| It was that fluorometric assay method, HPLC method and microbiological method were used in this paper, and the biotin content in the materials of glutamic acid fermentation were determined respectively by these methods. A simple, quick and accurate method for the determination of biotin content was found through these experiments and a reliable theoretical basis could be provided by this method for the control of biotin content in the glutamic acid fermentation.Firstly, microbiological method was used to determine the biotin content. Biotin of different concentration was added to the medium. Thalli was cultivated to the logarithmic growth phase by shake flask, and the absorbancy should be determined immediately. Cultivating conditions: pH of medium was 7.0, 30 mL medium was cased to 250 mL shake flask, cultivating 12h at 32℃and 200 r/min. Taking biotin concentration as abscissa and absorbancy as longitudinal coordinates, standard curve was drawed. The linear regression equation was Y=0.1857X+0.0049, R2= 0.9982. Corn steep liquor, molasses and starch hydrolysis sugar samples were diluted and added to the medium, then cultivated for 12 h. Biotin content in the samples could be caculated through the biotin standard curve. Biotin content in the corn steep liquor, molasses and amylohydrolysis saccharide were 927μg/L, 2106μg/L and 604μg/L.Secondly, fluorometric assay method was used to determine the biotin content. The treatment of experiment samples and the mensuration conditions were discussed in this experiment. Treatment of corn steep liquor sample: taking a certain amount of corn steep liquor, 1.5 times 6mol/L hydrochloric acid was added to the sample, then hydrolyzed at 121℃for 100 min; Treatment of molasses sample: taking a certain amount of molasses, same volume water and 1.5 times 6mol/L of hydrochloric acid were added to the molasses, then hydrolyzed at 121℃for 100 min. The hydrolyzed solution and the amylohydrolysis saccharide sample were centrifugated by centrifuge at 4000 r/min for 15 min. The pH of supernatant fluid was adjusted to 7.5 with 1 mol/L sodium hydroxide and then fixed with pH 7.5 phosphate buffer. Fluorometric assay conditions: excitation wavelength was 496 nm and emission wavelength was 516 nm; pH 7.5 phosphate was used as buffer solution; reaction temperature was below 20℃; complete the determination within 10 min. Biotin content in the corn steep liquor, molasses and amylohydrolysis saccharide were 940μg/L, 2180μg/L and 617μg/L. Thirdly,in the experiment of HPLC method, decoloration conditions of the corn steep liquor and molasses samples were explored, and the mensuration conditions were also explored. Treatment of corn steep liquor and molasses samples: 180 mL 6mol/L HCL was added to 120 mL corn steep liquor and 120mL molasses (adding 120mL distilled water ) respectively, then hydrolyzed at 121℃for 100 min; solution hydrolyzed was centrifugated by centrifuge at 4000 r/min for 15 min; the pH of supernatant fluid was adjusted to 3.0 with sodium hydroxide and then decolorized with activated carbon. Decolorization conditions of activated carbon: pH 3.0, 80℃, 30 min. Concentrated filter: the treated sample and 120mL amylohydrolysis saccharide sample were enriched to 2-3 mL by rotary evaporation apparatus respectively and filtrated by 0.45μm membrane, then analysised by HPLC. Chromatographic conditions: Hypersil ODS (25μm, 5.0 mm×250 mm) column; mobile phase was methanol-0.1% trifluoroacetic acid solution; pH 3.5; flow rate was 1 mL/min; UV detection wavelengh was 210 nm; sample volume was 30μL. Biotin content in corn steep liquor, molasses and amylohydrolysis saccharide were 770μg/L, 1950μg/L and 610μg/L.At the same time, the influence of biotin concentration to glutamic acid fermentation was explored through the experiment of shake flask fermentation. The best initial concentration of biotin was 2-5μg/L. When the initial concentration of biotin was 2mg/mL, yield of glutamic acid could reach the highest, up to 64.36mg/mL; yield of lactic acid was relatively low, 6.68mg/mL.
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