ENZYME IMMOBILIZATION 固定酶.docx

Title ：Report of Fermentation：Studies on the Production of an Antimicrobial Peptide Produced by Staphylococcus warneri in the 2L and 10L FermenterName： Zhang Xiaoyun X00097360Partners：Wu Ting， Wang Ting， Zhou Han，Wayne Travers， Sanhanath MeadowsDate：7th ,21st OctAbstract：The effect of different conditions of fermenters like air flow and agitation speed on the antimicrobial activity of S.Warneri bacteriocins were studied in this experiment. The best conditions were determined by antimicrobial activity which decided by the zone size and highest dilution demonstrating activity of the bacteriocins after different periods. The experiment has been done for 2 weeks. 10 different conditions have been tested. The highest airflow(4L/min) and agitation speed (450RPM) were decided as the optimized operating condition.And the concentration of dissolved oxygen and turbidity were also studied by the curve.Introduction/BackgroundBacteriocin are antibiotic polypeptide，protein or protein complex produced by some bacteria. In 1982，Konisly definited it as the protein which has antimicrobial activities generated by the ribosomes of some bacteria. Generally，most of bacteriocins will only be toxic to the bacteria having close genetic relationship. Sources of bacterioncins are very wide like Gram-positive bacteria(G+)（E-coli）,Gram-negative bacteria(G-),and archaea. The bacteriocin produced by Lactic acid bacteria like nisin has high grade of safety which have been generally applied at food, pharmaceutical industry because of the great potential of the produced bacteriocins as food additives. In recent years，other bacteriocins like bacteriocins produced by staphylococcal species have been studied more and more. Staphylococcus bacteriocins have many advantages such as difficult to have bacterial resistance，no residue，non-toxic side effects on body etc. It is meaningful to treat the infection caused by Staphylococus.Difference between antibiotics and bacteriocins.Some articles didnt separated the antibiotics and bacteriocins . It will limited the appropriate application of bacteriocins in food industry. Although they are both produced by microorganisms, there are many difference on the production methods and mechanisms of action etc. 1. Bacteriocins are primary microbial product synthesized by ribosomes which belonging to proteins while the antibiotics are secondary microbial product synthesized by multi enzyme complexes. 2. In addition , bacteriocins have relatively narrow antibacterial spectrum which only affect the bacteria having close genetic relationship while the antibiotics have wide antibacterial spectrum. 3. Antibiotics are applied on the clinical therapy while the bacteriocins are applied on food industry. The mechanism of action of bacteriocins：Bacteriocins absorbed to the cell surface non-specifically but the specific binding with cell is depended on the structure of the cell wall and plasma membrane. The absorption capacity of G-positive bacteriocins is relative weak，may not need to combine with specific receptor and can affect directly. Compared with G-positive bacteriocins，G-negative bacteriocins should combine firstly belonging to receptor-mediate absorption. G-positive bacteriocins destroy the structure of membrane by forming a membrane channel on the plasma membrane of susceptible cell or affects the stability which leads to cell death by the leakage of ions, amino acids, and ATP. Different G-positive bacteriocins need different minimum membrane potential to react. In addition to form a film channel, some G-positive bacteriocins can also induce cell autolysis. Actually, it is similar to most of antibiotics in some ways. The usage of bacteriocinsBecause bacteriocins have many advantages such as high efficiency, wide range of application, no change to the flavor of product(good additives) and pathogen resistance. So bacteriocins have been applied to food, medicine, and fodder etc.In food industry:Nisin has been used as important natural food preservative in world . It main application areas include milk products(including fresh milk, powdered milk, yogurt, cheese, etc.), canned food, beverage, meat, fish , and alcoholic beverages etc. Bacteriocins often combine with other preservation method and can greatly improve the effectiveness of preservative. Several factors need to be considered before adding bacteriocins. Firstly, the structure and composition of food need to be estimated because the specific component can affect the activity of bacteriocins. For example , bacteriocins can bond with fat to affect its activity. Secondly, temperature ,pH and exogenous enzymes(eg. Proteases) should also be considered. Bacteriocins can be byproducts of fermentation presented in cheese, yogurt, sausage. It can prevent and control the contamination caused by bacteria.In medicine:Due to the growing problem of antibiotic resistance, people constantly looking for something that can replace the antibiotics., which bacteriocins have been considered to have great potentials. Compared with the wide antibacterial spectrum of antibiotics, the bacteriocins antibacterial spectrum is narrow, with some specificity. But it is difficult to have drug resistance. At the same time, the species of bacteriocins are more than antibiotics and people normally able to find the relatively bacteriocin for any kind of bacterial pathogens. Currently, the treatment of bacterial infections used bacteriocins are generally applied producing bacterial strains. None of bacteriocins has been used as drug for clinical treatment directly, but only applied in laboratory.Bacteriocins have great potential in commercial production. But for large-scale production of bacteriocins, currently there are many limitations. Firstly, although the bacterioins have many species, only nisin has been used as a commodity. Secondly, the current application of nisin still has many difficult.1) most of the lactic acid bacteria has narrow spectrum. 2) The amount of bacteriocins are a little.MethodOct 7th 1. Prepared 4 agar plates（use TSA）TSA quantity： 40g for 1L 2.4fg for 60mlPut 2.4g TSA and 60ml into tube 4 x 60ml1. Put them into autoclave to sterile （high temp. and high pressure） 2. Once sterile，prepare Groassay plates Add 1.2ml of a 50:50 mixture of tween 20 water and 3ml of a freshly grown broth3. Shaked the tube until it is not very hot.4. Poured the contents into a 150mm sterile Petri dish.5. Allowed the contents of the plate to solidify.6. Aseptically cut a series of wells in the agar plate using 0.5 ml volumes.The graph of inoculation.SamplePositivePositiveNegativeNegativeSample1243651 duplicate234567. Prepared a set of serial dilutions of the samples using TSB 6 microtubes containing 250ul TSB/time pt8. AT 13:00, 14:00, 15:00, 16:00 Repeat the following stepsprepare samplecentrifuged and filtered it. Diluted the sample into 6 six tubes and marked them from 1 to 6 Took 250ul neat into the first tube and centrifuged it followed by taking 250ul from the first tube into the second tube. And so on.Using pipette to place 100ul of sample (neat), nisin (as positive), broth(as negative ),and a series of dilution in to wells.Positive test if there is bacteria Negative test if there is contaminationOct 21st The procedure is same as the Oct 7th. Discussion QUAD 3：07/10/2014 pH7.0, 300RPM. 37，2L/minPH and temperature：The pH and temperature had been maintain all the time.Turbidity and oxygen：1. The graph shows that during the first 1h,the turbidity climbed slowly from almost 0AU to 0.2Au than 1h-5h. while the pO2 was decreasing steadily from 90 to 80 between 0h and 1h. It may mean that the bacteria was in lag phase，and oxygen was consumed2. Between 1h and 5h the turbidity climbed rapidly from almost 0.4Au to 1.8Au when dissolved oxygen dropped dramatically at first from 1h to 2.5h. It may means that at early exponential phase，the number of bacteria was increasing rapidly and the oxygen uptake rate of the bacteria was significant higher than the rate of fermenter support.3. Between 1h and 5h the turbidity climbed rapidly from almost 0.4Au to 1.8Au，when dissolved oxygen reached equilibrium from 2.5h to 5h and had remained stable at a low level. It means that when the bacteria was during the exponential phase(1h-5h)，the number of bacteria was increasing rapidly and bacteria growth required a lot of dissolved oxygen. In the fermentation process, when the dissolved oxygen in the culture medium(CL) was higher than the critical oxygen needed by cell growth, metabolic activities and respiration of cell ,the rate of growth wasnt affected and bacteria growth requires a lot of dissolved oxygen.4. After 5h，turbidity grow slowly and steadily from 1.8Au to 2.2 Au when the dissolved oxygen trend didnt change. It may mean that bacteria was during the deceleration and stationary phase，a lot of the dissolved oxygen was required by bacteria growth. QUAD 2：21/10/2014 pH7.0, 300RPM, 37，4L/minPH and temperature:The pH and temperature had been maintain all the time.Turbidity and oxygen:1. The graph shows that between 0h and 3h，the turbidity slightly rose and the pO2 slightly decreased. It may mean that the bacteria was during the lag phase and it needed oxygen.2. Between 3h to 12h,the turbidity grew rapidly from almost 0AU to 2.6AU and the pO2 increased rapidly during 3h-4h,then the curve became steady. It may mean that the bacteria was in exponential phase and needed a lot of oxygen at first but after 4h,the growth trend was steady and pO2 didnt affect the rate any more.3. Between 12h and 20h， the turbidity were both steady and the pO2 was fluctuated slightly. It means that the bacteria was in deceleration and stationary phase.4. Between 20h and 24h，the turbidity dropped a little when the pO2 didnt change. It may represent that the bacteria was in the death or decline phase.The staphylococcus warneri is aerobe. When the dissolved oxygen concentration is higher than the critical oxygen concentration of respiration, the metabolism can be carried out. the maximum bacteriocin can be synthesized. However，dissolved oxygen dont need to be too high because the purpose of fermentation is the product instead of bacteria growth，some anaerobe or facultative aerobe may need low dissolved oxygen to produce the bacteriocin. Comprehensive those two figure，it is obvious that under 4L/min air flow the bacteria grew quicker than under 2L/min. high air flow is good for the bacteria growth.Table1The diameter of each well at different time 07/10/201413:0014:0015:0016:00Positive 11918.42116Positive 222192113Negative10000Negative20000Neat 11920.42219.6Neat 22221.223201/2 dilution12120.219.519.51/2 dilution22219.82020.51/42019.517191/42119.21719.51/81817.820.2181/82018.120.2118.11/161616.912161/161616.51416.41/321214.81901/321413.62514.21/6400012.11/6400012.4The table 1 shows the diameter of each wells inoculated at 13:00,14:00,15:00,16:00. From the table, it indicated that generally with the increase of dilution， the diameter of the wells was reduced. And at 1/64 dilution, the antimicrobial activity is little. Activity Table 07/10/2014 and the table of Fermentation conditions for each vessel. Oct 7th and 8th 20141. Compared vessel 1 and vessel 2It indicated that vessel 1 and vessel 2 had same conditions expect the air flow rate. Compared the activity of vessel 1 and vessel 2 , it is obvious that the activity of vessel 2 is better than vessel 1 during the all time by comparing the zone size(mm) and the highest dilution demonstrating activity AU/ml at different time. The larger the highest dilution demonstrating activity is ,the more active the bacteria is. air flow: 4L/min is better than 2L/min under agitation speed of 150RPM in the S.warnerii fermentation. 2. Compared vessel 1 and vessel 3It indicated thait vessel 1 and vessel 2 had same conditions expect the agitation speed(RPM). It is obvious that the activity of vessel 3 is better than vessel 1 all the time by comparing the zone size and the highest dilution demonstrating activity. agitation speed: 300RPM is better than 150RPM under the air flow of 2L/min in the S.warnerii fermentation.3. Compared vessel 2 and vessel 4It indicated that vessel 2 and vessel 4 had same conditions expect the agitation speed(RPM). It is hard to decide which is better. 4. Compared 10L vessel and vessel 4Although the 10L vessel has same pH, agitation speed, temperature as vessel 4, and air flow of 10 L vessel is larger than vessel 4, the antimicrobial activity was lower than vessel 4. It indicates that the volume of fermenter affect the bacteriocins. The larger the volume is ,the lower the antimicrobial activity is. Activity Table 21/10/2014 and the fermentation conditions.5. Compare the vessel 2 and vessel 4It indicated that vessel 2 and vessel 4 had same conditions expect the agitation speed（RPM）. It is obvious that vessel 4 is better than vessel 2 all the time.agitation speed：450RPM is better than 300RPM.6. Compare the vessel 3 and vessel 4It indicated that vessel 3 and vessel 4 had same conditions expect the air flow. No results.7. Compared the vessel 3 week2 and vessel 1 week 4:It indicated that vessel 3 week2 and vessel 1 week4 had same conditions expect the pH. It is oblivious that vessel 4 week 2 is better than vessel 1 week4 by comparing the zone size and highest dilution demonstrating activity.pH： pH controlled at 7 is better than no control in the S.warnerii fermentation.pH may affect the surface charge of the membrane and the permeability affect the absorption capacity of the material.8. Compared the vessel 4 week 2 and vessel 2 week 4They have same conditions but the antimicrobial activities are different at all.9. Compared the 10L vessel and other vessel.Even the conditions of the 10Lvessel are good but the antimicrobial activity is still lower than the 2L. In summary, the larger air flow is , the larger antimicrobial activity is. The larger agitation speed is, the larger antimicrobial activity s.Agitation speed may affect the dissolution, transport and uniform distribution of oxygen and nutrients. affect the product either. Proper agitation speed can promote bacteria growth.Air flow rate is very important for the aerobic bacteria. It directly has influence on the dissolved oxygen. Keeping enough dissolved oxygen is cirtical for ferment process.Comparison of turbidity week 4It is apparent that the curve of the turbidity are similar for different conditions. It mean