterium grew with an optimum at pH 7.0–8.0 and 30–35oC, where maximum bacterial growth, keratin degradation, keratinolytic activity and soluble protein concentration were also observed. The keratin degradation rate obtained 47% and 46% when the bacterium was cultivated in a chicken feather medium contains 0.5% (w/v) skim milk and yeast extract. Additionally, corn meal had a positive influence on keratin degradation; the treatment degraded 48% chicken feather meal, and showed 36.8% higher than the control. The amount of chicken feather degraded higher in a medium contains 1.2-1.8% (w/v) chicken feather meal. Besides, the quantity of keratinolytic enzyme production depended on chicken feather meal concentrations. Bacillus subtilis k15 degraded 3040% chicken feather meal after 5 days of incubation at 150 rpm; bacterial growth, keratinolytic enzyme and soluble protein concentration also got the highest value at that time. The bacterium effectively degraded chicken feather meal and duck feather meal, whereas hair human and pig fur showed relatively low degradation rates. In short, Bacillus subtilis k15 presented high keratinolytic activity and was very effective in keratin degradation, providing potential use for biotechnological processes of keratin degradation. Keywords: Bacillus subtilis, chicken feather meal, keratin degradation, keratinolytic enzyme i CONTENTS Abstract ............................................................................................i CONTENTS....................................................................................ii 1. INTRODUCTION....................................................................... 1 2. MATERIALS AND METHODS ................................................ 3 2.1. Materials .............................................................................. 3 2.2. Methods ............................................................................... 3 2.2.1. Effect of medium temperature on the keratin degradation of Bacillus subtilis k15. ...................................... 3 2.2.2. Effect of medium pH on the keratin degradation of Bacillus subtilis k15 ............................................................... 4 2.2.3. Effect of different nitrogen sources on the keratin degradation of Bacillus subtilis k15 ....................................... 4 2.2.4. Effect of different carbon sources on the keratin degradation of Bacillus subtilis k15 ....................................... 5 2.3.5. Effect of different chicken feather concentration on the keratin degradation of Bacillus subtilis k15 ..................... 5 2.2.6. Time course of keratin degradation .............................. 6 2.2.7. The keratin degradation capability of Bacillus subtilis k15 degraded the different keratin sources............................. 6 2.2.8. Methods determined characteristic samples in the experiments............................................................................. 6 3. RESULTS AND DISCUSSION ............................................... 10 3.1. Effect of temperature on keratin degradation .................... 10 3.2. Effect of pH on keratin degradation................................... 11 3.3. Effect of nitrogen sources on keratin degradation ............. 13 3.4. Effect of carbon sources on keratin degradation................ 14 ii 3.5. Effect of chicken feather concentrations on keratin degradation................................................................................ 16 3.6. Time course of keratin degradation ................................... 18 3.7. The ability of Bacillus subtilis k15 degrades different keratin sources. ......................................................................... 19 4. CONCLUSIONS AND SUGGESTIONS ................................. 20 4.1. Conclusion ......................................................................... 20 4.2. Suggestions ........................................................................ 20 REFERENCES.............................................................................. 21 iii 1. INTRODUCTION Environmental wastes are found in large quantities in many countries. Feathers are largely produced as a waste byproduct at poultry plants, reaching millions of tons per year worldwide. Additionally, the accumulation of some of these wastes in nature is considered to be a serious source of pollution and health hazards. Therefore, their proper disposal may be considered as a means of avoiding environmental pollution (Williams et al., 1991). Feathers are insoluble structural proteins cross-linked by disulfide, hydrogen and hydrophobic bonds but could represent a rich protein resource because they contain over 90% (w/w) keratins. Keratins cannot be degraded by the usual proteolytic enzymes such as pepsin, trypsin and papain (Kumar et al., 2007). The mechanical stability of keratin and its resistance to biochemical degradation depend on the tightly packed protein chains in α-helix (α-keratin) and β-sheet (β-keratin) structures. In addition, these structures are cross-linking by disulfide bridges in cystines residues (Riffel et al., 2007). Feathers are almost pure keratin protein, potential alternative to more expensive dietary ingredients for animal feedstuffs. Generally, they become feather meal used as animal feed after undergoing physical and chemical treatments. These processes require significant energy and also destroy certain amino acids (Papadoulos and Ketelaars, 1986). An alternative and attractive method for improving the digestibility of feathers is biodegradation by keratinolytic microorganisms. A number of keratinolytic microorganisms can produce keratinases which are 1 capable of degrading keratin. Various authors have reported that, among the keratinolytic microorganisms, some species of bacillus, actinomycetes and fungi are able to produce these keratinases and peptidases (Sangali and Brandelli, 1999). Bacteria are dependent on their environment to provide for their basic needs. There are several factors that influence the growth of bacteria: nutrition, oxygen, pH, temperature, and moisture. Adverse conditions can alter their growth rate or kill them. By understanding the factors affecting the growth of bacteria we can know how to create an optimum condition in which bacteria grow fastest and effectively degrade keratin. Objectives: Screening of cultural conditions affecting on the growth and keratin-degrading capability of Bacillus subtilis k15 2 2. MATERIALS AND METHODS 2.1. Materials − Bacillus subtilis k15 from the research of Nguyễn Thị Hồng Thẩm (2012), Biotechnology Research and Development Institute, Can tho university. − The feather medium used contained the following (g/l): 0.5 NH4Cl, 0.5NaCl, 0.3 K2HPO4, 0.4 KH2PO4, 0.1MgCl2.6H2O, and 10 chicken feather meals. The pH was adjusted to 7 before sterilization. The agar medium added 20 agar and 0.1 yeast extract. These samples were enriched in broth containing feather meal (0.5 NH4Cl, 0.5 NaCl, 0.3 K2HPO4, 0.4 KH2PO4, 0.24 MgCl2.6H2O, 0.2 yeast extract and 10 chicken feather powder (g/l), pH 7.5). − Chicken feathers were washed, dried, and hammer milled prior to being added to the medium. − Chemicals and equipments in laboratory 2.2. Methods 2.2.1. Effect of medium temperature on the keratin degradation of Bacillus subtilis k15. This activity examined the keratin degrading capability of bacteria at different temperature levels (25, 30, 35, 40, 45, and 50ºC). The capacity of degradation of keratin substrates was tested on the feather medium. Using 250 ml Erlenmeyer flasks added 50 ml medium, sterilized at 121ºC in 15 minutes, inoculated 1ml of bacterial suspension (107CFU/ml) into flasks, cultivated for 5 days with constant shaking at 150 rpm. After 5 days determined bacterial growth, keratinolytic activity and protein. 3 keratin degradation, 2.2.2. Effect of medium pH on the keratin degradation of Bacillus subtilis k15 This activity screened the keratin degrading capability of bacteria at different pH levels (4, 5, 6, 7, 8, 9, 10, 11). The capacity of degradation of keratin substrates was tested on the feather medium. 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature at experiment temperature and 150 rpm for 5days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein. 2.2.3. Effect of different nitrogen sources on the keratin degradation of Bacillus subtilis k15 The feather medium was supplemented with 0.5% (w/v) skim milk, yeast extract, urea, NH4Cl, NaNO3, soybean to study the effects of addition different nitrogen sources on keratin degradation. The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH. 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature and 150 rpm for 5 days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein. 4 2.2.4. Effect of different carbon sources on the keratin degradation of Bacillus subtilis k15 The feather medium was supplemented with 1% (w/v) glucose, sucrose, starch, molasses and corn meal to study the effects of addition different carbon sources on keratin degradation. The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH before sterilization. 50 ml of the medium was dispensed into each of 250 ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature and 150 rpm for 5days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein. 2.3.5. Effect of different chicken feather concentration on the keratin degradation of Bacillus subtilis k15 The feather medium was supplemented with 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.8%, 2%(w/v) chicken feather powder to study the effects of addition different chicken feather concentration on keratin degradation. The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH. 50 ml of the medium was dispensed into each of 250ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature and 150 rpm for 5 days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein. 5 2.2.6. Time course of keratin degradation The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH before sterilization. 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature and 150 rpm from 1 to 7 days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein every day. 2.2.7. The keratin degradation capability of Bacillus subtilis k15 degraded the different keratin sources. The feather medium was supplemented with 1%(w/v) duck feather, chicken feather, human hair, goat hair, pig fur to study the effects of addition different keratin sources on keratin degradation. The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH. 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of Bacillus subtilis k15 culture (107CFU/ml). Flasks were sterilized at 121ºC in 15 minutes before inoculating the bacteria. Cultivations were performed at optimum temperature and 150 rpm for 5 days. All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein. 2.2.8. Methods determined characteristic samples in the experiments. 6 a. Determination of bacterial growth (Nguyễn Đức Lượng et al., 2003) Bacterial growth was determined by total plate count on nutrient agar medium. b. Determination of keratin degradation Feather in cultures was harvested by filtration with filter paper, washed twice with distilled water and dried at 70oC to constant weight. The percentage of keratin degradation was calculated from the differences in residual feather dry weight between a control (feather without bacterial inoculation) and treated sample (bacteria were inoculated) c. Determination of kerinolytic activity Azokeratin (Joshi el at., 2007): Azokeratin was prepared by a similar method Ball-milled feather powder was prepared. 1 g portion of the chicken feather powder (the keratin source) was placed in a 100-ml round bottomed reaction flask with 20 ml of sterilized water. The suspension was mixed with a magnetic stirrer. Two ml of 10% NaHCO3 (w/v) were mixed into the feather suspension. In a separate 10-ml tube, 174 mg of sulfanilic acid were dissolved in 5 ml of 0.2 N NaOH. Sixty-nine mg of NaNO2 were then added to the tube and dissolved. The solution was acidified with 0.4 ml of 5 N HCl, mixed for 2 min and neutralized by adding in 0.4ml of 5 N NaOH This solution was added to the feather keratin suspension and mixed for 10 min. The reaction mixture was filtered and the insoluble azo-keratin was rinsed thoroughly with deionized water. The azo-keratin was suspended in water and shaken at 50°C, for 2 hr and filtered again. This wash cycle was 7 repeated until the pH of the filtrate reached 6.0-7.0 and the spectrophotometric absorbance of the washing at 450 nm was less than 0.01. Finally, the wash cycles were repeated at least twice using 50mM potassium phosphate buffer, pH 7.5. The azokeratin was washed once again with water and dried in vacuum overnight at 50°C. Extraction of Enzyme (Park and Son, 2009) Bacillus subtilis k15 incubated in feather medium at different culture conditions. Filtration: The culture medium was filtered through filter paper to remove undegraded residues. Centrifugation: The filtrate was then subjected to centrifugation at 12,000 rpm for 5 min to remove bacterial residue. It was used as the crude enzyme keratinase. This procedure tested the keratinolytic activity 5 mg of azokeratin was added to a 1.5 ml centrifuge tube along with 0.8ml of 50 mM potassium phosphate buffer, pH 7.5. This mixture was agitated until the azo-keratin was completely suspended. A 0.2ml aliquot of supernatant of crude enzyme was added to the azokeratin, mixed and incubated for 15 min at 50°C with shaking. The reaction was terminated by adding 0.2 ml of 10% trichloroacetic acid (TCA). Control sample was prepared by adding the TCA to a reaction mixture before the addition of enzyme. The absorbance of the filtrate was measured at 450. enzyme solution 1 unit of keratinase activity was defined as a 0.01 unit increase in the absorbance at 450 nm as compared to the control after 15 min of reaction. d. Protein determination 8 Protein concentration was measured by the method of Bradford (1976), using bovine serum albumin (BSA) as standard. 9 3. RESULTS AND DISCUSSION 3.1. Effect of temperature on keratin degradation 14 45 Degradation (%) 35 b a ab a 12 b 10 30 8 25 20 c b c 15 6 c 4 Growth (CFU/ml, 109) a 40 10 5 2 d d 45 50 0 0 25 30 35 40 Tem perature Feather Degradation Bacterial Growth Figure 1. Effect of temperature on feather degradation and bacterial growth Note: The different letters denote a statistically significant difference at 5%. As shown in figure 1, the higher keratin degradation rate was obtained from 25oC to 40oC. The degradation rate got the highest at 30oC and 35oC, were 38% and 37% after 5 days, respectively. Keratin degradation decreased gradually from 40oC50oC. The Bacillus subtilis k15 had ability to grow from 25 to 40oC and the optimum growth temperature was determined to be 30-35oC, the bacterial population was 1010CFU/ml at optimum conditions. At high temperature 45-50oC, bacterial growth was reduced so keratin degradation decrease. The highest bacterial population was observed at 30oC. The highest keratinolytic activity also got 14 U/ml at this condition. 10 At 30oC and 35oC, soluble protein gained the highest concentration, were 29 µg/ml and 26 µg/ml, respectively. The lowest keratinolytic activity and soluble protein were observed in too low or high temperature. The other study also showed that keratinase production and keratin degradation of Bacillus sp. were observed the highest during cultivation in the range 30–40oC (Tom et al., 2011). Table 5. Effect of temperature on keratinolytic activity and soluble protein. Temperature Keratinolytic activity o ( C) ( U/ ml) c Protein (µg/ml) 25 6 10c 30 14a 29a 35 11b 26a 40 9b 17b 45 4c 20b 50 2d 12c Note: The different superscript letters denote a statistically significant difference at 5%. 3.2. Effect of pH on keratin degradation The optimum pH for keratin degradation was determined by growing Bacillus subtilis k15 at pH 4.0–11.0 and 30oC. As shown fig. 2, the keratin degradation increased gradually up to pH 7, after that it decreased from pH 8-11. At the optimum pH 7-8, 40% feather was completely destroyed. Besides, the bacterial population at pH 7-8 was significantly higher at pH 4-5 and pH 11 10-11. The highest population growth was obtained between pH 7 and pH 8, was nearly 1010CFU/ml. 45 18 a a b Degradation (%) 35 c 30 e 14 12 a cd a de 25 20 16 10 b cb cd b 8 c 15 10 6 d 4 d 5 Growth (CFU/ml, 109) 40 2 0 0 4 5 6 7 pH 8 9 Feather degradation 10 11 Bacterial growth Figure 2. Effect of pH on feather degradation and bacterial growth Note: The different letters denote a statistically significant difference at 5%. The keratinolytic activity was also higher in neutral range of pH 7-8; the highest keratinolytic enzyme production was obtained at pH 7 and 8, was 12-14 U/ml. However, the highest soluble protein was determined at pH 10-11; in alkaline condition, protein was produced effectively. B. licheniformis PWD-1 exhibited best keratinase activity and higher degradation ability under neutral conditions (Wang and Shih, 1999), additionally Bacillus sp. tended to do best in pH 6-8 (Pandian et al., 2012). The results indicated that Bacillus subtilis k15 grew best in neutral pH, which can be more 12 efficiently applied to degrade keratin in the neutral pH. This bacterium was inhibited in high alkaline and acid condition. Table 6. Effect of pH on keratin degradation and bacterial growth pH Protein Keratinolytic activity (µ µg/ml) (U/ml) 4 11 f 4c 5 17fe 5c 6 28cd 9b 7 35c 14a 8 37bc 12ab 9 24ed 10b 10 63a 6c 11 45b 6c Note: The different superscript letters denote a statistically significant difference at 5%. 3.3. Effect of nitrogen sources on keratin degradation The effect of nitrogen sources on keratin degradation in Table 7. Bacillus subtilis k15 degraded 32% chicken feather meal when it cultivated in medium containing none nitrogen complements. This result suggests that chicken feather as the sole carbon and energy source supports bacterial growth. Additionally, yeast extract, NH4Cl, skim milk, soybean had a positive influence on keratin degradation, resulting in 46%, 36%, 47%, 41% whereas control was only 32%. On the other hand, urea and NaNO3 were no effect of keratin degradation. Table 7 shows that keratinolytic enzyme production increased with 13 complementing skim milk, 29 U/ml. The bacterial growth and soluble protein concentration obtained the highest in the media containing yeast extract and skim milk. Park and Son (2009) reported that the present of skim milk and yeast extract in the medium increase keratin degradation and keratinolytic activity of Bacillus megaterium F7-1. Table 7: Effect of nitrogen sources on keratin degradation Keratin Bacterial kertinolytic degradation growth enzyme (0.5% w/v) (%) (CFU/ml, 108) ( U/ ml) (µg/ml) yeast 51e 118c 26d 91e Urea 36b 88ab 18bc 27bc NH4Cl 41c 99b 15ab 31c Skim milk 52e 113c 29d 174f soybean 46d 91b 21c 78d NaNO3 37b 76a 12a 4a None 37b 87ab 14a 18b Nitrogen Control_none bacteria Protein 5a Note: The different superscript letters denote a statistically significant difference at 5%. 3.4. Effect of carbon sources on keratin degradation The influence of the addition of various carbon sources supplement is shown in Table 8. The result showed glucose 14 slightly increase or no effect of keratin degradation. Molasses was inhibited bacterial growth, so it decreased keratin degradation. However, starch, soybean and sucrose were positive effect of keratin degradation. Similar result was also reported for streptomyces albidus E4 (Kansoh et al., 2009). Additionally, soybean obtained the appreciable level of degrading keratin, resulting 48%. The bacterial growth and keratinolytic activity in the medium containing soybean were significantly higher compare to the other carbon sources. Table 8: Effect of carbon sources on feather degradation Keratin Bacterial kertinolytic degradation growth enzyme (1% w/v) (%) (CFU/ml,108) ( U/ ml) (µg/ml) Glucose 41c 81a 14ab 34a Sucrose 49d 94ab 17c 55b Starch 48d 123cd 20d 63b Molasses 34b 82a 13a 50b Corn meal 53e 135d 20d 81c None 40c 107bc 16bc 55b carbon Control_none bacteria Protein 5a Note: The different superscript letters denote a statistically significant difference at 5%. 15
- Xem thêm -