ENZYME IMMOBILIZATION 固定酶文档格式.docx

1、Partners：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 determ

2、ined 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 dec

3、ided 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 a

4、ntimicrobial 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 bacteri

5、ocin 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

6、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

7、 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

8、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 relation

9、ship 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 i

10、s 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

11、 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

12、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, wid

13、e 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 produc

14、ts（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 bac

15、teriocins. 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 co

16、nsidered. 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., w

17、hich 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

18、 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 d

19、irectly, 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 curren

20、t 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 autoclav

21、e 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 t

22、o solidify.6. Aseptically cut a series of wells in the agar plate using 0.5 ml volumes.The graph of inoculation.7. 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

23、 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

24、）,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

25、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

26、 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.

27、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 bacte

28、ria 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 lo

29、t 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, 3

30、00RPM, 37，4L/minPH and temperature: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

31、 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