Bo WANG, Yanfeng HAN, Yanwei ZHANG

1. Teaching Laboratory Management Center ,Guizhou Normal College, Guiyang 550018, China ;

2. Institute of Fungus Resources, College of Life Sciences, Guizhou University, Guiyang 550025, China;

3. Guizhou Bioresource Development and Utilization Key Laboratory, Guizhou Normal College, Guiyang 550018, China

Paecilomyces marquandii is a kind of fungi. There has been currently scarce concern on Paecilomyces marquandii, except the aspects of pollution elimination,metabolism and biological control.Slaba et al.[1-3]studied the adsorption of zinc and lead ions by Paecilomyces marquandii stains and the relevant mechanisms, as well as the degradation effect of Paecilomyces marquandii on herbicide alachlor and possible degradation way. They found that Paecilomyces marquandii had potential industrial application prospects in eliminating pollution caused by zinc ion and alachlor.Ahuja et al.[4]isolated a fungi strain with phosphate-solubilizing ability from phosphate-deficient soil,and the isolated strain was identified as Paecilomyces marquandii.Dosio et al.[5]isolated a antibiotic composed of nine peptides from the culture of Paecilomyces marquandii. If bound with AR-3 monoclonal antibody,the isolated antibiotic had anti-cancer effect. In the screening of strains for biocontrol of wheat cereal cyst nematodes, Chen et al.[6]screened a Paecilomyces marquandii strain (09F11),which parasitized in the eggs and larvae, and could kill the second instar larvae and reduce the egg hatchability of cereal cyst nematodes.

Studies have primarily discussed the effects of medium composition and culture conditions on natural vitamin A and vitamin E(referred to as VAand VEhereafter) production by a Paecilomyces marquandii strain isolated from soil[7]. In this study, the culture conditions of VA- and VE-producing Paecilomyces marquandii strain were further discussed so as to find a more economical and convenient culture method.

Material and Methods

Material

Tested strain The Paecilomyces marquandii strain was provided by the Institute of Fungus Resources,College of Life Sciences, Guizhou University.

Equipment and reagents In this study, the used equipment included Agilent fluorescence spectrophotometer; the used reagents includedethanol, petroleum ether, sodium sulphate anhydrous,vitamin C(of analytical grade,made in China),60%sulfuric acid, VAstandard and VEstandard(Sigma Corporation).

Methods

Culture of sample The PDA medium was selected as basic medium. To investigate the effects of nitrogen source(3 g/L),pH value and Fe2+(0.01 g/L)on the VA-and VE-producing ability of Paecilomyces marquandii strain,a three-factor and two-level orthogonal experiment was designed (Table 1).Meanwhile, the blank control (PDA medium, Treatment 5), VAcontrol(optimum medium for VAproduction,Treatment 6) and VEcontrol (optimum medium for VEproduction, Treatment 7) were arranged. The Paecilomyces marquandii strain was respectively inoculated in the liquid medium of each treatment group, and then incubated(shaking) at 26 ℃for 25 d. Subsequently, the mycelia were removed and dried out with filter for indicator determination.

Treatment of sample Before the determination, the samples were first treated as described by Liu et al.[7]and Kan et al[8].

Determination of VA and VE contents

(1) VAfluorescence intensity measurement.

The fluorescence intensities of petroleum ether solution samples were measured by fluorescence spectrophotometer at λex340 nm/λem478 nm. At the same time, the fluorescence intensities of blank solution and standard solution were also measured.

(2) VEfluorescence intensity measurement.

The fluorescence intensities of upper-layer petroleum ether solution samples were measured by fluorescence spectrophotometer at λex295 nm/λem330 nm. At the same time, the fluorescence intensities of blank solution and standard solution were also measured.

(3)Content calculation.

The VAand VEcontents (μg/g)were all calculated in accordance with the following formula:

Wherein, R1refers to the fluorescence intensity of the standard solution; R2refers to the fluorescence intensity of the solution sample;R0refers to the fluorescence intensity of the blank solution; S refers to the final concentration of standard solution (VA,15 μg/ml;VE,20 μg/ml);W refers to the weight of sample (g); H refers to the moisture content in the sample(%).

Results and Analysis

Determination results of VA and VE contents

The VAand VEcontents in all the treatment groups were shown in Table 2. The VAyield was highest (448.25 μg/g)when 3 g/L of sodium nitrate was used as nitrogen source, pH value of medium was 7 and Fe2+was not added in the medium; while the VEyield was highest (32.16 μg/g) when 3 g/L of peptone was used as nitrogen source,pH value of medium was 7 and Fe2+was added in the medium.

Results of orthogonal experiment

The range analysis (Table 3)showed that for VAproduction by Paecilomyces marquandii strain,nitrogen source showed the greatest effect,followed by Fe2+, and the effect of pH was weakest. The optimum combination was A2B2C2, i.e., the nitrogen source was 3 g/L of sodium nitrate,the pH of medium was 6 and the Fe2+was not added. For VEproduction, nitrogen source showed the greatest effect,followed by Fe2+, and the effect of pH was weakest. The optimum combination was A1B1C1, i.e., the nitrogen source was 3 g/L of peptone,the pH of medium was 7 and the 0.01 g/l of Fe2+was added. The variance analysis results indicated that there was significant difference between the two nitrogen sources (P = 0.022 ＜0.05), and the peptone was more suitable; there were no significant differences in pH(P=0.809 ＞0.05)or Fe2+(P=0.183 ＞0.05).

Table 1 Factors and levels

Table 2 VA and VE contents in treatment groups μg/g

Table 3 Effects of culture composition on VA and VE contents

Discussion

The VAand VEextraction methods were improved. The extended extraction at room temperature was adopted.Although the extraction duration was extended, the operation was easier and the effect of petroleum ethervolatilization was avoided to some extent compared to the water extraction.

When PDA medium was used as basic medium, the addition of nitrogen source produced great effect on VAand VEcontents. The addition of sodium nitrate and slightly acidic culture conditions were conducive to the production of VA. The VAcontent in the Treatment 3 was nearly 4 times higher than that in the black control, and was also nearly 4 times higher than that in the VA control.It suggests that the optimization of medium by adding components can greatly improve the yield of VAproduced by strains. For VEproduction by Paecilomyces marquandii strain, the nitrogen source showed great effect,but the pH of medium and addition of Fe2+all showed no significant effects.After the optimization,the VEcontent in the Treatment 1 was only increased slightly compared to those in the blank control and VEcontrol.The optimization effect was not obvious,which was presumably due to the PDA medium as a basic medium. Compared with Czapek medium, the nutrients in PDA medium are more comprehensive and complex. The VEcontent determined in this study was much lower than those obtained in previous studies, which might be because of different extraction agents and extraction methods. The optimum nitrogen sources for VAand VEproduction by Paecilomyces marquandii strain were sodium nitrate and peptone,respectively.

[1]SLABA M,SZEWCZYK R,PIATEK MA,et al. Alachlor oxidation by the filamentous fungus Paecilomyces marquandii[J]. Journal of Hazardous Materials,2013,261(15):443-450.

[2]SLABA M,DLUGONSKI J.Efficient Zn2+and Pb2+uptake by filamentous fungus Paecilomyces marquandii with engagement of metal hydrocarbonates precipitation[J].International Biodeterioration&Biodegradation,2011,65(7):954-960.

[3]SLABA M, SZEWCZYK R, BERNAT P,et al. Simultaneous toxic action of zinc and alachlor resulted in enhancement of zinc uptake by the filamentous fungus Paecilomyces marquandii [J]. Science of the Total Environment, 2009, 407(13):4127-4133.

[4]AHUJA A, GHOSH SB, DSOUZA SF.Isolation of a starch utilizing, phosphate solubilizing fungus on buffered medium and its characterization[J]. Bioresource Technology,2007,98(17):3408-3411.

[5]DOSIO F, RICCI M, BRUSA P, et al.Antibody-targeted leucinostatin A [J].Journal of Controlled Release,1994,32(1):37-44.

[6]CHEN L (陳莉).Screening of biocontrol strains against wheat cereal cyst nematode and preliminary studies on the biocontrol mechanism(小麥禾谷胞囊線蟲病生防菌株篩選及防病機(jī)理的初步研究) [D]. Zhengzhou: Henan Agricultural University(鄭州: 河南農(nóng)業(yè)大學(xué)),2010.

[7]LIU X(劉訊),DONG L(董利),FENG XX(豐曉雪),et al.Effects of different medium on natural vitamin A and vitamin E produced by strain Paecilomyces marquandii (不同培養(yǎng)基成分對(duì)1 株馬昆德擬青霉產(chǎn)天然VA及VE的影響)[J].Guizhou Agricultural Sciences (貴州農(nóng)業(yè)科學(xué)),2013,41(9):115-117.

[8]KAN JQ (闞健全).Simultaneous determination of vitamin A and E in foods by fluorometry (熒光法同時(shí)測(cè)定食品中維生素A 和維生素E 的含量)[J]. Acta Nutrimenta Sinica(營(yíng)養(yǎng)學(xué)報(bào)),1990,12(1):46-51.