| Feruloyl oligosaccharides (FOs) largely exists in Gramineous plants, which is a kind of functional oligosaccharide with significant antioxidant activity formed by the carboxyl esterification of ferulic acid (FA) and sugar hydroxyl. Wheat bran from the production of flour is an important source of FOs. The ferulic acyl and hydrophilic oligosaccharides group in the molecular structure of FOs makes it water soluble and heat resistant,therefore,it has wide application prospects in health food. But, the efficient preparation technology of FOs remains to be researched further.In this study, the strains which can ferment wheat bran to produce FOs were screened out.Effects of culture medium and fermentation conditions on FOs production and hemicellulase biosythensis were investigated. One-stage and two-stage controlling pH and temperature on FOs preparation by Auerobasidium pullulans was compared. The differences of the compositions and structures of FOs produced by the two fermentation processes were identified. In addition, their antioxidant and antitumor activities in vivo and in vitro were compared. The main results are shown below:1. The strain 2012 producing FOs was isolated from the surrounding soil of the flour factory. It was identified as Aureobacidium pullulans not secreting melanin by study of morphology and ITS sequence and phylogenetic analysis. At pH 5.5 and 60 g/L of wheat bran liquid, the strain 2012 could produce 545 nmol/L FOs. Activities of xylanase (Xyln) and Ferulic acid ester-ase (FAE) was 64.12 IU/mL and 0.14 IU/mL, respectively. The structure of wheat bran broke down, loosed, formed the cavity,and its internal structure appeared obvious broken phenomenon after fermentation.2. When Aureobacidium pullulans was cultivated in the media with wheat bran, hemicellulases decompose wheat bran were induced. Xyln was mainly distributed in extracellular matrix, induced by wheat bran, xylose and xylan while inhibited by glucose and sucrose. Xylosidase was mainly in the hyphal filaments, induced by wheat bran, xylose and xylan while inhibited by sucrose, glucose and lactose. Arabinofuransidase was distributed in both the culture filtrate and the hyphal filaments cultivated in the media with wheat bran and xylan, and wasn’t produced in other carbon sources. FAE was mainly in the culture filtrate, and wasn’t produced in the media with glucose and sucrose. The production of FOs and the activity of Xyln has significant positive correlation (r=0.992). Xylosidase and the production of FAE has significant positive correlation (r=0.966). The correlation between FOs production and activity of xylosidase, arabinofuransidase, FAE was positive effect.Aureobacidium pullulans was cultivated in the media with wheat bran as only carbon source, with its concentration increased, Xyln activity and FOs yield increased, the best mass concentration of wheat bran fluid was 50-60 g/L,it was helpful to Xyln synthesis and preparation of FOs. A moderate amount of xylan and peptone can promote Xyln synthesis and FOs production. The additions of metal ion and surface active agent failed to improve the yield of FOs. The best medium composition for FOs preparation were 10 g/L xylan and 1 g/L peptone added 60 g/L wheat bran fluid.Under the medium, the yield of 774 nmol/L FOs was produced.3. Effects of fermentation conditions on FOs production by Aureobacidium pullulans were investigated. The factors influencing FOs yield positively were initial pH, inoculation quantity and fermentation temperature screened by P-B tests. Optimized by D-Optimal design, the optimal fermentation conditions were pH 6.0 initial pH,4.50%inoculation quantity and 29℃ fermentation temperature. Under this condition,904 nmol/L FOs was produced in the fermentation liquor of Aureobacidium pullulans, which was consistent with predicted value.Two-stage controlling pH and temperature on FOs production was investigated. In the conditions of initial fermentation pH 4.0, initial temperature 33℃, fermentation to 36 h, pH 6.0 and temperature 29℃, FOs reached the peak value of 1123 nmol/L in fermentation to 84 h, which was increased by 24% than one-stage, and the peak’s appearance was 12 h ahead.4. The one-stage fermentation process (FOs 1) and two-stage fermentation process (FOs 2) were isolated and purified by precipitation of ethnol, absorption of resin Amberlite XAD-2. The analysis of FOs’structure by liquid chromatography and gas chromatography showed that they were both composed of xylose, arabinose and ferulic acid. Ultraviolet spectrum analysis showed the maximum absorbance wave of two kinds of FOs in MOPS buffer were at 325nm. Infrared spectrum analysis showed the two kinds of FOs has characteric groups such as hydroxyl, ester bond and arabinofuran. ESI-MS showed degree of polymerization of FOs 1 was 5,4,3 and 2, the molecular weight was 986,854,722 and 590 respectively, consisted of four components like FAX5 FAX4, FAX3, FAX2. Degree of FOs 2 was 6,5,4, the molecular weight is 1118,986 and 854 respectively, consisted of FAX6, FAX5, FAX4, whose component purity and degree of polymerization were higher than that of FOs 1.5. Effects of FOs 1 and FOs 2 on hemolysis of rat RBC, rat liver homogenate and MDA formation in rat liver mitoehondria in vitro were studied. Hemolysis of rat RBC, rat liver homogenate, MDA formation of rat liver mitoehondria were inhibited in a dosage-dependent manner by FOs 1 and FOs 2 in the tested concentration of 0.5-10 mg.mL-1. The results showed that FOs 1 and FOs 2 had antioxidate activity in vitro, and the effect of FOs 2 is better than that of FOs 1.With the increasing of the dose, FOs 1 and FOs 2 could increase the activity of SOD and GSH-Px in serum of S180 tumor-bearing mice, reduce the level of MDA and thus improved the activity of antioxidant in vivo. When dose reached 250 mg·kg-1·d-1, FOs 2 was more advantageous to improve the capability of antioxidant of tumor-burdened mice than 5-FU and FOs 1 in vivo.MTT assay was used to analysis the anti-tumor activity of FOs 1 and FOs 2 on human liver cell HepG2, human gastric cancer cell BGC-823 and human lung cancer cell A549. Results indicated that both FOs 1 and FOs 2 could inhibit the three tumor cells, and the inhibition of FOs 2 was more superior than FOs 1, especially had the best effect to human liver cell HepG2. The inhibition of HepG2 reached 50.8% when the concentration of FOs 2 was 1 mg/mL. Morphological observation of tumor cell after treated by FOsl and FOs 2 showed that cellular shrinkage to round and being smaller, cells’floating and intercellular loosing, therefore, the proliferation rate decreased.Antitumor activity of FOs 1 and FOs 2 in vivo was studied through investigating the effect of Tumor inhibition rate, Thymus index, Spleen index, immune function factor IFN-y and IL-3 of S180 tumor-bearing mice,and HE dyeing observation. The results showed that FOs 1 and FOs 2 could remarkably inhibit the tumor’s growth of S180 tumor-bearing mice. The inhibition rate of 250 mg·kg-1·d-1 group was 44.55%. HE dyeing figure appeared a whole piece of red dye area and a few white cavity, tumor cells’ apoptosis, no red dye structure. With the increasing of the dose, inhibition rate of tumor improved, presented dosage-dependent manner. The low immune function factors of the immune organs in tumor-bearing mice were stimulated to effective promotion, so as to improve the immune mechanism, achieve antitumor effect, effect of FOs 2 was better than that of FOs 1 with same doses. |
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