黑曲霉液体发酵产植酸酶条件的研究

试验以黑曲霉(Aspergillusniger)30132为材料,研究了碳源、氮源、培养基、起始pH值、接种量、温度对该菌株产植酸酶活力的影响。结果表明:黑曲霉30132的最适发酵温度为28℃,产酶pH值为5,最佳接种量为10%;在此条件下,以8%麸皮为碳源、0.5%硫酸铵为氮源时,产酶活力最高,发酵3d后发酵液中植酸酶活力高达35.5U/ml。     

复合饲用酶菌株A3产果胶酶的研究

本试验对黑曲霉A3菌株产果胶酶的液体发酵条件进行了初步研究。不同的碳源、氮源对产果胶酶均有影响,试验结果表明,该菌株产果胶酶较适培养基为:碳源为麸皮70 g／L和可溶性淀粉10 g／L、氮源为(NH4)2SO410 g／L和NH4NO3 3．5 g／L;诱导物以果胶20 g／L为最佳;适宜产酶条件为培养基起始pH 3．0,培养温度为25℃,发酵 72 h达到产酶高峰。     

饲用β-葡聚糖酶的发酵工艺研究

通过对本实验室保藏的12株黑曲霉菌株采用培养、选择性培养基分离,筛选出一株相对酶活较高的菌株作为出发菌株,然后通过紫外线、硫酸二乙酯等复合诱变筛选出一株β-葡聚糖酶高产菌株An08-752。以复合碳源(麸皮+花生壳粉+大麦粉)为碳源;有机氮源为豆粕粉,无机氮源为硫酸铵、硝酸钠;添加无机盐磷酸氢二钾(K+)、硫酸镁(Mg2+);料水比为1:1。固态发酵优化条件为:初始pH值6.8;装料量50 g/500 ml;发酵温度30℃;发酵时间37 h。酶活力达到了1.23×105 U/g。     

MB22木聚糖酶发酵条件的研究

通过因素轮换试验和正交试验,对MB22菌产木聚糖酶的培养基配方和发酵条件进行了研究。结果显示最佳培养基组成是:以玉米芯粉和麸皮为碳源,麸皮120g/l,玉米芯280g/l,(NH4)2SO44g/l,CaCO31.5g/l,Tween80,2.0g/l,MgSO4·7H2O1.5g/l;最佳发酵条件为:起始pH5.0,摇床培养温度30℃,转速160r/min,振荡培养84h。在最佳发酵条件下,MB22菌的最高产酶活力可达898.23U/ml。虽然与一些高产菌株相比该菌株的产酶能力还有待于进一步提高,但该试验结果为进一步进行微生物生产木聚糖酶的研究提供了理论依据。     

响应面法优化里氏木霉固体发酵产α-半乳糖苷酶培养条件

本试验采用响应面法对里氏木霉(Trichoderma reesei)固体发酵产α-半乳糖苷酶的培养条件进行了优化。首先根据单因素实验设计Plackett-Burman试验筛选出影响产酶的3个主要因素:玉米芯与甘蔗渣的碳源配比、牛肉膏与硫酸铵的氮源配比和发酵初始pH。在此基础上运用最陡爬坡路径法逼近最大响应值区域,最后利用响应面分析法确定主要因子之间的交互作用及最佳培养条件。结果表明,里氏木霉固体发酵产α-半乳糖苷酶的优化培养条件为:碳源配比即玉米芯与甘蔗渣比值为3.86,氮源配比即牛肉膏与硫酸铵之比1.17,初始pH为9.12。优化后,里氏木霉固体发酵产α-半乳糖苷酶的酶活可达到342.98U/g,与模型预测值353.43U/g接近,比优化前的酶活提高113.91%。综上,影响里氏木霉固体发酵产α-半乳糖苷酶最重要因素分别为碳源、氮源及pH,三者之间相互影响,分别在碳源配比3.86、氮源配比1.17及初始pH 9.12时α-半乳糖苷酶酶活最大。     

产朊酵母菌利用烤鳗蒲烧废水产单细胞蛋白的研究

以产朊酵母菌(Candida utilis)为出发菌,利用烤鳗蒲烧废水生产单细胞蛋白(SCP)。试验考察了有关接种量、发酵时间、温度、酸碱度、氮源、磷源及无机盐金属离子等影响因素。结果表明:接种量15%、发酵时间为48h、pH值5.0、温度25℃、5.04g/l硝酸钾、40ml的装液量、0.1g/l磷酸氢二钠、0.1g/l硫酸锌为其最佳条件。     

响应面法优化里氏木霉固体发酵产α-半乳糖苷酶培养条件

本试验采用响应面法对里氏木霉（Trichoderma reesei）固体发酵产α-半乳糖苷酶的培养条件进行了优化。首先根据单因素实验设计Plackett-Burman试验筛选出影响产酶的3个主要因素：玉米芯与甘蔗渣的碳源配比、牛肉膏与硫酸铵的氮源配比和发酵初始pH。在此基础上运用最陡爬坡路径法逼近最大响应值区域,最后利用响应面分析法确定主要因子之间的交互作用及最佳培养条件。结果表明,里氏木霉固体发酵产α-半乳糖苷酶的优化培养条件为：碳源配比即玉米芯与甘蔗渣比值为3.86,氮源配比即牛肉膏与硫酸铵之比1.17,初始pH为9.12。优化后,里氏木霉固体发酵产α-半乳糖苷酶的酶活可达到342.98 U/g,与模型预测值353.43 U/g接近,比优化前的酶活提高113.91%。综上,影响里氏木霉固体发酵产α-半乳糖苷酶最重要因素分别为碳源、氮源及pH,三者之间相互影响,分别在碳源配比3.86、氮源配比1.17及初始pH 9.12时α-半乳糖苷酶酶活最大。     

Thermoascus aurantiacus产耐热木聚糖酶水解活性的培养条件优化

在前期研究的基础上,利用SPSS进行主成分分析,构建了催化水解玉米芯的综合水解能力指数(XCHI);然后以XCHI为自变量,水解玉米芯的还原糖得率为因变量进行回归分析,回归分析模型证明了综合水解能力XCHI与糖得率之间存在相关关系,以XCHI为响应值,正交实验优化得到T.aurantiacus CGMCC11334产Xyn水解玉米芯能力的最适培养基组成为:麸皮16 g/l、微晶纤维素7 g/l、牛肉浸膏4 g/l、玉米浆3 g/l、小麦蛋白胨4 g/l、KH_2PO_4 1.0 g/l、MgSO_4 2.0 g/l、CuSO_4 0.00 g/l、Fe-SO_4 0.05 g/l、VB1 0.15 g/l;在此条件下,Xyn、CMC和FPA的活性分别为135.12、1.95 U/ml和1.68 U/ml,XCHI为1 649.44。     

产β-葡聚糖酶黑曲霉固态孢子增殖工艺研究

试验旨在提高固态培养黑曲霉孢子增殖。以平板单菌落计数的方法计算单个孢子增殖孢子数目,通过单因素筛选及正交试验对黑曲霉产孢培养基以及培养条件进行优化研究。确定最优产孢培养基配方为:葡萄糖40 g/l、(NH_4)_2SO_4 3.0 g/l、KH_2PO_4 1.5 g/l、Mg SO_4 1.0 g/l、Mn Cl_2 3.0 mg/l、Zn SO_4 15.0 mg/l;最佳培养条件为:培养基体积150 ml/500 ml,接种量10~3个孢子/瓶。在此优化条件下,黑曲霉单孢子产孢量达7.38×10~6个,是未优化条件下的1.8倍。     

屎肠球菌CCTCCM2017191发酵培养基的优化

为了实现屎肠球菌CCTCCM2017191的高密度工业化生产,必须提高该屎肠球菌的发酵活菌量,因此对其发酵培养基进行了优化筛选。首先在MRS培养基的基础上进行单因素试验以确定适合该屎肠球菌生长的最优pH值和培养温度以及该菌的最适发酵碳源和氮源;再利用Plackett-Bur-man试验设计分析筛选培养基成分中影响屎肠球菌发酵活菌数的显著影响因子,设计最陡爬坡试验逼近显著因子的最大活菌数响应区域,最后采用中心复合序贯试验设计(central composite circum-scribed design,CCC)和响应面分析法得到显著影响因子的最优配比浓度。获得发酵优化培养基配方为:麦芽糊精20 g/l、酵母浸粉77.22 g/l、结晶乙酸钠6.42 g/l、柠檬酸二铵2.0 g/l、磷酸氢二钾2.0 g/l、无水硫酸镁0.2 g/l、硫酸锰0.04 g/l、吐温-80 0.99 g/l。培养基pH值为8.0,培养温度为37℃。经验证,优化后培养基的发酵菌液活菌数可达6.136×109CFU/ml是相同条件下MRS培养基发酵菌液活菌数的4.63倍,优化方案及所得回归模型切实可靠,成功实现了该屎肠球菌CCTCCM2017191的高密度发酵培养。     

固态混菌发酵生产饲用复合酶制剂营养条件和培养条件的研究

黑曲霉变种(A.niger v.Tiegh)CGMCC1182、黑曲霉MA-56(A.niger MA-56)CGMCC2722和黑曲霉XY-1(A.niger XY-1)CGMCC1182分别为α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶生产菌株。为获得高产α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶的复合酶制剂,通过单因素实验,研究了黑曲霉三种菌株在固态发酵条件下产复合酶制剂的培养基组成和培养条件。结果表明,黑曲霉混菌发酵生产复合酶的最适培养基组成为:麸皮∶豆粕为7∶3(m/m),在此基础上(以麸皮和豆粕总量为10 g计算)添加玉米芯1.0 g,魔芋粉0.1 g,葡萄糖0.5 g,(NH4)2SO4 0.2 g,NaNO3 0.1 g,MgSO4 0.1 g,KH2PO4 0.2 g,H2O 11 mL。产酶最适培养条件为:培养温度30℃,固形物与加水比1∶1,α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶接种比例为5∶6∶6,接种混合孢子悬浮液2.5 mL(以一支菌种斜面加30 mL无菌水为标准),300 mL三角瓶中装量8 g培养基,发酵60 h时,复合酶产量达到最优,α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶三种酶制剂的活力分别可以达到221、894、10188 IU/g。     

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation (SSF) as a potential feed additive.The results of the first trial showed that there were at least 5 non-starch polysaccharide enzymes:xylanase,β-glucanase,pectinase,mannase and carboxy methyl cellulase (CMCase) contained in the compound.Xylanase and β-glucanase showed good activities at pH 2.5-7.0,which were in the range of 649-1046 U/g and 444-648 U/g,respectively.Pectinase showed good activity in acidic solution (pH 2.5-3.0),which ranged from 195 to 917 U/g.Mannase showed high activity of 235-298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0,which was in the range of 38.2-78.6 U/g.The second trial was aimed to test the stability of the enzymes in gastric liquor (pH 2.6) of finishing pigs and Na2HPO4-gastric liquor (pH 5.5).After 6 h incubation at 40℃ in gastric liquor,the retained activity of xylanase,β-glucanase,pectinase,mannase and CMCase was 26.3%,65.0%,71.0%,74.8% and 85.6%,respectively.While after 6 h incubation at 40℃ in Na2HPO4-gastric liquor,the retained activity of xylanase,β-glucanase,pectinase,mannase and CMCase was 87.9%,91.1%,92.3%,95.0%,and 97.5%,respectively.The third trial was carried out in a jejunum liquor (pH 5.8,200 mL),which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran,respectively.After 8 h incubation at 40℃,18.7% of soybean hull and 20.1% of wheat bran could be degraded to soluble saccharide,respectively.Compared with the traditional methods for feed enzyme testing which involve feeding animals for 1-3 months,enzyme assay in this way was relatively convenient.     

Exogenous phytase and xylanase exhibit opposing effects on real-time gizzard pH in broiler chickens

ABSTRACT

1. The current study was conducted to evaluate the influence of high phytase doses and xylanase, individually and in combination, on performance, blood inositol and real-time gastric pH in broilers fed wheat-based diets.

2. In a 42-d experiment, a total of 576 male Ross 308 broiler chicks were allocated to 4 dietary treatments. Treatments consisted of a 2 × 2 factorial arrangement, with 500 or 2500 FTU/kg phytase and 0 or 16 000 BXU/kg xylanase, fed in two phases (starter 0–21; grower 21–42 d). Heidelberg pH capsules were administered to 8 birds from each treatment group, pre- and post-diet phase change, with readings captured over a 5.5-h period.

3. At 21 and 42 d, birds fed 500 FTU/kg phytase without xylanase had on average 127 and 223 g lower weight gain than all other treatments, respectively (P < 0.05). At 42 d, body weight-corrected feed conversion ratio (_bwcFCR) was reduced (P < 0.05) by supplementing 2500 FTU/kg phytase or xylanase, with the combination giving a 12 point reduction in _bwcFCR compared to birds fed 500 FTU/kg phytase without xylanase. Inositol content of plasma was twice that of the erythrocyte (P < 0.001), with 2500 FTU/kg phytase tending to increase (P = 0.07) inositol content in both blood fractions.

4. Across all treatments, capsule readings ranged from pH 0.54 to 4.84 in the gizzard of broilers. Addition of 2500 FTU/kg phytase to the grower diet reduced (P < 0.05) average gizzard pH from 2.89 to 1.69, whilst feeding xylanase increased (P < 0.001) gizzard pH from 2.04 to 2.40. In contrast, digital probe measurements showed no effect of xylanase on gizzard pH, while addition of 2500 FTU/kg phytase increased (P = 0.05) pH compared to 500 FTU/kg phytase with or without xylanase.

5. These findings suggested that xylanase and high phytase doses have opposite effects on real-time gastric pH, while similarly improving performance of broilers.     

Effect of exogenous xylanase on rumen in vitro gas production and degradability of wheat straw

The objective of this study was to determine effects of xylanase on in vitro gas production (GP) and in sacco degradability of wheat straw. Rumen fluid was obtained from three Mongolian native goats fitted with permanent rumen cannulas. The trial consisted of five doses (0, 0.5, 1.0, 1.5, 2.0 μL/g of substrate) of a commercial xylanase (Dyadic^® xylanase PLUS, Dyadic International, Inc., Jupiter, FL, USA). For the in sacco degradability, different levels of xylanase enzyme were added directly onto 2 g of wheat straw in nylon bags and incubated in the rumen for 3, 6, 12, 24 and 48 h to estimate degradability of wheat straw. Total GP increased (P < 0.001) at all times of incubation at intermediate levels of xylanase. Methane production had a similar pattern at 3 and 12 h of incubation; increased linearly at 24 h of incubation, and was unaffected at 6 and 48 h of incubation. Rumen NH₃‐N concentration increased linearly at 3 h and the highest values were observed with intermediate enzyme levels. All ruminal volatile fatty acids increased linearly with intermediate levels of the fibrolytic enzyme. The in sacco rate of dry matter degradation decreased linearly (P = 0.020) with increasing enzymes. Intermediate levels of xylanase improved rumen kinetic fermentation and degradability. The outcome of this research indicated that the application of xylanase enzyme could improve in vitro GP fermentation of wheat straw.     

饲用木聚糖酶活力的综合评价

试验采用综合分析方法评价了1种饲用木聚糖酶的活力。评价内容包括耐酸性试验、耐胃液试验、热稳定性试验和实际底物降解试验。在pH5.4～6.8条件下,该酶活力较稳定,保持在5000U/g以上,但随着pH的降低,活力迅速下降;在胃液中作用一段时间后该酶的活力损失很快,1.5h后其活力就下降到原有的37.4%;热稳定性试验中,该酶在90℃的环境中作用5min后,酶活损失不超过5%;实际底物降解试验中,反应4h后,无淀粉麦麸的降解率可以达到5%以上。试验结果表明,以上几个指标能够较为充分、准确的评价该木聚糖酶的可应用性,从而为饲料酶活性的系统评价提供了参考,为饲料加工和动物生产提供了更为可靠的数据。     

变温操作对黑曲霉A-25产木聚糖酶的影响

试验研究不同温度对黑曲霉生物量和木聚糖酶产量的影响,并通过单位质量菌丝的产酶量和酶谱探究木聚糖酶产量提高的原因。结果显示:黑曲霉A-25在33℃培养时获得最大的生物量,而在29℃时获得最高胞外木聚糖酶分泌量。因此,变温操作采用0~48 h在33℃下培养、之后降为29℃,木聚糖酶活力可达721 U/mL,比29℃恒温发酵提高18%,同时发酵周期缩短8 h。进一步研究发现,0~48 h阶段内变温发酵条件下黑曲霉菌丝体生物量明显增加,并且单位质量菌丝体的产酶能力也有不同程度的提高;木聚糖酶的酶谱检测发现,变温发酵促进木聚糖酶XynⅢ的提前表达,同时提高木聚糖酶XynⅠ和木聚糖酶XynⅡ的表达量。     

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation (SSF) as a potential feed additive. The results of the first trial showed that there were at least 5 non-starch polysaccharide enzymes: xylanase, b-glucanase, pectinase, mannase and carboxy methyl cellulase (CMCase) contained in the compound. Xylanase and b-glucanase showed good activities at pH 2.5-7.0, which were in the range of 649-1046 U/g and 444-648 U/g, respectively. Pectinase showed good activity in acidic solution (pH 2.5-3.0), which ranged from 195 to 917 U/g. Mannase showed high activity of 235- 298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0, which was in the range of 38.2-78.6 U/g. The second trial was aimed to test the stability of the enzymes in gastric liquor (pH 2.6) of finishing pigs and Na₂HPO₄-gastric liquor (pH 5.5). After 6 h incubation at 40°C in gastric liquor, the retained activity of xylanase, b-glucanase, pectinase, mannase and CMCase was 26.3%, 65.0%, 71.0%, 74.8% and 85.6%, respectively. While after 6 h incubation at 40°C in Na₂HPO₄-gastric liquor, the retained activity of xylanase, b-glucanase, pectinase, mannase and CMCase was 87.9%, 91.1%, 92.3%, 95.0%, and 97.5%, respectively. The third trial was carried out in a jejunum liquor (pH 5.8, 200mL), which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran, respectively. After 8 h incubation at 40°C, 18.7% of soybean hull and 20.1% of wheat bran could be degraded to soluble saccharide, respectively. Compared with the traditional methods for feed enzyme testing which involve feeding animals for 1-3 months, enzyme assay in this way was relatively convenient.     

耐热木聚糖酶水解法制备木寡糖的研究

为探讨耐热木聚糖酶水解自制甘蔗渣木聚糖制备木寡糖的工艺条件和分析水解液的寡糖成分,试验以产耐热木聚糖酶的基因工程菌1020为原料,水解自制的甘蔗渣木聚糖,分别研究水解过程中酶的用量、木聚糖的浓度及酶解温度对酶解反应的影响,并采用高压液相色谱和质谱联用技术对水解液成分进行分析。试验结果表明,2%的木聚糖浓度,水解温度95℃,酶液用量100U/g,酶解3h后DP值可降至2.94,满足生产木寡糖对聚合度的要求;并且水解液的成分主要为木糖、木二糖和木三糖,没能检测到木四糖。耐热木聚糖酶在高温下酶解木聚糖对酶法制备木寡糖具有重要的应用价值。     

木聚糖酶对小麦中非淀粉多糖体外酶解效果的影响研究

为了探究木聚糖酶对小麦非淀粉多糖的降解效果。试验采用单因子设计,用气相色谱法测定不同木聚糖酶水平,在一定水分(料水比为1:10)、温度(40℃)和pH值(6.0)条件下酶解2 h,小麦非淀粉多糖含量变化。在酶水平为每克小麦60 000 IU时,降解效果最大;以可溶性非淀粉多糖、不可溶性非淀粉多糖、总非淀粉多糖含量变化为指标,每千克小麦的适宜木聚糖酶添加量分别为0.718 g、0.732 g、0.717 g。添加木聚糖酶对小麦非淀粉多糖有显著的降解效果。