Fermentation assays

Monensin was tested in a rumen fermentation assay at the Lilly Laboratories, and it was found to produce the desired shift in the fermentation (9). Monensin has been licensed in the U.S. for use in beef cattle for improved feed efficiency, where it is administered at 5 to 30 g/ton in a complete feed. In this application, the rate of growth is not increased, but the cattle consume about 10% less food. It is also licensed for increased rate of weight gain in cattle weighing more than 400 lb. and on pasture, where it is fed in a supplement at a rate of 50 to 200 mg per head per day. Lasalocid and salinomycin have also been licensed for use in cattle. 

American Society of Brewing Chemists (ASBC). (2011). Methods of analysis, 12th ed., Yeast-14 Miniature fermentation assay. St. Paul, MN The Society. 

MacroHdes are obtained by controUed submerged aerobic fermentations of soil microorganisms. Although species of Streptomjces have dominated, species of Saccharopoljspora Micromonospora and Streptoverticillium are also weU represented. New techniques such as enzyme-linked immunosorbent assay (ELISA) based assays may prove beneficial for discovering new stmctures (464). 

Bioprocess Control An industrial fermenter is a fairly sophisticated device with control of temperature, aeration rate, and perhaps pH, concentration of dissolved oxygen, or some nutrient concentration. There has been a strong trend to automated data collection and analysis. Analog control is stiU very common, but when a computer is available for on-line data collec tion, it makes sense to use it for control as well. More elaborate measurements are performed with research bioreactors, but each new electrode or assay adds more work, additional costs, and potential headaches. Most of the functional relationships in biotechnology are nonlinear, but this may not hinder control when bioprocess operate over a narrow range of conditions. Furthermore, process control is far advanced beyond the days when the main tools for designing control systems were intended for linear systems. 

The common indices of the physical environment are temperature, pressure, shaft power input, impeller speed, foam level, gas flow rate, liquid feed rates, broth viscosity, turbidity, pH, oxidation-reduction potential, dissolved oxygen, and exit gas concentrations. A wide variety of chemical assays can be performed product concentration, nutrient concentration, and product precursor concentration are important. Indices of respiration were mentioned with regard to oxygen transfer and are particularly useful in tracking fermentation behavior. Computer control schemes for fermentation can focus on high productiv-... 

A subsequent patent, U.S. Patent 2,828,246 described a commercial process for bacitracin production. A 1,230 gallon portion of a medium containing 10% soybean oil meal, 2.50% starch and 0.50% calcium carbonate having a pH of 7.0 was inoculated with a culture of bacitracin-producing bacteria of the Bacillus subtilis group and the inoculated medium incubated for a period of 24 hours with aeration such that the superficial air velocity was 12.1. An assay of the nutrient medium following the fermentation revealed a yield of bacitracin amounting to 323 units/ml. This was more than twice the yields previously obtained. 

A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27 C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coll showed the presence of 1,200 units of polymyxin per cubic centimeter. The polymyxin was extracted and purified by removing the mycelia, adsorbing the active principle on charcoal and eluting with acidic methanol. 

As mentioned earlier, a-D-carba-galactopyranose (1) has been found in a fermentation broth of Streptomyces sp. MA-4145, as an antibiotic. The potency of the antibiotic was rather low. A concentration of—125 fig/mh is required in order to produce a standard inhibition zone of 25-mm diameter against Klebsiella pneumoniae MB-1264, using 13-mm assay discs in a disc-plate assay. A sample of the synthetic a-DL-carba-galactopyranose (17) was... 

PG activity was assayed from cells grown in a medium containing 1% glucose in one-litre self-induced anaerobic fermentation for 5 days by increase of reducing sugars. Enzyme activity increased from pH 3.0 to pH 5.0 (citrate buffer) and decreased drastically above 5.0 (phosphate buffer), but activity was not affected differentially by the two buffers used (data not shown). PG activity increased almost linearly from 20°C to 40°C but above this optimum, activity was lost rapidly and the enzyme was completely inactivated at 60°C and 70°C after 10 and 6 min, respectively (data not shown). No PL, PGL or PME were detected. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

The culture broth was recovered after 72 h of fermentation, the biomass removed and the total protein content measured. Broth aliquots with a protein content of 1 mg were collected and their pH regulated at different values ranging from 3.5 to 8.0. To each broth fraction, 50 mg of the microspheres sample, previously equilibrated at the corresponding pH, was added and the suspension left under stirring overnight. Then, the microspheres were removed by centrifugation and the protein content and the PG activity were assayed on the resulting supernatant. 

Rittenberg and coworkers used a colorimetric assay for tryptophane to determine tyrothricin in fermentation broth152. Kreuzig described a semi-automated colorimetric assay for gramicidin utilizing the reaction of... 

Ivashkiv has used this reaction to assay gramicidin and tyrocidine in fermentation broth155. The antibiotics are... 

Dimick reported that the hemolytic assay can be used to measure the concentration of antibiotic in fermentation broth162. Other authors have reported on modified hemolytic methods163 164 165. 

Nekola published a thin layer chromatographic assay for gramicidin in fermentation broth192. The... 

Recently an isotope dilution method has been reported O for assaying neomycin sulphate. However, it is first necessary to prepare 14C-labelled neomycin sulphate. This is accomplished by adding l4C-labelled glucose to a small-scale fermentation of 5. nad-iat. l4C-labelled neomycin can then be extracted by solvent-extraction or by ion-exchange chromatography. 

C Red No. 2) has been utilised by Hill2 and by Bufton and Saddlerl42 to assay neomycin in aqueous solution. Amaranth may also be used to determine neomycin in production samples from fermentation-recoveryl68. The dye, Orange II, has been similarly described(A max. = 484 nm). Complex formation between neomycin and the dye is very dependant on the ionic strength of the solution, thus necessitating careful control of reaction conditions to ensure complete precipitation of the complex during the assay procedure. Reaction of sodium 1,2-naphtho-quinone-4-sulphonate with... 

After mild hydrolysis, neomycin has been shown to give a positive Elson-Morgan reaction , a reaction characteristic of amino-sugarsl52 A method involving this reaction has been made the basis of a quantitative assay for neomycinl l which has been used to determine the neomycin content of fermentation broths. ... 

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