茶薪菇发酵制备麦麸膳食纤维与阿魏酰低聚糖及其生物活性研究
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摘要
麦麸是小麦制粉过程中形成的副产物,主要由皮层和部分糊粉层构成,富含纤维素、半纤维素等成分,还含有0.4-0.7%的阿魏酸,且多以共价键的形式与细胞壁多糖连接。因而麦麸可作为制备膳食纤维(Dietary Fibre, DF)和阿魏酰低聚糖(Feruloyl oligosaccharides, FOs)的原料。
本研究以麦麸为原料,采用食药用真菌发酵法制备膳食纤维和FOs。通过比较食药用真菌在麦麸培养基中的生长特性、产酶情况及生成膳食纤维和FOs产量,筛选得发酵菌株；研究培养基成分及发酵条件对发酵菌株生成膳食纤维、FOs和胞外酶的影响,确定产酶诱导物及最佳发酵条件；比较不同发酵条件下菌株产酶情况,揭示发酵法制备膳食纤维和FOs的机理；通过乙醇沉淀和大孔树脂吸附及柱层析法对FOs分离纯化,采用气相色谱、液相色谱、红外和质谱等方法鉴定FOs的组成及结构；比较发酵前后不溶性膳食纤维(Insoluble dirtary fibre, IDF)的物理特性,不同来源的可溶性膳食纤维(Soluble dietary fibre, SDF)和不同结构的FOs抗氧化、抗肿瘤及其对肠道微生物作用。主要研究结果如下：
1、比较了平菇、灵芝、荼薪菇等食药用真菌在麦麸培养基中生长、产酶及生成膳食纤维和FOs的情况。结果发现,茶薪菇适于发酵法制备膳食纤维和FOs,经该菌发酵后膳食纤维含量达68.49%,其中含有19.24%的SDF, FOs产量为35.4μM。
比较了各菌株在麦麸培养基和PDA培养基中的生长速度,结果表明：食药用真菌在麦麸培养基中的生长情况优于PDA培养基,其中以猴头菇和平菇最明显。测定各菌株在麦麸培养基中的产酶情况可知,各菌均能生成淀粉酶、蛋白酶、纤维素C1酶和木聚糖酶等,其中猴头菇亦可生成阿魏酸酯酶；酶活测定结果显示发酵过程中茶薪菇生成淀粉酶、蛋白酶和纤维素C1酶和木聚糖酶活性高于其他菌株。膳食纤维和FOs产量与纤维素酶和木聚糖酶的相关性分析表明,膳食纤维产量与β-葡萄糖苷酶活性负相关(r=-0.71),FOs产量与木聚糖酶活性正相关(r=0.912)。
2、比较了氮源和无机盐种类对茶薪菇发酵后膳食纤维品质(SDF/(SDF+IDF))的影响,由结果得知：酵母膏、MgSO4·7H2O和VB1与膳食纤维品质显著正相关。采用D-Optimal实验设计方法研究了上述三基质组分添加量对膳食纤维品质的影响,结果表明：随酵母膏、MgSO4·7H2O和VB1添加量的增大,膳食纤维品质先上升后下降,当三基质组分浓度分别为2.50g/L、1.10g/L和0.14g/L时,发酵后膳食纤维品质最好,SDF占总膳食纤维的22.3%。
研究了发酵条件对茶薪菇发酵后膳食纤维品质的影响,结果显示,装液量、pH值、接种量和发酵温度与发酵时间均显著影响膳食纤维品质,其中装液量对膳食纤维品质影响呈负效应；pH值和接种量对膳食纤维品质的影响趋势相似,随pH值和接种量的增大,膳食纤维品质先上升后下降最后变化迟缓；发酵温度的升高和发酵时间的延长使膳食纤维品质先上升后下降。茶薪菇最佳发酵条件为：发酵温度25℃、装液量60 mL、pH值5.5、接种量12%和发酵时间6 d,在该条件下获得的膳食纤维中含有24.83%的SDF。
比较了培养基及发酵条件对茶薪菇产酶的影响,结果表明在优化的培养基及发酵条件下,茶薪菇生成淀粉酶、蛋白酶和纤维素cl酶的活性均显著高于其他培养基及发酵条件,在发酵4 d,淀粉酶分别提高了30%和20%,蛋白酶提高了20%和27%,纤维素cl酶提高了45%。
3、考察了碳源、氮源和金属离子及表面活性剂种类对茶薪菇生成FOs和木聚糖酶的影响,由结果得知：在麦麸培养基中添加10g/L的葡萄糖、10g/L的乳糖和1g/L的蛋白胨,可促进茶薪菇木聚糖酶的合成,超出上述浓度则抑制荼薪菇木聚糖酶的合成。在设定范围内,FOs产量随葡萄糖和乳糖添加量的增加而增大,采用Taguchi OA实验,以木聚糖酶和FOs产量为指标,经Design Expert软件分析得,荼薪菇发酵制备FOs的最佳培养基组成为：在50g/L的麦麸处理液中添加15g/L的葡萄糖和乳糖及1g/L的蛋白胨,该培养基组成下,FOs产量和木聚糖酶活达最大值,分别为0.335mM和1.27 U/mL。
发酵条件中摇瓶装液量、发酵时间对茶薪菇生成木聚糖酶影响显著,随装液量增加,木聚糖酶活性先上升后下降,发酵时间的延长使木聚糖酶活性先升高后趋于平缓。摇瓶装液量对FOs产量影响呈负相关,FOs产量随pH值升高、接种量增大和发酵时间的延长先增加后减少。经Design Expert软件分析确定茶薪菇发酵制备FOs的最佳发酵条件为：摇瓶装液量90 mL、接种量12%、pH5.5、发酵时间6 d,在该条件下,FOs产量为0.428 mM。麦麸纤维结构观察结果显示,发酵后麦麸纤维纵向及横向结构断裂,表层出现褶皱,出现溶解现象,部分结构脱离纤维主体。
4、采用乙醇沉淀、Amberlite XAD-2树脂吸附和葡聚糖LH-20分离纯化FOs,并对其结构进行解析,结果表明：乙醇浓度大于50%时,可析出FOs,60-90%乙醇析出的FOs对乳酸菌增值效果最佳；再经Amberlite XAD-2树脂吸附和葡聚糖LH-20分离得FOs-1、FOs-2和FOs-3三组分,单糖组成及结构分析表明FOs由木糖、阿拉伯糖和阿魏酸组成,且三组分为不同聚合度的FOs,其中组分FOS-1聚合度为3-6,FOs-2中含有聚合度为2-3的阿魏酰低聚木糖和阿魏酰阿拉伯糖基木糖,FOs-3组分中含有阿魏酰阿拉伯糖和阿魏酰阿拉伯糖基木三糖。
5、茶薪菇发酵后IDF的持水力、溶胀性和持油力分别比发酵前提高8.85%、23.13%和15.21%,吸附胆酸钠、脱氧胆酸钠和牛磺胆酸钠的能力均比发酵前提高10%以上。
SDF和FOs对DPPH自由基抑制率均大于60%,对羟自由基抑制率均达80%以上；FOs总还原能力高于SDF。可见,SDF和FOs均具有较强的抗氧化能力。
SDF和FOs对乳酸菌具有显著增值作用,其中以FOs-2组分和发酵上清液中提取的SDF效果最好。FOs-2组分可抑制大肠杆菌和金黄色葡萄球菌的生长,发酵上清液中提取的SDF可抑制大肠杆菌的生长。
采用MTT法检验了SDF和FOs对人结肠癌细胞株HCT-116的抗肿瘤活性,结果表明,两种物质对肿瘤细胞均有抑制作用,且FOs的抑制效果优于SDF,并以FOs-1组分效果最好。当其浓度为1 mg/mL时,抑制率可达54.7%。发酵法制得的SDF对肿瘤细胞的抑制效果显著高于麦麸原样中提取的SDF,浓度为1 mg/mL时,抑制率可达45%。对给药后的肿瘤细胞进行可见光形态学观察后发现,结肠癌细胞株给药后出现贴壁减少、细胞变圆、浮起等现象。
Wheat bran is the by-product of wheat milling, which is composed of episperm, peel and aleurone. And it contains some starch, protein, cellulose and hemicellulose. In addition, there are 0.4～0.7 g ferulic acids in 100 g wheat bran, importantly, these acids mostly bound in bran in the form of covalent linkage with cell wall polysaccharides. Therefore, wheat bran is good material for preparation of dietary fiber (DF) and feruloyl oligosaccharides (FOs).
In this study, wheat bran was used as the material, DF and FOs was obtained by submerged fermentation of edible mushrooms. The comparison of different mushrooms' growth rates, biosythensis of enzymes and the production of DF and FOs from wheat bran were carried out, and then the optimal strain for fermentation was screened out. Effects of culture medium and fermentation conditions on yields of DF and FOs, and on biosythensis of xylanase were investigated. The mechanism of DF and FOs preparation by fermentation was revealed by detecting the enzymes secreted by mushroom in different culture conditions. FOs were puried by precipitating of ethanol, absorption of resin and chromatography of column. The compositions and structures of FOs were analyzed through HPLC, GC, IR and MS. Lastly, the biological activities of DF and FOs were studied, their antioxidant and antitumor and effects on the growth of intestinal microorganisms were compared. The main results and findings were shown as follows:
1. Agrocybe chaxingu, the common edible mushroom, was suitable for the preparation of DF and FOs. The yield of DF reached 68.49% after fermentation,of which, SDF was 19.24%. The production of FOs by fermentation was the highest yield among these edible mushrooms, and it was 35.4μM.
The growth rates of mushrooms in medium of wheat bran and PDA were determined, and results showed that all of the mushrooms grew well in these medium, and the growth rate in wheat bran was higher than that in PDA medium. Hericium erinaceus and Pleurotus ostreatus were the best strains, which grew well in wheat bran medium. Enzymes detection indicated that mushrooms used in our study could secrete amylase, protease, cellulase and xylanase. Hericium erinaceus also could secrete ferulic acid esterase, which was the first reported to our knowledge. The activities of these enzymes were different from the species of the mushrooms. Agrocybe chaxingu was the only strain, which could secrete higher amylase, protease, cellulase and xylanase during fermentation. In the end, the correlation of DF and FOs production with activities of enzymes was analyzed, and results proved that correlation between DF yield and cellulase activity was negative effect (r=-0.711), and xylanase activity positively influenced FOs production (r=0.912).
2. Effects of nitrogen source and inorganic salt on the quality of DF were investigated. Results showed that yeast poast, MgSO4·7H2O and VB1 influenced DF quality positively. And then the optimal additions of the three substances were optimized by D-Optimal design, and results were analyzed by Design Expert. The optimal fermentation medium was determined:under the condition of 2.50 g/L yeast posat,1.10 g/L MgSO4·7H2O and 0.14 g/L VB1 in wheat bran of 50 g/L, the best quality of DF, which contains 22.3% SDF in total DF, was obtained.
The culture conditions of fermentation temperature, volume capacity, pH and inoculum quantity and fermentation time influenced DF quality effectively. The effect of volume capacity on DF quality was linearity. DF quality decreased with the increase of volume capacity, and the optimal value was 60 mL/250 mL. The influence of inoculum quantity on DF quality was similar with the effect of pH on DF, both curves fitted well with Guass equation. By analyzing the experiments' results, the optimal culture condition was obtained. Under the condition of fermentation temperature 25℃, volume capacity 60 mL/250 mL, pH value 5.5, inoculum quantity 12% and fermentation time 6 d, the best quality DF was obtained, and it contained 24.83% SDF.
Effects of culture conditions on the secretion of enzymes from Agrocybe chaxingu during fermentation were investigated, results indicated that activities of amylase, protease and cellulase were increased under the optimal medium and condition. Among them, amylase and protease improved by 30% and 20% than the based medium, respectively, and cellulase of C1 got up to 145% of the initial activity.
3. Effects of medium components on the bio-synthesis of xylanase and FOs yield were studied, and results showed that carbon and nitrogen source could regulated the yields of xylanase and FOs secreted by Agrocybe chaxingu. Glucose, lactose and peptone improved the bio-synthesis of xylanase, and their additions influenced the enzyme activity effectively. The best additions of glucose, lactose and peptone were 15 g/L,15 g/L and 1 g/L, respectively. Under this medium, the yield of 0.335 mM FOs and 1.27 U/mL xylanase was produced.
Volume capacity and fermentation time influenced the bio-synthesis of xylanase significantly; and the activity of xylanase first improved and then decreased with the increase of volume capacity. With the prolonging of fermentation time, the biosynthesis of xylanase first increased and then unchanged. The influence of culture condition on FOs production was not as same as the effect on bio-synthesis of xylanase, especially, the effect of volume capacity. For FOs, the yield was decreased with the increase of volume capacity. FOs yield first improved and then decreased with the raise of pH, increase of inoculum volume and prolonging of fermentation time. Under the conditions of volume capacity 90 mL, inoculum quantity 12% and pH 5.5, and feremtnation time 6 d, the highest yields of FOs (0.428 mM) and xylanase (1.36 U/mL) were obtained.
The structure of wheat bran fiber before and after fermentation by Agrocybe chaxingu was detected by scanning electron microscopy. Results indicated that the fiber structure changed significantly by fermentation. Originally, the fiber combined tightly with smooth surface, while, they were not smooth and broke dnown after fermentation.
4. The FOs were isolated and purified by precipitation of ethnol, absorption of resin Amberlite XAD-2 and Sephadex LH-20. Results showed that concentration of ethnol influenced FOs precipitation significantly. FOs started to precipitate when ethnol concentration was higher than 50%. The precipitation of 60～90% ethnol increased the growth of lactic acid higher than the precipitations under other concentrations. And then, the precipitation of 60～90% was purified by Amberlite XAD-2 and Sephadex LH-20, and three components of FOs were obtained. The analysis of FOs'structure showed that they were composed of xyln, arabinose and ferulic acid, and the degree of FOs-1 was 3 to 6, FOs-2 degree was 1 to 3 and FOs-3 was 0 and 3.
5. The physical characteristics of IDF were improved by fermentation. The water hoding power, swelling ability and oil hoding power of IDF increased by 8.85%,23.13% and 15.21%, respectively. The adoption of bile salt was also improved by fermentation.
DPPH radical-scavenging activities of FOs and SDFs were higher than 60%. The hydroxyl radical scavenging activity of SDF from wheat bran was the highest among FOs and other SDF samples, the inhibition rate was 89%, followed by FOs and SDF-Out. The FOs have the highest antioxidant capacity among SDFs and FOs, followed by SDF-In and SDF-Ou, while the lowest was SDF-0. In a word, both SDFs and FOs showed high antioxidant ability.
FOs and SDFs could increase the growth of lactic bacteria and influence the growth of E. coli and Staph. aureu. FOs-2 was the best among them, which only increased the growth of lactic bacteri, but also inhibited the growth of E. coli and Staph aureu. SDF-Out could increase the growth of lactic bacteri and inhibit the growth of E.coli, there was no function on Staph. aureu.
MTT assay was used to analyses the effects of FOs and SDFs on human colon cancer cell of HCT-116. Results indicated that both FOs and SDFs could inhibit the growth of HCT-116, and the inhibition of FOs was higher than SDFs, especially FOs-1. The inhibition of HCT-116 reached 54.7% when the concentration of FOs-1 was 1 mg/mL. SDFs obtained by fermentation inhibited HCT-116 higher than that from wheat bran, and the inhibition was 45% when their concentrations were 1 mg/mL. Morphological observation of HCT-116 cell after treated by FOs-1 showed that cellular shrinkage to round, and cell floated, therefore, the cell closing to wall decreased.
本研究以麦麸为原料,采用食药用真菌发酵法制备膳食纤维和FOs。通过比较食药用真菌在麦麸培养基中的生长特性、产酶情况及生成膳食纤维和FOs产量,筛选得发酵菌株；研究培养基成分及发酵条件对发酵菌株生成膳食纤维、FOs和胞外酶的影响,确定产酶诱导物及最佳发酵条件；比较不同发酵条件下菌株产酶情况,揭示发酵法制备膳食纤维和FOs的机理；通过乙醇沉淀和大孔树脂吸附及柱层析法对FOs分离纯化,采用气相色谱、液相色谱、红外和质谱等方法鉴定FOs的组成及结构；比较发酵前后不溶性膳食纤维(Insoluble dirtary fibre, IDF)的物理特性,不同来源的可溶性膳食纤维(Soluble dietary fibre, SDF)和不同结构的FOs抗氧化、抗肿瘤及其对肠道微生物作用。主要研究结果如下：
1、比较了平菇、灵芝、荼薪菇等食药用真菌在麦麸培养基中生长、产酶及生成膳食纤维和FOs的情况。结果发现,茶薪菇适于发酵法制备膳食纤维和FOs,经该菌发酵后膳食纤维含量达68.49%,其中含有19.24%的SDF, FOs产量为35.4μM。
比较了各菌株在麦麸培养基和PDA培养基中的生长速度,结果表明：食药用真菌在麦麸培养基中的生长情况优于PDA培养基,其中以猴头菇和平菇最明显。测定各菌株在麦麸培养基中的产酶情况可知,各菌均能生成淀粉酶、蛋白酶、纤维素C1酶和木聚糖酶等,其中猴头菇亦可生成阿魏酸酯酶；酶活测定结果显示发酵过程中茶薪菇生成淀粉酶、蛋白酶和纤维素C1酶和木聚糖酶活性高于其他菌株。膳食纤维和FOs产量与纤维素酶和木聚糖酶的相关性分析表明,膳食纤维产量与β-葡萄糖苷酶活性负相关(r=-0.71),FOs产量与木聚糖酶活性正相关(r=0.912)。
2、比较了氮源和无机盐种类对茶薪菇发酵后膳食纤维品质(SDF/(SDF+IDF))的影响,由结果得知：酵母膏、MgSO4·7H2O和VB1与膳食纤维品质显著正相关。采用D-Optimal实验设计方法研究了上述三基质组分添加量对膳食纤维品质的影响,结果表明：随酵母膏、MgSO4·7H2O和VB1添加量的增大,膳食纤维品质先上升后下降,当三基质组分浓度分别为2.50g/L、1.10g/L和0.14g/L时,发酵后膳食纤维品质最好,SDF占总膳食纤维的22.3%。
研究了发酵条件对茶薪菇发酵后膳食纤维品质的影响,结果显示,装液量、pH值、接种量和发酵温度与发酵时间均显著影响膳食纤维品质,其中装液量对膳食纤维品质影响呈负效应；pH值和接种量对膳食纤维品质的影响趋势相似,随pH值和接种量的增大,膳食纤维品质先上升后下降最后变化迟缓；发酵温度的升高和发酵时间的延长使膳食纤维品质先上升后下降。茶薪菇最佳发酵条件为：发酵温度25℃、装液量60 mL、pH值5.5、接种量12%和发酵时间6 d,在该条件下获得的膳食纤维中含有24.83%的SDF。
比较了培养基及发酵条件对茶薪菇产酶的影响,结果表明在优化的培养基及发酵条件下,茶薪菇生成淀粉酶、蛋白酶和纤维素cl酶的活性均显著高于其他培养基及发酵条件,在发酵4 d,淀粉酶分别提高了30%和20%,蛋白酶提高了20%和27%,纤维素cl酶提高了45%。
3、考察了碳源、氮源和金属离子及表面活性剂种类对茶薪菇生成FOs和木聚糖酶的影响,由结果得知：在麦麸培养基中添加10g/L的葡萄糖、10g/L的乳糖和1g/L的蛋白胨,可促进茶薪菇木聚糖酶的合成,超出上述浓度则抑制荼薪菇木聚糖酶的合成。在设定范围内,FOs产量随葡萄糖和乳糖添加量的增加而增大,采用Taguchi OA实验,以木聚糖酶和FOs产量为指标,经Design Expert软件分析得,荼薪菇发酵制备FOs的最佳培养基组成为：在50g/L的麦麸处理液中添加15g/L的葡萄糖和乳糖及1g/L的蛋白胨,该培养基组成下,FOs产量和木聚糖酶活达最大值,分别为0.335mM和1.27 U/mL。
发酵条件中摇瓶装液量、发酵时间对茶薪菇生成木聚糖酶影响显著,随装液量增加,木聚糖酶活性先上升后下降,发酵时间的延长使木聚糖酶活性先升高后趋于平缓。摇瓶装液量对FOs产量影响呈负相关,FOs产量随pH值升高、接种量增大和发酵时间的延长先增加后减少。经Design Expert软件分析确定茶薪菇发酵制备FOs的最佳发酵条件为：摇瓶装液量90 mL、接种量12%、pH5.5、发酵时间6 d,在该条件下,FOs产量为0.428 mM。麦麸纤维结构观察结果显示,发酵后麦麸纤维纵向及横向结构断裂,表层出现褶皱,出现溶解现象,部分结构脱离纤维主体。
4、采用乙醇沉淀、Amberlite XAD-2树脂吸附和葡聚糖LH-20分离纯化FOs,并对其结构进行解析,结果表明：乙醇浓度大于50%时,可析出FOs,60-90%乙醇析出的FOs对乳酸菌增值效果最佳；再经Amberlite XAD-2树脂吸附和葡聚糖LH-20分离得FOs-1、FOs-2和FOs-3三组分,单糖组成及结构分析表明FOs由木糖、阿拉伯糖和阿魏酸组成,且三组分为不同聚合度的FOs,其中组分FOS-1聚合度为3-6,FOs-2中含有聚合度为2-3的阿魏酰低聚木糖和阿魏酰阿拉伯糖基木糖,FOs-3组分中含有阿魏酰阿拉伯糖和阿魏酰阿拉伯糖基木三糖。
5、茶薪菇发酵后IDF的持水力、溶胀性和持油力分别比发酵前提高8.85%、23.13%和15.21%,吸附胆酸钠、脱氧胆酸钠和牛磺胆酸钠的能力均比发酵前提高10%以上。
SDF和FOs对DPPH自由基抑制率均大于60%,对羟自由基抑制率均达80%以上；FOs总还原能力高于SDF。可见,SDF和FOs均具有较强的抗氧化能力。
SDF和FOs对乳酸菌具有显著增值作用,其中以FOs-2组分和发酵上清液中提取的SDF效果最好。FOs-2组分可抑制大肠杆菌和金黄色葡萄球菌的生长,发酵上清液中提取的SDF可抑制大肠杆菌的生长。
采用MTT法检验了SDF和FOs对人结肠癌细胞株HCT-116的抗肿瘤活性,结果表明,两种物质对肿瘤细胞均有抑制作用,且FOs的抑制效果优于SDF,并以FOs-1组分效果最好。当其浓度为1 mg/mL时,抑制率可达54.7%。发酵法制得的SDF对肿瘤细胞的抑制效果显著高于麦麸原样中提取的SDF,浓度为1 mg/mL时,抑制率可达45%。对给药后的肿瘤细胞进行可见光形态学观察后发现,结肠癌细胞株给药后出现贴壁减少、细胞变圆、浮起等现象。
Wheat bran is the by-product of wheat milling, which is composed of episperm, peel and aleurone. And it contains some starch, protein, cellulose and hemicellulose. In addition, there are 0.4～0.7 g ferulic acids in 100 g wheat bran, importantly, these acids mostly bound in bran in the form of covalent linkage with cell wall polysaccharides. Therefore, wheat bran is good material for preparation of dietary fiber (DF) and feruloyl oligosaccharides (FOs).
In this study, wheat bran was used as the material, DF and FOs was obtained by submerged fermentation of edible mushrooms. The comparison of different mushrooms' growth rates, biosythensis of enzymes and the production of DF and FOs from wheat bran were carried out, and then the optimal strain for fermentation was screened out. Effects of culture medium and fermentation conditions on yields of DF and FOs, and on biosythensis of xylanase were investigated. The mechanism of DF and FOs preparation by fermentation was revealed by detecting the enzymes secreted by mushroom in different culture conditions. FOs were puried by precipitating of ethanol, absorption of resin and chromatography of column. The compositions and structures of FOs were analyzed through HPLC, GC, IR and MS. Lastly, the biological activities of DF and FOs were studied, their antioxidant and antitumor and effects on the growth of intestinal microorganisms were compared. The main results and findings were shown as follows:
1. Agrocybe chaxingu, the common edible mushroom, was suitable for the preparation of DF and FOs. The yield of DF reached 68.49% after fermentation,of which, SDF was 19.24%. The production of FOs by fermentation was the highest yield among these edible mushrooms, and it was 35.4μM.
The growth rates of mushrooms in medium of wheat bran and PDA were determined, and results showed that all of the mushrooms grew well in these medium, and the growth rate in wheat bran was higher than that in PDA medium. Hericium erinaceus and Pleurotus ostreatus were the best strains, which grew well in wheat bran medium. Enzymes detection indicated that mushrooms used in our study could secrete amylase, protease, cellulase and xylanase. Hericium erinaceus also could secrete ferulic acid esterase, which was the first reported to our knowledge. The activities of these enzymes were different from the species of the mushrooms. Agrocybe chaxingu was the only strain, which could secrete higher amylase, protease, cellulase and xylanase during fermentation. In the end, the correlation of DF and FOs production with activities of enzymes was analyzed, and results proved that correlation between DF yield and cellulase activity was negative effect (r=-0.711), and xylanase activity positively influenced FOs production (r=0.912).
2. Effects of nitrogen source and inorganic salt on the quality of DF were investigated. Results showed that yeast poast, MgSO4·7H2O and VB1 influenced DF quality positively. And then the optimal additions of the three substances were optimized by D-Optimal design, and results were analyzed by Design Expert. The optimal fermentation medium was determined:under the condition of 2.50 g/L yeast posat,1.10 g/L MgSO4·7H2O and 0.14 g/L VB1 in wheat bran of 50 g/L, the best quality of DF, which contains 22.3% SDF in total DF, was obtained.
The culture conditions of fermentation temperature, volume capacity, pH and inoculum quantity and fermentation time influenced DF quality effectively. The effect of volume capacity on DF quality was linearity. DF quality decreased with the increase of volume capacity, and the optimal value was 60 mL/250 mL. The influence of inoculum quantity on DF quality was similar with the effect of pH on DF, both curves fitted well with Guass equation. By analyzing the experiments' results, the optimal culture condition was obtained. Under the condition of fermentation temperature 25℃, volume capacity 60 mL/250 mL, pH value 5.5, inoculum quantity 12% and fermentation time 6 d, the best quality DF was obtained, and it contained 24.83% SDF.
Effects of culture conditions on the secretion of enzymes from Agrocybe chaxingu during fermentation were investigated, results indicated that activities of amylase, protease and cellulase were increased under the optimal medium and condition. Among them, amylase and protease improved by 30% and 20% than the based medium, respectively, and cellulase of C1 got up to 145% of the initial activity.
3. Effects of medium components on the bio-synthesis of xylanase and FOs yield were studied, and results showed that carbon and nitrogen source could regulated the yields of xylanase and FOs secreted by Agrocybe chaxingu. Glucose, lactose and peptone improved the bio-synthesis of xylanase, and their additions influenced the enzyme activity effectively. The best additions of glucose, lactose and peptone were 15 g/L,15 g/L and 1 g/L, respectively. Under this medium, the yield of 0.335 mM FOs and 1.27 U/mL xylanase was produced.
Volume capacity and fermentation time influenced the bio-synthesis of xylanase significantly; and the activity of xylanase first improved and then decreased with the increase of volume capacity. With the prolonging of fermentation time, the biosynthesis of xylanase first increased and then unchanged. The influence of culture condition on FOs production was not as same as the effect on bio-synthesis of xylanase, especially, the effect of volume capacity. For FOs, the yield was decreased with the increase of volume capacity. FOs yield first improved and then decreased with the raise of pH, increase of inoculum volume and prolonging of fermentation time. Under the conditions of volume capacity 90 mL, inoculum quantity 12% and pH 5.5, and feremtnation time 6 d, the highest yields of FOs (0.428 mM) and xylanase (1.36 U/mL) were obtained.
The structure of wheat bran fiber before and after fermentation by Agrocybe chaxingu was detected by scanning electron microscopy. Results indicated that the fiber structure changed significantly by fermentation. Originally, the fiber combined tightly with smooth surface, while, they were not smooth and broke dnown after fermentation.
4. The FOs were isolated and purified by precipitation of ethnol, absorption of resin Amberlite XAD-2 and Sephadex LH-20. Results showed that concentration of ethnol influenced FOs precipitation significantly. FOs started to precipitate when ethnol concentration was higher than 50%. The precipitation of 60～90% ethnol increased the growth of lactic acid higher than the precipitations under other concentrations. And then, the precipitation of 60～90% was purified by Amberlite XAD-2 and Sephadex LH-20, and three components of FOs were obtained. The analysis of FOs'structure showed that they were composed of xyln, arabinose and ferulic acid, and the degree of FOs-1 was 3 to 6, FOs-2 degree was 1 to 3 and FOs-3 was 0 and 3.
5. The physical characteristics of IDF were improved by fermentation. The water hoding power, swelling ability and oil hoding power of IDF increased by 8.85%,23.13% and 15.21%, respectively. The adoption of bile salt was also improved by fermentation.
DPPH radical-scavenging activities of FOs and SDFs were higher than 60%. The hydroxyl radical scavenging activity of SDF from wheat bran was the highest among FOs and other SDF samples, the inhibition rate was 89%, followed by FOs and SDF-Out. The FOs have the highest antioxidant capacity among SDFs and FOs, followed by SDF-In and SDF-Ou, while the lowest was SDF-0. In a word, both SDFs and FOs showed high antioxidant ability.
FOs and SDFs could increase the growth of lactic bacteria and influence the growth of E. coli and Staph. aureu. FOs-2 was the best among them, which only increased the growth of lactic bacteri, but also inhibited the growth of E. coli and Staph aureu. SDF-Out could increase the growth of lactic bacteri and inhibit the growth of E.coli, there was no function on Staph. aureu.
MTT assay was used to analyses the effects of FOs and SDFs on human colon cancer cell of HCT-116. Results indicated that both FOs and SDFs could inhibit the growth of HCT-116, and the inhibition of FOs was higher than SDFs, especially FOs-1. The inhibition of HCT-116 reached 54.7% when the concentration of FOs-1 was 1 mg/mL. SDFs obtained by fermentation inhibited HCT-116 higher than that from wheat bran, and the inhibition was 45% when their concentrations were 1 mg/mL. Morphological observation of HCT-116 cell after treated by FOs-1 showed that cellular shrinkage to round, and cell floated, therefore, the cell closing to wall decreased.
引文
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