A feeding trial was conducted for nine weeks to investigate the effects of partially replacing Ca(H2PO4)2 with neutral phytase on the growth performance, phosphorus utilization, nutrient digestibility, serum biochemical parameters, bone and carcass mineral composition, and digestive-enzyme-specific activity in crucian carp (Carassius auratus). The diets prepared with 0.8%, 0%, and 1.8% Ca(H2PO4)2 (1%=1 g/100 g) supplements were regarded as the P1E0, negative control (NC), and positive control (PC) groups, respectively; the other three experimental diets were prepared with the addition of 200, 300, and 500 U/kg of neutral phytase, respectively, based on the P1E0 group. Three hundred and eighty-four fish ((1.50±0.01) g) were randomly distributed in the six treatments with four replicates each. The fish were initially fed with 2%–3% diets of their body weight per day, with feeding twice daily (08:00 and 16:00), under a 12-h light/12-h dark cycle at the temperature of (27.56±0.89) °C. The results showed that supplemental phytase at different levels in the diet improved the final body weight, average daily gain, feed conversion ratio, phosphorus utilization, and protein efficiency ratio of crucian carp (P<0.05). Phytase supplementation increased the mineral content in serum (P), bone (P, Ca), and carcass (P, Ca, Zn, Na, and Mg) (P<0.05); the trypsin and chymotrypsin activity soared when fed with the phytase-supplemented diets (P<0.05). We may conclude that supplemental dietary neutral phytase improved the growth performance, phosphorus utilization as well as nutrient utilization in crucian carp, and it can be considered an important nutritional replacement for Ca(H2PO4)2.比较研究在无机磷 (磷酸二氢钙) 添加不足时, 添加不同剂量中性植酸酶对鲫鱼生长和磷利用的影响, 为水产养殖更好地应用植酸酶提供依据并减少磷的排放, 保护环境。试验采用的中性植酸酶能够耐受饲料加工温度, 并从营养物质消化及磷利用等方面研究中性植酸酶对鲫鱼的影响, 探究低磷日粮在水产养殖的应用。384 条鲫鱼 ((1.50±0.01) g) 随机分为 6 组, 分别饲喂对照组 (未添加中性植酸酶) 跟试验组 (添加不同剂量中性植酸酶) 日粮 63 天, 比较 3 组间各项指标差异。在以植物蛋白为主的日粮中, 添加中性植酸酶能够促进植酸磷水解, 提高鲫鱼对饲粮中磷、 粗蛋白等营养物质的利用, 促进钙、 磷等矿物质在鲫鱼体中的沉积, 降低粪磷排出量, 促进鲫鱼生长。说明可以在鲫鱼日粮中添加中性植酸酶, 减少无机磷的添加, 降低粪磷排放, 从而减轻环境污染。

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0–8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km (apparent Michaelis-Menten constant) and Vmax (maximal reaction velocity) for p-nitrophenyl-β-d-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The Km and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.分离纯化土曲霉来源的β-葡萄糖苷酶(At-Bgl), 探究其酶学特性及其对大豆异黄酮的水解效果。成功将At-Bgl 分离纯化出来, 并首次将分离纯化后的At-Bgl 应用于水解大豆异黄酮糖苷。利用超滤、透析、阴离子交换柱层析和聚丙烯酰氨凝胶电泳(SDS-PAGE)等手段分离纯化土曲霉来源的β-葡萄糖苷酶(表1), 并用解析电离串联飞行时间质谱技术(MALDI-TOF/TOF MS)鉴定蛋白条带。以对硝基苯基β-d-葡萄糖苷(pNPG)为底物进行酶学特性研究; 以pNPG和纤维二糖为底物, 进行酶动力学参数研究(表2); 以胃蛋白酶和胰蛋白酶模拟动物胃肠道酸性环境, 进行酸耐受性研究。通过高效液相色谱(HPLC)检测At-Bgl 对大豆异黄酮糖苷的水解效果(表3)。At-Bgl 属糖苷水解酶第三家族(GH3), 分子量约为120 kDa(图1), 最适酶解条件为pH 5.0 和65 °C, 具有良好的热稳定性和pH 稳定性(图2), 且胃蛋白酶和胰蛋白酶耐受性强(图3)。At-Bgl可将大豆异黄酮糖苷高效转化为异黄酮苷元(图4)。综上所述, At-Bgl 在增强动物胃肠道对大豆异黄酮的水解方面具有重要应用价值。

The growth inhibition and induction of apoptosis brought by amygdalin and activated with β-d-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC50 of stand-alone amygdalin and activation with β-d-glucosidase on the proliferation of HepG2 cells for 48 h were 458.10 mg/mL and 3.2 mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (p < 0.01). Together, these findings indicate that amygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with β-d-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity.Highlights► Amygdalin activated with β-d-glucosidase shows superior anti-HepG2 activity. ► Activated amygdalin could inhibit HepG2 cells proliferation and promote apoptosis. ► Injection for amygdalin is more reliable than oral administration. ► Our findings highlight the combination strategy with higher anti-tumors activity.

The study was conducted to investigate the effects of replacing antibiotics using multi-enzyme preparations on growth performance, coefficient of total tract apparent digestibility, digestive enzyme activity, and antioxidant property in piglets. A total of 160 piglets ((21.35±0.22) kg) were randomly assigned to five dietary treatments: 1) basal diet supplemented with antibiotics (AC), 2) antibiotic diet supplemented with 0.5 g kg−1 multi-enzyme preparations (AC+0.5EP), 3) antibiotic diet supplemented with 1.5 g kg−1 multi-enzyme preparations (AC+1.5EP), 4) basal diet supplemented with a half dosage of antibiotics and 1.5 g kg−1 multi-enzyme preparations (AH+1.5EP), and 5) basal diet supplemented with 1.5 g kg−1 multi-enzyme preparations (BC+1.5EP). The results showed that AC+1.5EP significantly improved the feed efficiency, apparent digestibility of ether extract (EE) and crude ash (CA), lipase activity in pancreas and duodenum content, maltase and lactase activity in jejunum and ileum mucosa, glutathione peroxidase (GSH-Px) concentration in serum and liver, and decreased malondialdehyde (MDA) concentration in serum and liver compared with piglets receiving AC (P<0.05). Piglets receiving BC+1.5EP showed no significant difference in growth performance (P>0.05) but had lower MDA concentration than piglets receiving AC (P<0.05). The apparent digestibility of EE and crude fiber (CF), duodenal lipase activity, jejunum mucosa maltase, and ileum mucosa lactase activity of piglets receiving AH+1.5EP or BC+1.5EP were significantly improved compared with piglets receiving AC (P<0.05). These results indicated an additive growth promotion effect between antibiotics and multi-enzyme preparations on piglets, and the multi-enzyme preparations may be used as substitutes for antibiotics for improving piglet production performance and health status.