Fermentation experiment of side spore cytobacteria in batches

experimental purpose 1, familiar with the batch feed fermentation experimental operation process2, learn and master the sampling operation during fermentation3, understand the various methods of reducing sugar measurement, master the DNS method of determination of the operation methods and precautions4 and master the method of amino nitrogen determination in fermentation liquidexperimental principle1, sampling operation in fermentation processsampling operation is an important part of the on-line control in the fermentation process;operation should pay attention to the coordination of action, fast, can not cause the contamination of the fermentation tank, in strict accordance with the corresponding proceduresThe vessels in which samples are placed should also be clean and sterile to prevent effects on the assay2, the process of fermentation of reduced sugar determinationmonosaccharides and some oligosaccharides contain free aldehyde sylor or ketogenic, reductive, belong to reduced sugarPolysaccharides and sucrose are non-reductive sugars, and the properties of polysaccharides that can be hydrolysised into monosaccharides can be determined by determining the monosaccharides content after hydrolysisfermentation process control is achieved through a series of process parametersThese parameters include physical parameters, i.etemperature, pressure, etcSecond, the chemical control coefficient, i.epH, sugar concentration, etc , the concentration of sugar is one of the important parameters in the process of controlling fermentation Because the carbon source has a great influence on the growth of bacteria and product synthesis in the fermentation process Therefore, it is important to measure the change of reduced sugar concentration in fermentation process there are many methods of reducing sugar, which generally have the filin reagent method, the enzyme method and the DNS method A, filin reagent Philin reagent by the mass concentration of 0.1g/mL of NaOH solution and mass concentration of 0.05g/mL of CuSO4 solution, the two mixed, immediately produce light blue Cu (OH)2 precipitation Cu (OH) 2 reacts with aldehyde-based under heating conditions, is reduced to brick red Cu2O precipitation, and the aldehyde base is oxidized into argon the use of filin solution and reduced sugar boiling, the formation of copper oxide precipitation reaction, to secondary methyl blue as an indication of liquid, to the sample or hydrolyzed sample titration boiling Philin solution, to reach the end point, slightly excess of reduced sugar will be blue secondary methyl deposit reduced to colorless, to show the end point The total sugar or reduced sugar content is obtained according to the sample consumption when the soluble reduced sugar is identified with filin reagents, the color change process of the solution is: light blue, brown, brick red (precipitation) B, enzyme also known as glucose oxidase-peroxidase method Glucose oxidase oxidizes glucose into gluconate and produces hydrogen peroxide, which is produced by phenol and Antibilin under the action of peroxidase, the concentration of glucose is proportional to the red depth can generally use glucose determination kit, the measurement method is simple, fast C, DNS method also known as 3,5-dinitrosic salicylic acid method In alkaline solutions, reduced sugar becomes oleglycol, which can be oxidized into glyceic acid by 3,5-dinitrosalilic Heating further produces a brown-red amino compound Within a certain concentration range, the amount of reduced sugar is proportional to the depth of the brown-red substance, and the content of reduced sugar is determined by this ratio the three methods have different applications and scope of use, which should be selected on a case-by-case basis For example, the filin method is easily disturbed by penicillin and other reductive substances and the titration endpoint is not easy to judge, the error is large, the enzyme method is specific and accurate, but the cost is high, suitable for accurate measurement, and the DNS law results are accurate and the operation is relatively simple, and the application is relatively extensive in production and research 3, amino acid determination the amino acid - NH3 - base of the pk value is often above 9.0, can not be used in general acid-base indicators (including phenolic), sodium hydroxide solution for titration measurement However, it can be measured by formaldehyde titration Under pH neutrality and room temperature conditions, formaldehyde interacts rapidly with amino acid amino acids, moving the titration endpoint to pH9.0 or so, in which the indicatant phenol is not related to formaldehyde pH9.0 is the discoloration range of phenolic molybdenum, therefore, can be used phenol as an indicator, sodium hydroxide solution to titat - NH3 plus on the base of H-plus, each release of a hydrogen ion, is equivalent to an amino acid R-NH3-R-NH2 R-NH2-2CH3CHO 2OH) 2 experimental materials 1, analytical reagents DNS color rendering agent per 1000mLDNS solution contains 182g of potassium liconate, waterless sulphate 3g, sodium hydroxide 20g, 3,5-dinitrohydrosic water yalnate 6.3g, heavy distilled phenol After the post-filtering is placed for half a month Methyl red indicator, phenolic agent, standard NaOH solution, 12% neutral formaldehyde solution, 10% ZnSO4 solution 1% glucose standard solution: accurately call 100 mg of glucose, with a small amount of distilled water dissolved to 100mL, refrigerator storage 2, equipment mechanical mixing fermentation tank, photophotometer, sugar tube, pipette, alkali titration tube, capacity bottle, beaker, electric furnace and so on experimental steps 1, batch feed fermentation experimental operation steps (demonstration experiments) 2, reduced sugar determination in fermentation liquid (i) the drawing of the standard glucose curve take 9 fixed sugar tubes (with lids, and tied with rope), respectively, in accordance with the order of the following table1 to add a variety of reagents, boiling water bath heating 5min, immediately after the flow of water cooling The solution in the tube is mixed, the light density value (O.D.) is measured by the blank tube solution, the glucose content is the horizontal coordinate, and the light density value is the horizontal length of the glucose solution (ii) sampleofthet sampleaccording to the operating procedures of the fermentation tank sampling and placed in a clean triangular bottle (iii) the preparation of the fermentation fluid to detect the preparation of the sample take a certain volume of fermentation liquid at 12000 rpm centrifugal removal of the production of bacteria The exact amount of 5mL fermentation on-liquid (depending on the amount of sugar, the amount of sampling in different periods of the fermentation cycle should be different) in the 100mL capacity bottle, add 10mL 10% ZnSO4 solution, with the alkali (NaOHmol 3/L) to adjust the alkaline, diluted to scale, shake; Follow Table 2 by adding the corresponding reagent to react 520nm measures the light density value, and finally calculates the amount of reduced sugar contained in the fermentation solution according to the standard glucose curve Each tube is measured three times to average Note: all test tubes in the 1) experiment should be clean and all test doses should be added to be accurate 2) reagent can not pour out the reagent bottle, with a clean pipette to absorb, with the back cap of the cork, anti-return to the original place 3) The mouth of the fixed sugar tube should not be facing the person when heated, so as not to cause the sugar liquid to boil excessively splashing hurt 4) The glucose standard curve should be plotted accurately and statistically significant as possible (R2?gt;97%) If the concentration is over-dispersed when drawn, it needs to be reassembled in turn 5) Used in the photomemeter: the solution should not be poured too much; (iv) the results 1) fill out Form 1 and draw a glucose standard graph 2) The concentration of reduced sugar in the fermentation solution is calculated according to the glucose standard curve By the standard curve can be seen that the reduction sugar concentration of fermentation days is mg/mL, fermentation hours mg/mL 3, the determination of ammonia nitrogen in the fermentation liquid (1) will filter the fermentation liquid, take the filter 2.5 ml in 250ml triangular bottle, add distilled water about 50 ml, add methyl red indicator 2 to 3 drops, add 1mol/LHCl solution 1 to 2 drops, make the red 3 min (2) titration to just improved orange-yellow with a standard NaOH solution, read the titration tube scale at this time, add 12% neutral formaldehyde 10ml, and place for 5-10 minutes (3) add phenolic agent 1 ml, with a standard NaOH solution titration to a slight red as the end point, read at this time titration tube scale The difference between the two readings is the number of NaOH ml consumed (4): NNaOH x VNaOH x 14.01 NH-N% x 100 x mg/100ml