A Thermotolerant Lactic Acid Bacteria's Screening And Lactic Acid Extraction From Its Fermentation Broth

Lactic acid, molecular formula is C3H6O3.It is also called a-hydroxypropionic acid or 2-Hydroxy propionic acid, which contains L- Lactic acid and D-Lactic acid. Lactic acid is the most popular carboxylic acid. It is widely used in food, beverage, fodder, morden medicine, daily chemical, papermaking, electron industry and so on. With the development of Poly-lactic acid, lactic acid, especially high optical purity lactic acid gets more and more attention in the market. In this paper, Acid-producing bacterias were screened from soil. The strain is proved to be lactic acid producing bacteria by metabolite quality and quantitative test. Its genus were determined by physiological and biochemical property test and 16SrDNA analysis, which is Bacillus coagulans. After UV mutagenesis to improve lactic acid yield, Aaueous two phase system was added into fermentation to extract lactic acid. After reextraction of the top phase, calcium lactate was got. The conclusion is as follows:1. Five thermotolerant (50℃) lactic acid producing bacterias were got. Rf of paper chromatography was as followed:The Rf of nominal sample is 0.78, the Rf of Y3 is 0.78, the Rf of Y6 is 0.78, the Rf of Y9 is 0.77, the Rf of Y11 is 0.77, the Rf of Y12 is 0.77. The Rf of screening sample was the same as the nominal sample. As a result, Y3, Y6, Y9, Y11, Y12 can produce lactic acid. Strain Y3 and Y6 was fermented under the condition of 50℃.48 hours later, lactic acid concentration of Y3 and Y6 were 78.5g/L and 66.7g/L. Transformation efficiency of glucose is 65.4%,55.6%; L-lactic acid concentration is 73.4g/L and 63.1g/L respectively. The optical purity of lactic acid is 93.5%,94.6% respectively.2. As a result of quality and quantitative test, the colony form of Y3 on solid medium is round, ivory white, center oncoides, surface smooth and moist, verge neat. The morphological character of Y3 is long rod shape and straight. The colony form of Y6 on solid medium is the same as Y3, white and opacity. The morphological character of Y6 is rod shape, single align, minor bend. Both Y3 and Y6 produces spore, can move. Y3 and Y6 can hydrolyze starch, produce acetoin from glucose. Y3 and Y6 can not reduce nitrate, not produce hydrogen sulfide, not liquify gelatin, not reduce urea; Y3 and Y6 can metabolize glucose, saccharose, D (+)maltose, xylose and glycerine into acid.3. Using 16SrDNA sequence phylogenetic analysis, the sequence of Y3 is 99% similar to Bacillus coagulans NRIC 1527. The sequence of Y6 is 99% similar to Bacillus coagulans IDSp. They belong to Firmicutes, Bacilli, Bacillales, Bacillacea, Bacillus. The sequence of Y3 and Y6 are 98% similar to Lactobacillus thermophilus. So, Y3 and Y6 are Bacillus coagulans which can produce L-lactic acid.4. Y3 was mutagenized to improve lactic acid yield by UV mutagenesis method. After three mutagenesis, YB3-3 was got. Its physiology character is stable. Under the condition of 50℃, The mutagenized Y3 was fermentated 48 hours, the lactic acid concentration of fermentation broth is 87.9g/L. Compareing with the parent strain, the improvement of lactic acid produced were up to 12%.5.5/15,10/11 and 15/10 of PEG 4000/MgSO4 aqueous two-phase (ATP) were added into fermentation broth. The strain YB3-3 was fermented under the condition of 50℃200r/min 48h. The extraction rate of ATP is 19.27%,43.31%,56.78% individly. As the increasing of the value of PEG 4000/MgSO4, extraction rate was enhanced. Add PEG 4000 and MgSO4 into fermentation broth in triangular flask to make the final concentration up to 15% and 10%. Fermentation was coupled with extraction. The top phase was extradted every 24 hours. CaCO3 and PEG 4000 solution containing glucose was then added into broth to form continuous fermentation condition. As a result, strain is well isolated from extract phase. Lactic acid concentration in top phase is 97.5,96.8,96.2,95.4,95.6(g/L) by the 5 times' determination. It suggested that the lactic acid concentration in top phase was not reduced obviously. The lactic acid producing vigor of the strain YB3-3 didn't decline during the fermention of 120 hours. The yield of lactic acid in the ATP extraction and fermentation coupled system is more than 2.2 times than ordinary batch fermentation. Finally,3.5g calcium lactate was got by acetidin extracting the top phase.