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Synthesis of ACES and its application in Legionella culture

2019-03-25 来源:亚科官网
N-(2-aminoamino)-2-aminoethanesulfonic acid (ACES) is a zwitterionic buffer. It was first synthesized by Good et al. in 1966 and became a bio-organism due to its special physical and chemical properties. A very important buffer in the field of research. Especially in 1980, when Pasculle et al. first used ACES in Legionella culture, it was found that the growth of Legionella was obviously good. At this point, the biological buffer ACES became an indispensable component in Legionella culture.
At present, there is no research report on the preparation of ACES in China, and the synthesis method of ACES in foreign literature is cumbersome, and the raw materials are difficult to purchase. In view of the above situation, We studied the synthetic route and synthesis conditions of ACES in the laboratory, and explored a simple, effective and economical synthesis method. The ACES and imported ACES synthesized by this method were respectively in the Legion. The effects on the culture of the bacteria were compared.
synthesis conditions
The reagents used in the experiment are: chloroacetamide (chemically pure, Shanghai Tingxin Chemical Plant), taurine (biochemical reagent, Shanghai Boao Biotechnology Co., Ltd.), 95% ethanol (chemically pure, analytically pure, Shanghai Zhenxing Chemical Factory).
The instruments used in the experiment are: three-necked flask, spherical condenser tube, cylindrical funnel, suction filter bottle, Buchner funnel, agitator, vacuum dryer, qualitative filter paper (9cm), CDR-1 type differential thermal meter (Shanghai Tianping Instrument Factory) ), PE580-B type infrared spectrum analyzer (US PE company) and so on.
synthetic path
A certain amount of an aqueous mixture of chlorhexidine and taurine is refluxed, and the reaction solution is adjusted to a certain pH. When the reaction was completed, the reaction liquid was cooled, and then adjusted to be acidic, and white crystals were obtained in an alcohol solution in a yield of 65% to 70%. After recrystallization, a white product was obtained which was dried in a vacuum drier. The yield of recrystallization is from 45% to 55%.
The ACES synthesized in our laboratory was analyzed by CDR-1 type differential calorimeter, and its decomposition temperature was 285 °C. The titration curve showed that the pKa was 6.67 at 0.1 mol/L and 25 °C. The decomposition temperature of US ACES is 290 °C, and its pKa is 6.75 at 0.1mol/L and 25°C, which are very close. The infrared spectrum and nuclear magnetic resonance spectrum of the substance are consistent with the US ACES. It is reported in foreign literature that the synthesis of ACES is carried out by reacting sodium 2-succinyl sulfonate with glycine amide hydrochloride, but these two substances are rarely available on the market, and their synthesis is difficult, and 2-Sodium olethane sulfonate is toxic, and the productivity reported in the literature is also low, only 50%. In contrast, the raw materials used in this method are cheap and easy to obtain, the synthesis route is simple, and the yield is high (65% to 70%).
1) Comparison of pure strain growth
The growth of Legionella on BCYEa medium prepared from two different sources of ACES as a buffer is very close, and the number of colonies and colony diameter are not significantly different (P>0.05). In addition, the appearance indicators such as colony color and shape are also very close. The colonies are grayish white, moist, shiny, and soft to the touch. Legionella also grew at the same rate on both media.
2) Growth simulated air conditioning cooling water
Based on the comparison of pure strain culture, this experiment further compared the difference between the culture medium prepared from two sources of ACES in the culture of simulated air-conditioning cooling water.
The growth of Legionella pneumophila serotype 1 standard strain, after 3 to 5 days of culture, is very close to the growth of BCYEa-American ACES and BCYEa-made ACES medium, and the number of colonies per plate is generally Between 198 and 20, the colony diameter was between 1.75 and 3.0 mm; while the growth of Legionella on the CYE medium was significantly inferior to the two BCYEa-ACES media. Because this experiment simulates the isolation and culture of Legionella from air-conditioned water contaminated by Legionella, the results are applicable to the isolation and culture of Legionella pneumophila in external environmental specimens.
From the above two experiments, we can also see that although the BCYE a medium and the CYE medium differ only in the composition of the buffer ACES, they have a very different effect on the culture of Legionella, indicating that ACES is an indispensable factor in the growth of Legionella culture.
Related links: ACES
Edited by Suzhou Yacoo Science Co., Ltd.