Sampling of grapevines
Wine grape Cabernet Sauvignon, 5 years old, was collected at the junction of the third zone and fourth zone in Zhangyu Chateau in Shihezi city on a sunny day without wind at 8:00–10:00 on April 25, July 8 and September 20, 2011, respectively. The separation materials collected, the root, stem, leaf and fruit of Cabernet Sauvignon (the root, stem and leaf collected only on April 25), were immediately transported to the laboratory with the sterile bag at 4 °C, respectively. The materials were immediately rinsed with aseptic water on a sterile operating table to remove the dust, dirt and some microorganisms on the sample surface. After drying at room temperature, the root, stem, leaf and fruits collected were preserved at 4 °C, respectively.
The surface sterilization of the samples and the pure culture of the endophytes
The samples above were soaked by 75 % alcohol for 3 min before washing with sterile water and drying using filter paper, respectively, and then treated by 10 % effective chlorine of sodium hypochlorite for 20 s prior to drying employing filter paper again, and finally rinsed by aseptic water for 5 times. Following slice of 5–8 mm long segment in term of the phloem of the root and stem, a 5–8 mm × 5–8 mm of the tablet as for the leaf, and 5 × 5 × 5 mm of diamonds without the fruit peel for the sarcocarp under the aseptic operation, which were laid upon the surface of the culture medium and pressed slightly to better integration between the culture and the sample, after the sterilizing surface and peeling epidermi, respectively. The samples placed on the middle of each Petri dish, keeping a certain distance of 4–5 blocks, were cultured under the suitable temperature. The three parallel Petri dishes were prepared for each tissue. The colonies cultivated were picked out and transferred carefully to a new sterile plate for re-culturing when the new hyphae or fungi culture appeared. After repeating the procedure several times, the endophytes of pure culture in different grapevine organs were just obtained.
Medium and chemical agents
The potato dextrose agar (PDA), the separation and purification culture media of endophytic fungi, was prepared as follows: the 200 g of fresh potato filtrate through four layers of gauze, after boiling for 30 min, was added to 1000 mL with supple water, and supplemented 20 g of the filtrate agar and 20 g of glucose, respectively. Then the mixture, adjusted to pH 7.0, was subjected to 121 °C high pressure steam sterilization for 20 min. Agar was not added in the fermentation liquid of PDA. Gaus No. l medium acting as the isolation and purification culture of endophytic actinomycete was prepared according to the method described by Cao et al. (2004), and nutrient broth–yeast extract medium employed for the isolation and purification culture of the endophytic bacteria was also made up according to the method of Zinniel et al. (2002).
Standard resveratrol was purchased from Beijing century Aoke Biological Technology Co. Ltd. While the chemical agents as analytical reagents, such as methanol, acetonitrile, beef extract, peptone, agar, soluble starch, sodium hypochlorite, ethanol, glucose, sodium chloride, KNO3, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, ethyl acetate, ferric chloride, potassium ferricyanide, toluene, acetic acid, and ethyl acetate, were provided by Beijing Baiyi Innovation Technology Co. Ltd.
Preliminary screening of resveratrol-producing endophytes
Endophytic fungi purified were inoculated to 250 mL of Erlenmeyer flask including 100 mL liquid medium, respectively, and incubated at 115 rpm for 3 days at 28 °C simultaneously. Then the fermentation liquid was centrifuged at 4000 rpm for 10 min. Preliminary screening of chromogenic reaction: the endophytic fungus producing resveratrol could be screened according to the resveratrol property with ferric chloride–potassium ferricyanide color reaction. While the color reaction of the fermentation liquor was as follows: the mixture, 0.1 % FeCl3: 0.1 % K3[Fe(CN)6] = 1:1 (v/v), was served as chromogenic agent. 2 mL of coarse extraction liquid per sample concentrated above was mixed with the same volume of methanol, and then added two drops of chromogenic agent, meanwhile the color changes were recorded, respectively. The polyphenols containing resveratrol would be indicated by the indication of the blue color.
Re-screening of resveratrol-producing endophytes
Thin layer chromatography (TLC)
The steps of TLC analysizing resveratrol qualitatively included:
Ten microlitre of the strains fermentation liquid above after the centrifugation and 5 μg mL−1 resveratrol standard solution was spotted into the same silica gel plate (20 × 20 cm) in chromatography cylinder, then expanded vertically upward with the function of the developing solvent (toluene: ethyl acetate: acetic acid = 15:3:1, v/v), respectively. The thin layer plate was immediately removed and blowed dry by blower after the developing solvent launched to 1 cm of location close to the top of the thin layer plate.
After the silica gel plate above was sprayed uniformly by the color developer, 0.1 % FeCl3: 0.1 % K3[Fe(CN)6] = 1: 1 (v/v) and dried, the distance was measured immediately between the blue spot center and origin as well as between the origin and solvent. Finally, the Rf value (rate of flow or retention factor) was calculated. The Rf value is a constant for a given component under the same experimental conditions. The Rf value may be calculated from the following equation. The Rf value itself is unitless.
where a is distance of the center of the sample spot from the origin; b is distance of the solvent front from the origin.
Extraction, separation and purification of resveratrol in the fermented liquid
Extraction and separation of crude resveratrol was carried out by a slight modification of the method of Zeng et al. (2012).
Thirty-six strains of endophytic fungi purified were inoculated to 250 mL of Erlenmeyer flask including 100 mL liquid medium, respectively, and incubated at 115 rpm for 3 days at 28 °C simultaneously. Then the collected supernatant of fermentation liquid centrifuged at 4000 rpm for 10 min, after vacuum concentration at 60 °C, was extracted 3 times by 50 mL of ethyl acetate finally. The raffinate in the ethyl acetate layer were collected and washed three times by 3 % (v/v) of NaHCO3 solution, before concentrated under the vacuum at 50 °C the extract liquor discarded NaHCO3 solution layer were dehydrated by the anhydrous magnesium sulfate and filtered by filter paper, then blowed dry by pure nitrogen, dissolved by 2.0 mL of methanol, and filtered by 0.45 μm of microporous membrane, successively, thus crude resveratrol products were obtained.
Purification of resveratrol in the crude products: the method of Jiang (2008) and Cai (2010) could be used in the trial by a slight modification. The steps were as follows: (1) crude extract of resveratrol was preliminarily purified and separated by AB-8 type macroporous adsorption resin; (2) 0.5–1.0 mg mL−1 of the samples were desorbed by 70 % ethanol (pH 7) at the rate of l mL min−1; (3) the samples isolated preliminarily was detached with the eluent of chloroform: methanol (15:1, v/v) after separating with silica gel chromatography, and the final residual liquid dried by nitrogen gas after vacuum concentration at 50 °C was just purified resveratrol, with a total process recovery above 77.3 %, of more than 90 % purity.
Ultraviolet wavelength scanning
Five milligram of purified resveratrol sample, weighed accurately, was diluted with aseptic water to the scale and shook well after completely dissolved by a small amount of methanol in a 50 mL of clean brown volumetric flask, which was 0.10 mg mL−1 of the resveratrol solution. Afterwards, 5 mL of accurate resveratrol solution, diluted with methanol to the scale in 25 mL of volumetric flask, was scanned by ultraviolet/visible spectroscopic measurements UV-1700 (Shimadzu) in 200–500 nm of the spectral range with methanol as CK. Thus the maximum absorption wavelength could be obtained from the absorption peak of the corresponding spectrum. It was obvious that the resveratrol existed in the extract liquid when the maximum absorption was at 306 nm.
Quantitative analysis of resveratrol by liquid chromatography (LC)
The methods of Zeng et al. (2012) and Standards Press of China (2006) were referred in the trial.
Preparation of resveratrol standard solution (0.5, 1, 2.5, 5, 10, 15 and 20 μg mL−1). Firstly, 5 mg of the resveratrol standard weighed accurately was dissolved into 50 μg mL−1 of standard stock solution with the methanol. Secondly, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 mL of the standard stock solution from the above were precisely drawn and put into a 10 mL volumetric flask, respectively. Then, shaken well, sealed, stored after it was set with methanol to constant volume and finally, they were analyzed in order.
Chromatographic condition: liquid chromatography (LC-2010 A HT type, Shimadzu) was accepted. A two-element of gradient elution protocols was employed on Waters XTeera MS C18 (4.6 × 250 mm, 5 μm) chromatographic column: 5 % acetonitrile in 95 % distilled water for the first 5.0 min; 60 % acetonitrile in 40 % distilled water for the second 28 min; 85 % acetonitrile in 15 % distilled water for the further 33 min; 5 % acetonitrile in 95 % distilled water for the final 40 min. The column temperature, flow rate and ultraviolet wavelength were 35 °C, 0.2 mL min−1 and 306 nm, respectively. The injection volume was 10 μL. Under such chromatographic conditions, resveratrol was eluted, and basically achieved baseline separation; retention time was 22.696 min.
Drawing of standard curve: the resveratrol standard solutions above, triplicate per concentration, were detected according to (B), respectively. Therefore, the regression equation of the resveratrol, Y = 9 × 10−6X + 0.298 (R2 = 0.9993), based on the regression curve according to the integral value of peak area (Y) as ordinate and solution concentration (X, g L−1) of the standard sample as the abscissa, showed good linear relationship between 0.5 and 20 g L−1.
Determination of resveratrol content: resveratrol purified was separated from the sample and diluted after dissolved with methanol to 25 mL, 1.0 mL of which was filtrated to automatical sample vials with 0.22 μm filter membrane, and 10 μL of the final filtrate was injected in LC before detection. The chromatographic peak of resveratrol in the samples was qualified by the retention time, meanwhile the resveratrol content was calculated through external standard method based on the peak area of the samples.
Genetic stability of the resveratrol production
C2J6 was grown in PDA medium with shaking (150 rpm, 28 °C) for 3 days. The content of resveratrol produced by C2J6 was detected by LC as described above, and genetic stability of the resveratrol production was determined by successively subculturing five generations strain in PDA.
Identification of resveratrol-producing endophytes
The resveratrol-producing endophytes were identified using morphological characteristics and molecular tools. The experiments of the morphological identification were carried out as described by Kim and Baek (2011). The strain was identified by sequencing the internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS2) according to White et al. (1990) and the D1/D2 domain at the 5′ end of the LSU rRNA gene according to Kurtzman and Robnett (1998). The DNA from fungi cell suspensions grown in YPD for 48 h was extracted employing NucleoMag 96 Plant Kit (Macherey-Nagel, Oensingen, Switzerland) and Kingfisher magnetic particle processor (Thermo Labsystems, Basingstoke, UK) following the manufacturers’ protocols. The ITS regions were amplified by means of genomic DNA as a template and universal primers ITS1 and ITS4, while the D1/D2 domains were augmented through the primers NL-1 and NL-4 on the genomic DNA.
Twenty microlitre of PCR contained 1 µL DNA template (50 ng), 200 mM of each deoxynucleotide triphosphate, 2 µL of tenfold buffer (Taq DNA Polymerase, Qiagen, Chatsworth, CA, USA), 0.7 mM each primer, and 1.0 U Taq DNA Polymerase (Qiagen).
PCR program for ITS regions followed: 95 °C, 3 min; 34 cycles; 94 °C, 15 s; 55 °C, 45 s; 72 °C, 55 s; 72 °C, 7 min. Meanwhile, the program for D1/D2 domain was: 95 °C, 10 min; 30 cycles: 94 °C, 30 s; 55 °C 30 s; 72 °C, 45 s; 72 °C, 7 min. A 10 µL aliquot of PCR products from each reaction, electrophoresed in TBE buffer including 2.0 % agarose gel, was stained with SYBR SAFE (Invitrogen, Eugene, OR, USA). Gel images were finally acquired with a Gel Doc 1000 System (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplification products were cloned into the PCR4 TOPO vector (Invitrogen) using the TOPO TA cloning kit following the manufacturer protocol and sequenced by Zhong-mei-Tai-he Sequencing Company (Beijing, China) and an Illumina HiSeq-2000 Sequencer (Illumina, USA).