Determination Of Ergosterol From Lactarius Hatsudake Tanaka And Its Preliminary Study On Microorganisms

Edible mushrooms are widely distributed,and their secondary metabolites are abundant,showing a variety of good physiological activities.Ergosterol is recognized as a major sterol in fungi due to its good anti-inflammatory,anti-tumor and immunomodulatory effects.In this paper,the extraction,determination,separation and purification of ergosterol from Lactarius hatsudake,as a specialty medicine and food dual-purpose mushrooms of Hunan Province,and its microbial activity were studied.Firstly,the ITS sequencing analysis method was used to identify the collected samples.DNA was detected and quantified by agarose gel electrophoresis and UV spectrophotometer.The corresponding primers were designed with ITS2 as the target region for PCR amplification.Miseq was sequenced on the platform.The results showed that among the seven samples tested,the five red samples were Lactarius hatsudake,and the two white samples were Lactarius akahatsu.A method for detecting ergosterol content in edible fungi was established by HPLC analysis.The content of ergosterol in Lactarius from 30 different producing areas in Hunan Province was detected.The concentration of ergosterol is in the range of 5?400)?g/mL and the regression equation is:Y=32529·X+6006.6.R2 = 0.9999,under the condition that the mobile phase is methanol,the flow rate is 1mL/min,and the detection wavelength is 282 nm.In the method,the theoretical number of ergosterol plates is greater than 5,000,the resolution is greater than 2,and the tailing factor is less than 2.When the detection limit is 30 ng/mL,the S/N>3,when the limit of quantification is 100 ng/mL,the S/N>10.The recovery rate of the method is in the range of 98-101%and the relative standard deviation(RSD)of the recovery rate is less than 2%.It is a reliable and effective method with great repeatability,reproducibility and precision for detecting ergosterol content in edible fungi.It was found that the content of ergosterol in Lactarius hatsudake in Hunan Province was about 2 mg/g,and the content in different fields was different.Through the single factor experiment on extractant,extraction times,ultrasonic time and liquid-to-liquid ratio,the optimal conditions for ultrasonic extraction of ergosterol from Lactarius hatsudake were as follows:using methanol as extractant,the ratio of material to liquid was 1:40 and ultrasonic extraction 90 min for 1 time.The crude crystals of ergosterol were purified by solvent crystallization,and the monomeric compounds were obtained by preparative high performance liquid chromatography.The prepared substance is compared with an ergosterol standard by analytical means such as ultraviolet,infrared,mass spectrometry,and the compound is determined to be a ergosterol monomer compound having a purity of 90%or more.It was proved by perforation test that ergosterol had no obvious inhibitory effect on three kinds of intestinal bacteria(Escherichia coli Staphylococcus aureus and Salmonella typhimurium)and four fungi(Aspergillusflavus,Monascus purpureus Went,Penicillium sp.and Aspergillus parasiticus Speare).And the double dilution test showed that ergosterol had no significant inhibitory effect on Mycobacterium tuberculosis H37Ra too.However,transformation experiments have shown that all four bacteria can utilize and transform ergosterol.The transformation of Staphylococcus aureus and Mycobacterium tuberculosis H37Ra was significant.Staphylococcus aureus utilized 27.88%±3.88%for 24 hours,and the conversion rate increased to 36.51%±8.36%after 48 hours,while the utilization rate of Salmonella was lower,and the utilization rates of 24 h and 48 h were 10.36%±5.10%and 22.36%±4.47%,respectively.The conversion rate of E.coli was the lowest.After 24 h of culture,the content of ergosterol was only reduced by 3.70%±1.00%,and after 48 h,it was only transformed by 16.37%±3.89%.The growth cycle of Mycobacterium tuberculosis was longer.So after 6 days of cultivation,the conversion rate of ergosterol reached 26.56%±5.58%.The transformation and utilization of ergosterol by Staphylococcus aureus and Mycobacterium tuberculosis is more obvious.Combined with the existing research,the reason may be that ergosterol is extremely difficult to dissolve in water,and Staphylococcus aureus is a Gram-positive bacterium.The cell surface contains more lipophilic ingredients,which makes it more efficacious for ergosterol.The cell wall peptidoglycan of Mycobacterium tuberculosis is surrounded by a large amount of lipids,which is also beneficial for its contact and utilization of ergosterol.