CN114249736A - Extraction method of dihydromaltophilin - Google Patents
Extraction method of dihydromaltophilin Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 39
- 230000004151 fermentation Effects 0.000 claims abstract description 39
- 239000011347 resin Substances 0.000 claims abstract description 31
- 229920005989 resin Polymers 0.000 claims abstract description 31
- 241000589634 Xanthomonas Species 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- 239000000047 product Substances 0.000 claims description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000001704 evaporation Methods 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 229920002101 Chitin Polymers 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 230000006837 decompression Effects 0.000 claims description 6
- 230000008020 evaporation Effects 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 230000008961 swelling Effects 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims 1
- 239000012071 phase Substances 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 241000863030 Lysobacter enzymogenes Species 0.000 description 2
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- 238000005406 washing Methods 0.000 description 2
- 102100024308 Ceramide synthase Human genes 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- -1 Dihydromaltophilin Natural products 0.000 description 1
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- 238000009825 accumulation Methods 0.000 description 1
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- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
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- 108010061814 dihydroceramide desaturase Proteins 0.000 description 1
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- 239000000543 intermediate Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- SPROEVQSLDXJAP-RLCDWEJWSA-N maltophilin Chemical compound OC1CCNC(=O)\C=C/C[C@@H]2[C@H]3C[C@H]4C[C@H](CC)[C@H](C)[C@H]4[C@@H]3C(=O)C[C@H]2\C=C\C(=O)C2=C(O)NC1C2=O SPROEVQSLDXJAP-RLCDWEJWSA-N 0.000 description 1
- ZIFOVGPOLIICJX-UHFFFAOYSA-N maltophilin Natural products CCC1CC2CC3C4CC=C/C(=O)NCCC(O)C5NC(=O)C(=C5O)C(=O)C=CC4CC(=O)C3C2C1C ZIFOVGPOLIICJX-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000004260 plant-type cell wall biogenesis Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/08—Bridged systems
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- C12N1/20—Bacteria; Culture media therefor
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Abstract
The invention provides a method for extracting dihydromaltophilin. In the extraction method, the supernatant of the fermentation liquor of the xanthomonas bacteria is separated by macroporous resin and then separated by HPLC. The method has high extraction efficiency and high extraction purity.
Description
Technical Field
The invention relates to an extraction method of dihydromaltophilin.
Background
Bacteria of the xanthomonas family are widely present in soil, water and deep sea sponges. Two strains in the family have better colonization activity, and the two strains are lysobacter enzymogenes and stenotrophomonas maltophilia respectively. The two kinds of bacteria have the common characteristic of secreting various extracellular enzymes and various secondary metabolites and have good control effects on various pathogenic fungi, pathogenic bacteria, algae, nematodes and the like. The lysobacter enzymogenes can produce an important antifungal secondary metabolite, namely Dihydromaltophilin, which is separated from streptomyces sp. Dihydromaltophilin can act on ceramide synthase of fungi, interfere synthesis of sphingolipid intermediates, activate cell wall synthesis pathways, cause excessive accumulation of cell wall synthetic substances to cause thickening and swelling of hyphae, also can cause degradation of fungal cell nucleus and induce apoptosis, and has no influence on body cells.
Disclosure of Invention
The invention aims to provide an extraction method of dihydromaltophilin.
In order to realize the purpose, the technical scheme is as follows: a method for extracting dihydromaltophilin comprises the following steps:
(1) preparing fermentation liquor of bacteria in xanthomonas;
(2) centrifuging the fermentation liquor of the bacteria in the xanthomonas family obtained in the step (1), and collecting supernatant;
(3) adding macroporous resin into the supernatant collected in the step (2) for incubation;
(4) putting the incubated macroporous resin in the step (3) into a chromatographic column, and eluting the macroporous resin with water until the eluent is clear;
(5) sequentially eluting the macroporous resin obtained in the step (4) by using a proper amount of 30%, 50% and 60% methanol;
(6) eluting the macroporous resin obtained in the step (5) by using 100% methanol until the eluent is colorless, and eluting the dihydromaltophilin into 100% methanol;
(7) concentrating the 100% methanol eluent obtained in the step (6) under reduced pressure and evaporating to dryness;
(8) dissolving the fermentation product which is subjected to decompression concentration and evaporation by using methanol and dichloromethane, and performing decompression concentration and evaporation by using the dissolved fermentation product to obtain a crude fermentation product;
(9) dissolving the crude fermentation product obtained in the step (7) with methanol and DMSO, centrifuging the dissolved crude fermentation product, collecting the supernatant, performing HPLC separation, and collecting dihydromaltophilin according to the retention time of the dihydromaltophilin.
Preferably, the specific steps for preparing the fermentation broth of bacteria of the family xanthomonas in the step (1) comprise:
inoculating bacteria of xanthomonas to an LB solid culture medium for activation culture; inoculating activated and cultured xanthomonas to an LB liquid culture medium for amplification culture to obtain a seed solution; inoculating the seed solution into a chitin liquid culture medium by 3-5% of inoculation amount for fermentation culture to obtain the xanthomonas bacterium fermentation liquor.
Preferably, the activation culture conditions are: the activation culture temperature is 28-30 ℃, and the activation culture time is 1-2 days; and (3) expanding culture conditions: the expanding culture temperature is 28-30 ℃, the expanding culture time is 1-2 days, and the rotating speed of a shaking table is 170-200 rpm; fermentation culture conditions: the fermentation culture temperature is 28-30 ℃, the fermentation culture time is 2-3 days, and the rotating speed of a shaking table is 170-200 rpm.
Preferably, the chitin liquid medium is: 1% chitin, 0.2mM indole, pH 7.2.
Preferably, the centrifugation speed in the step (2) is 6000rpm, and the centrifugation time is 10 minutes; in the step (8), the centrifugal speed is 13000rpm, and the centrifugal time is 10 min.
Preferably, the macroporous resin is pretreated before the macroporous resin is used in the step (3), wherein the pretreatment mode is that the macroporous resin is soaked in ethanol and stands for 24 hours to swell the macroporous resin; then, the swollen macroporous resin was washed with distilled water, and ethanol was completely washed. If the macroporous resin floats on the surface of the distilled water, the macroporous resin is not completely swelled, and the macroporous resin is continuously swelled by the ethanol until the macroporous resin is completely sunk in the water.
Preferably, the incubation conditions in step (3): the incubation temperature is 28-30 ℃, the rotation speed of a shaking table is 170-200 rpm, and the incubation time is 1 day. And incubating for 1 day to ensure that the compounds in the fermentation liquor are fully adsorbed to the macroporous resin.
Preferably, the flow rate of elution in the step (5) is 1 s/drop; the flow rate at the time of elution in the step (6) was 1 s/drop.
Preferably, the temperature for concentrating and evaporating to dryness under reduced pressure in the step (7) is 30-35 ℃; the temperature for decompression concentration and evaporation in the step (8) is 30-35 ℃.
Preferably, the HPLC conditions in step (8):
mobile phase A: water to formic acid 100: 0.1, mobile phase B: acetonitrile and formic acid (100: 0.1), flow rate of 4mL/min, detection wavelength of 320nm, and elution gradient as follows:
| Time | mobile phase B/%) |
| 0 | 44 |
| 11 | 44 |
| 12 | 100 |
| 14 | 100 |
| 15 | 44 |
| 17 | 44 |
。
Has the advantages that:
the invention provides an extraction method of dihydromaltophilin, which has high extraction efficiency and high extraction purity.
Drawings
FIG. 1: schematic diagram of extraction process of dihydromaltophilin.
FIG. 2: the change in yield of dihydromaltophilin during the extraction was detected by HPLC.
FIG. 3: process for preparing dihydromaltophilins1H NMR detection result chart.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
This example provides a method for extracting dihydromaltophilin, and the schematic diagram is shown in FIG. 1.
1. Preparation of fermentation broth of Xanthomonas bacteria
(1) The bacteria of xanthomonas (lysobacter or stenotrophomonas maltophilia) preserved at-80 ℃ are respectively inoculated on an LB solid culture medium, cultured for 2 days at 28 ℃, then single colonies are selected and transferred to a new LB solid culture medium, and cultured for 2 days at 28 ℃.
(2) Washing the thallus on the plate with sterile normal saline, inoculating to LB liquid culture medium, shake culturing at 30 deg.C and 200rpm for 1d to obtain seed liquid.
(3) Inoculating the seed solution to chitin liquid culture medium at an inoculum size (volume ratio) of 5%, performing fermentation culture, and shake culturing at 30 deg.C and 200rpm for 2d to obtain fermentation liquid.
2. Extraction and purification of dihydromaltophilin
(1) And (3) centrifuging the fermentation liquor at 6000rpm for 10min, collecting supernatant, and detecting the yield of the dihydromaltophilin in the supernatant to ensure normal fermentation.
(2) Adding macroporous resin into the supernatant, paving the bottom, incubating at 30 deg.C and 200rpm for 1 day to make the compounds in the fermentation liquid fully adsorbed to the macroporous resin.
(3) Standing for precipitation, discarding supernatant, collecting macroporous resin, loading into chromatographic column, eluting macroporous resin with ultrapure water until eluate is clear, and washing to remove residual culture medium. The macroporous resin was then eluted sequentially with 500ml of 30%, 50%, 60% methanol at a flow rate of 1 s/drop.
(4) The macroporous resin was eluted with 100% methanol until the eluent was colorless at a flow rate of 1 s/drop and the dihydromaltophilin was eluted into 100% methanol.
(5) Transferring 100% methanol eluate to rotary evaporation bottle, concentrating under reduced pressure at 30 deg.C, evaporating to dryness, dissolving the fermentation product in the inner wall of rotary evaporation bottle with 5ml methanol and 1ml dichloromethane, transferring to 100ml round bottom flask, concentrating under reduced pressure at 30 deg.C again, evaporating to dryness to obtain crude fermentation product. Dihydrophilus maltophilin production was tested by dissolving a small amount of crude product in methanol and DMSO.
(6) The crude fermentation product on the inner wall of the rotary evaporation bottle is dissolved by proper amount of methanol and DMSO, transferred into an EP tube, centrifuged at 13000rpm for 10min, transferred into a liquid phase vial for HPLC preparation, and the sample must be ensured to be completely dissolved and have no precipitate. 10-90. mu.l of the treated sample is loaded each time, the retention time of the dihydromaltophilin is about 15min, and the samples are collected according to the retention time. Transferring the collected liquid into rotary evaporation bottle, concentrating under reduced pressure, evaporating to obtain pure dihydromaltophilin, dissolving a small amount of pure product with methanol and DMSO, and detecting purity by HPLC, or using1H NMR was used to determine the purity of the pure product. Storing the rest at-80 deg.C.
In the extraction process, fermentation liquor, crude fermentation product and pure product are selected for HPLC detection, and the yield change of the dihydromaltophilin is observed, as shown in figure 2.
The elution gradient in HPLC detection is shown in Table 1, the flow rate is 1mL/min, and the detection wavelength is 320 nm; the HPLC preparation was carried out with an elution gradient as shown in Table 2, a flow rate of 4mL/min and a detection wavelength of 320 nm. The liquid chromatographic column used was YMC-Pack Pro C18, and the column temperature was 32 ℃.
TABLE 1 HPLC detection of elution gradients
| Time | Mobile phase B/%) |
| 0 | 20 |
| 5 | 35 |
| 12 | 75 |
| 20 | 90 |
| 23 | 100 |
| 27 | 100 |
| 28 | 20 |
| 30 | 20 |
TABLE 2 HPLC preparative elution gradient
| Time | Mobile phase B/%) |
| 0 | 44 |
| 11 | 44 |
| 12 | 100 |
| 14 | 100 |
| 15 | 44 |
| 17 | 44 |
By using1The purity of the pure product was checked by H NMR, and the results are shown in FIG. 3. The structural formula of the dihydromaltophilin prepared by the invention is as follows:
process for preparing dihydromaltophilins according to the invention1H NMR detection results:
1H NMR(600MHz.DMSO-d6)δ8.89(S，1H)，7.96(S，1H)，6.87(d，J＝15.5Hz，1H)，6.57(dd，J＝15.7，10.1Hz，1H)，5.91(t，J＝11.3Hz，1H)，5.71(d，J＝11.4Hz，1H)，5.09(S，2H)，3.99-3.70(m，3H)，3.52(S，1H)，3.25(td，J＝10.3，4.6Hz，2H)，2.65-2.52(m，2H)，2.35(m，1H)，2.01(m，3H)，1.89(d，J＝17.0Hz，1H)，1.83-1.71(m，2H)，1.65(m，1H)，1.56(m，1H)，1.46-0.98(m，10H)，0.97-0.73(m，5H).
finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A method for extracting dihydromaltophilin, which is characterized by comprising the following steps:
(1) preparing fermentation liquor of bacteria in xanthomonas;
(2) centrifuging the fermentation liquor of the bacteria in the xanthomonas family obtained in the step (1), and collecting supernatant;
(3) adding macroporous resin into the supernatant collected in the step (2) for incubation;
(4) putting the incubated macroporous resin in the step (3) into a chromatographic column, and eluting the macroporous resin with water until the eluent is clear;
(5) sequentially eluting the macroporous resin obtained in the step (4) by using a proper amount of 30%, 50% and 60% methanol;
(6) eluting the macroporous resin obtained in the step (5) by using 100% methanol until the eluent is colorless, and eluting the dihydromaltophilin into 100% methanol;
(7) concentrating the 100% methanol eluent obtained in the step (6) under reduced pressure and evaporating to dryness;
(8) dissolving the fermentation product which is subjected to decompression concentration and evaporation by using methanol and dichloromethane, and performing decompression concentration and evaporation by using the dissolved fermentation product to obtain a crude fermentation product;
(9) dissolving the crude fermentation product obtained in the step (7) with methanol and DMSO, centrifuging the dissolved crude fermentation product, collecting the supernatant, performing HPLC separation, and collecting dihydromaltophilin according to the retention time of the dihydromaltophilin.
2. The extraction method according to claim 1, wherein the step (1) of preparing the fermentation broth of bacteria of the family Xanthomonas comprises the following steps:
inoculating bacteria of xanthomonas to an LB solid culture medium for activation culture; inoculating activated and cultured xanthomonas to an LB liquid culture medium for amplification culture to obtain a seed solution; inoculating the seed solution into a chitin liquid culture medium by 3-5% of inoculation amount for fermentation culture to obtain the xanthomonas bacterium fermentation liquor.
3. The extraction method according to claim 2, wherein the activation culture conditions are: the activation culture temperature is 28-30 ℃, and the activation culture time is 1-2 days; and (3) expanding culture conditions: the expanding culture temperature is 28-30 ℃, the expanding culture time is 1-2 days, and the rotating speed of a shaking table is 170-200 rpm; fermentation culture conditions: the fermentation culture temperature is 28-30 ℃, the fermentation culture time is 2-3 days, and the rotating speed of a shaking table is 170-200 rpm.
4. The extraction method according to claim 2, wherein the chitin liquid medium is: 1% chitin, 0.2mM indole, pH 7.2.
5. The extraction method according to claim 1, wherein the centrifugation speed in the step (2) is 6000rpm, and the centrifugation time is 10 minutes; in the step (8), the centrifugal speed is 13000rpm, and the centrifugal time is 10 min.
6. The extraction method according to claim 1, wherein the macroporous resin is pretreated before being used in the step (3), and the pretreatment mode is as follows: soaking macroporous resin in ethanol, standing for 24h, and swelling the macroporous resin; then, the swollen macroporous resin was washed with distilled water, and ethanol was completely washed.
7. The extraction method according to claim 1, wherein the incubation conditions in step (3) are: the incubation temperature is 28-30 ℃, the rotation speed of a shaking table is 170-200 rpm, and the incubation time is 1 day.
8. The extraction method according to claim 1, wherein the flow rate at the time of elution in the step (5) is 1 s/drop; the flow rate at the time of elution in the step (6) was 1 s/drop.
9. The extraction method according to claim 1, wherein the temperature for concentrating and evaporating to dryness under reduced pressure in step (7) is 30-35 ℃; the temperature for decompression concentration and evaporation in the step (8) is 30-35 ℃.
10. The extraction process according to claim 1, wherein the HPLC conditions in the step (8) are:
mobile phase A: water to formic acid 100: 0.1, mobile phase B: acetonitrile and formic acid (100: 0.1), flow rate of 4mL/min, detection wavelength of 320nm, and elution gradient as follows:
。
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| CN107119088A (en) * | 2017-05-05 | 2017-09-01 | 江苏省农业科学院 | A kind of molten bacillus OH11 production active antibacterial materials HSAF of response phase method optimization producing enzyme method |
| CN110387389A (en) * | 2018-04-19 | 2019-10-29 | 江苏省农业科学院 | A method of improving antifungus active substance HSAF fermentation yield |
| CN112773778A (en) * | 2020-10-14 | 2021-05-11 | 中山大学中山眼科中心 | Medical material for treating fungal keratitis and preparation method thereof |
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2021
- 2021-09-24 CN CN202111123134.9A patent/CN114249736A/en active Pending
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| CN107119088A (en) * | 2017-05-05 | 2017-09-01 | 江苏省农业科学院 | A kind of molten bacillus OH11 production active antibacterial materials HSAF of response phase method optimization producing enzyme method |
| CN110387389A (en) * | 2018-04-19 | 2019-10-29 | 江苏省农业科学院 | A method of improving antifungus active substance HSAF fermentation yield |
| CN112773778A (en) * | 2020-10-14 | 2021-05-11 | 中山大学中山眼科中心 | Medical material for treating fungal keratitis and preparation method thereof |
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| ANDREW S. OLSON等: "Synthesis of a 2,4,6,8,10-dodecapentanoic acid thioester as a substrate for biosynthesis of heat stable antifungal factor (HSAF)", 《RSC ADV》 * |
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