An enhanced preparation and purification of the major antioxidants baicalein and wogonin from Scutellariae radix

A simple, rapid and improved method was developed to isolate high-purity baicalein and wogonin from Scutellariae radix. The method involves endogenous baicalinase-catalysed hydrolysis (EBCH), partition, automated low-pressure preparative chromatography (LPPC) and recrystallisation without multiple and tedious column chromatography. This process was optimised for large scale production of baicalein and wogonin with high yields, low costs and process automation. The transformation ratio of baicalin and wogonoside reached 98.21% and 96.60% after EBCH, leading to an increase of 5.41-fold in baicalein and 3.89-fold in wogonin, compared to a raw sample without hydrolysis. The purity of final products was more than 98% after one-step LPPC and recrystallisation. The experimental results show that EBCH–LPPC is an effective method for preparing high purity antioxidants.     

基于一测多评法对小儿解感颗粒中5个成分的质量控制

目的建立基于一测多评法(QAMS)测定小儿解感颗粒中黄芩苷、汉黄芩苷、黄芩素、汉黄芩素、甘草苷5个成分的含量的方法。方法采用高效液相色谱(HPLC),色谱柱为Waters SunFire C18柱(4.6 mm×250 mm,5μm);流动相为乙腈(A)-0.1%磷酸水溶液(B),梯度洗脱;流速1.0 ml/min;柱温30℃;检测波长274 nm。以黄芩苷为内参物,建立汉黄芩苷、黄芩素、汉黄芩素、甘草苷与内参物黄芩苷的相对校正因子(f),通过f计算小儿解感颗粒中5个成分的含量,并进行验证,将该方法与外标法的测定结果进行对比分析,以验证QAMS方法的准确性、重复性及可行性。结果黄芩苷、汉黄芩苷、黄芩素、汉黄芩素、甘草苷质量浓度分别在0.102~407.492μg/ml、0.067~84.147μg/ml、0.154~60.197μg/ml、0.054~50.634μg/ml、0.272~237.796μg/ml范围内线性关系良好;加样回收率分别为99.16%,100.54%,98.02%,97.84%,98.78%;以黄芩苷为内参物,测得的汉黄芩苷、甘草苷、黄芩素、汉黄芩素f值分别为0.823,0.674,0.549,1.873,且建立的QAMS法结果与外标法的结果一致。结论本实验建立的QAMS法可作为一种简便快捷的质量评价模式,用于小儿解感颗粒中5种成分的质量控制。     

Enzymatic hydrolysis of potato starches containing different amounts of phosphorus

The rapid hydrolysis of potato starches differing in phosphorus content, as well as sweet potato, cassava and yam starches, was accomplished by treatment of gelatinised starches with bacterial liquefying α-amylase at 50 °C for 1 h, followed by Bacillus licheniformis α-amylase at 55 °C up to 24 h, and then by glucoamylase at 40 °C for a further 24 h. Among the potato starches, the high-phosphorus starches showed higher starch resistant capacity than the medium-phosphorus starches, as well as other tuber and root starches. The hydrolysis rate of tuber and root starches was not greatly influenced by their amylose content and median granule size. Only glucose was detected in the almost completely hydrolysed tuber and root starch samples, indicating that the concomitant enzymes treatment could hydrolyse both the α-1,4 and α-1,6 linkages of the starches examined.     

Preparation and antihypertensive activity of peptides from Porphyra yezoensis

This research was to develop a antihypertensive peptide, an efficient angiotensin converting enzyme (ACE) inhibitor (ACEI), from Porphyra yezoensis. Seven commercial enzymes were screened and then enzymatic hydrolysis conditions were optimised. The results showed that alcalase was the most effective for hydrolysis and its optimum conditions for achieving the highest antihypertensive activity of peptide were 1.5% substrate, 5% alcalase, pH 9.0, and temperature of 50 °C at a hydrolysis time of 60 min. The antihypertensive peptide produced under the optimum conditions had a high ACE inhibition rate of 55.0% and a low IC₅₀ value of 1.6 g/l and remained at high stability at temperatures of 4, 25, and 37 °C, pH values of 2.0 and 8.0, and after pepsin and trypsin treatments. Major proteins from P. yezoensis were glutelin, albumin, and gliadin. The albumin and glutelin had higher hydrolysis rates than the gliadin, but the IC₅₀ value of glutelin was the lowest, which indicated that the antihypertensive peptide from glutelin was more functional.     

Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomycesthermophila

The β-glucosidase from Paecilomyces thermophila J18 was found to be capable of hydrolysing daidzin and genistin in a previous study. This report further evaluated the thermostability and hydrolysis of soybean isoflavone glycosides. The enzyme was found to be very stable at 50 °C, and retained more than 95% of its initial activity after 8 h at 50 °C. It converted isoflavone glycosides, in soybean flour extract and soybean embryo extract, to their aglycones, resulting in more than 93% of hydrolysis of three isoflavone glycosides (namely, daidzin, genistin and glycitin) after 4 h of incubation. Also, addition of the β-glucosidase greatly increased the contents of isoflavone aglycones in the suspended soybean flour and soymilk. The results indicate that the thermostable β-glucosidase may be used to increase the isoflavone aglycones in soy products. This is the first report on the potential application of fungal β-glucosidases for converting isoflavone glycosides to their aglycones in soy products.     

Extraction, thermal stability and kinetic behavior of pectinmethylesterase from hawthorn (Crataegus pubescens) fruit

Pectinmethylesterase (PME) extracted from hawthorn (Crataegus pubescens) fruit was evaluated for its thermal stability and kinetic behavior. The enzyme extraction process was established after studying different NaCl concentrations (0.5-3.0 moles/L). A maximum PME extraction of 51.61 units/mg protein was obtained using 2 moles/L NaCl. Kinetic parameters (K_m and V_max) were determined using a commercial citrus pectin and C. pubescens pectin as substrates. The effects of NaCl concentration, pH and temperature on PME activity were investigated. PME showed higher affinity for C. pubescens pectin (K_m and V_max of 2.84 mg/mL protein, and 64.10 units/mg protein, respectively) than for citrus pectin. C. pubescens PME extract showed maximum activity at 0.4 moles/L NaCl, pH 7.5, and 55 °C. The E_a and Q₁₀ for thermal activation were 36.27 kJ/mol and 2.01 (20-30 °C), respectively. About 50% of the activity still remained after heating for 25 min at 60 °C, and it was completely inactivated by incubation at 80 °C for 10 min. The Q₁₀ and E_a values for thermal inactivation reaction were 20.06 (70-80 °C) and 146.16 kJ/mol, respectively. These results provide useful information about the factors that affect the activity of C. pubescens PME, and might be used as a starting point for texture control during post-harvest handling and processing of this fruit.     

Advantages of a proteolytic extract by Aspergillus oryzae from fish flour over a commercial proteolytic preparation

Proteolytic extract (CPE) was produced by Aspergillus oryzae 2095 under solid state fermentation using a mix of fish flour with polyurethane foam (70:30, w/w) of particle size of 0.5 mm. The proteolytic activity from CPE was compared to a commercial protease (Flavourzyme 500 MG^®). The maximal activity for both extracts was found at pH 8 and 50 °C. The half-lives of CPE and Flavourzyme 500 MG^® were 52 and 25 min at 50 °C, respectively. Furthermore, the hydrolytic activity for both extracts was tested on muscle of giant sea bass (Epinephelus morio), CPE produced a higher degree of hydrolysis (DH) than Flavourzyme 500 MG^® (22.2 and 9.1%, respectively) under optimal experimental conditions for each extract.     

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35−68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Inactivation of Escherichia coli O157:H7 in liquid whole egg using combined pulsed electric field and thermal treatments

Inactivation of Escherichia coli O157:H7 in liquid whole egg using thermal and pulsed electric field (PEF) batch treatments, alone and in combination with each other, was investigated. Electric field intensities in the range from 9 to 15 kV/cm were used in the study. The threshold temperature for thermal inactivation alone was 50 °C. PEF enhanced the inactivation of E. coli O157:H7 when the sample temperature was higher than the thermal threshold temperature. The maximum inactivation of E. coli O157:H7 obtained using thermal treatment alone was ∼2 logs at 60 °C. However, combined heat and PEF treatments resulted in up to 4 log reduction of the pathogen. The kinetic rate constants k_TE for combined treatments at 55 °C varied from 0.025 to 0.119 pulse⁻¹ whereas the rate constants at 60 °C ranged from 0.034 to 0.228 pulse⁻¹. These results indicated a synergy between temperature and electric field on the inactivation of E. coli O157:H7 within a given temperature range.