Feng Hang

Composition and antioxidant activity of polysaccharides from jujuba by classical and ultrasound extraction

Crude Zizyphus Jujuba cv. Junzao polysaccharides (CZP) were extracted by hot water and ultrasound-assisted extraction respectively. Using a short application of ultrasound, the yield of polysaccharides increased from 6.23 to 7.95%, which represents an increase of about 27.6%. Both CZPU and CZPH were composed of arabinose, rhamnose, glucose, galactose, and mannose. However, the molar ratio of CZPU was 5.46: 4.96: 5.17: 2.63: 1, while it was 5.46: 4.89: 3.65: 2.54: 1 for CZPH. Furthermore, CZPU had stronger antioxidant activities than CZPH by evaluating in hydroxyl radical and ferric-reducing antioxidant power (FRAP) assay.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526

Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.

Purification and characterization of a novel milk-clotting metalloproteinase from Paenibacillus spp. BD3526

In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65°C and 60°C, respectively. The enzyme was stable over a pH range of 5.0-9.0 and at temperatures below 50°C. The MCA was completely inactivated when the enzyme was heated at 60°C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55°C and 60°C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis.

Effect of acids produced from carbohydrate metabolism in cryoprotectants on the viability of freeze-dried Lactobacillus and prediction of optimal initial cell concentration

For the industrial production of probiotics powder, various sugars have been used as cryoprotectants to preserve probiotics during freeze-drying. Some of these sugars can be metabolized by Lactobacillus with the production of acids during the mix. In this study, we investigated the effect of acids on ATPase, β-galactosidase, lactate dehydrogenase (LDH), integrity and fluidity of cell membrane and the survival rate of Lactobacillus during freeze-drying. In the presence of Lactobacillus, acids were produced from cryoprotectants containing fermentable sugars before freezing, resulting in a decrease in the pH of the bacterial suspension to below 5.0. During freeze-drying, the acids caused a loss of viability of Lactobacillus due to aggravated damage to ATPase, β-galactosidase and cell membrane fluidity, but not LDH and cell membrane integrity. This finding implied that cryoprotectants that do not lead to the production of acids are effective in improving the survival rate of freeze-dried Lactobacillus. Here, a new formula was proposed for a protectant containing whey protein isolate (WPI) and rhamnose, which were not metabolized. In addition, linear-regression analyses were performed on the proportion of cryoprotectants (M) against cell paste (m), total cell count (N), total surface area (St) and total volume (Vt) of bacteria for 100% survival rate. The total surface areas of bacteria were found to be highly correlated with the amount of proposed cryoprotectant. The following prediction equation was established for the optimal initial cell concentration for a 100% survival rate of freeze-dried Lactobacillus: N (4πr2+2πl)=(0.66±0.03)M.

Effect of Calcium on Absorption Properties and Thermal Stability of Milk during Microwave Heating

During heating, there are a lot of physical and chemical changes in milk components, which are mainly reflected in the changes of proteins. Calcium ions in milk react with proteins to precipitate or form gels, and the thermal stability of milk is affected by the type and content of calcium. In this study, different calcium-fortified milk systems were treated by rapid conventional heating (RCV) and microwave heating (MV) to evaluate the effects of forms and concentration of calcium in liquid milk on microwave absorption properties and thermal stability of milk. It was found that the concentration of calcium ions on microwave energy absorption is not a significant influence, while the forms affected the systems dramatically. The thermal stability of milk during MV is remarkably affected by the forms of calcium ions. When adding ionized calcium, the calcium-fortified milk systems had poor thermal stability and severe agglomeration of protein, while the addition of milk calcium had little effect and was almost free from protein coagulation. It could be speculated that the metal ions in the microwave field could create a strong vibration that could trigger protein agglomeration through the combination of the surrounding casein phosphorylates.

Effect of Microwave Heating on the Dielectric Properties and Components of Iron-Fortified Milk

With the iron-fortified milk as research object, this paper makes a research on the influence of iron on the dielectric properties and wave absorption properties and effect of nutritional components, such as casein and whey protein in milk, and thermostability in the process of microwave heating, and rapid heat transfer method in ferrous gluconate–milk and ferrous chloride–milk, respectively. The results show that the iron of ionic form has greater influence to convert microwave to heat energy and the effect of microwave absorption properties was greater for ferrous chloride than for ferrous gluconate at high concentration. The effect of different forms of iron on the composition of milk was different, and the composition of milk systems was more stable by microwave heating, but the rapid heat transfer method is superior in the aim of increasing the nutritional value of milk. The ferrous gluconate–milk system has a better thermal stability than ferrous chloride–milk system. From the aspect of dielectric induction, the paper discovers the response rules of iron and evaluates the microwave thermal safety of the traditional and the iron-fortified products by microwave heating.

Structural insight into a novel neutral metalloproteinase from Paenibacillus spp. BD3526: Implications for mechanisms of rapid inactivation and calcium-dependent stability

The proteinase with milk-clotting activity (MCA) from Paenibacillus spp. BD3526 is characterized as a neutral metalloproteinase of 35kDa. However, the rapid reduction of its MCA during separation and purification leads to low enzyme recovery. The effects of metal ions, inactivation kinetics, and concentration of calcium on the enzymatic activities and thermal stability of the BD3526 metalloproteinase were investigated. In the absence of calcium, the residual activities of the BD3526 metalloproteinase sharply declined during the first three hours, and continued to slowly decrease thereafter. The activities were well fitted by a double-exponential decay model. The inactivation rates were significantly inhibited by calcium and the residual enzyme activities were maintained at more than 80% for 30d at room temperature with 50-100mM calcium. An intermolecular autoproteolytic mechanism was responsible for BD3526 metalloproteinase inactivation. The target protein band with MCA remained largely undegraded in the enzyme solution that was supplemented with 100mM of calcium, but gradually diminished over time in the absence of calcium. N-terminal amino acid sequencing showed that cleavage at the His252-Ala253 peptide bond facilitated the conversion of the zymogen into the active enzyme. Sequence alignment revealed the presence of two highly conserved motifs, HEXXH and GXXNEXXSD, indicating that the enzyme belonged to the metalloproteinase family M4, also known as thermolysin-like proteinases (TLPs). Further structural analysis showed that the observed calcium-dependent stability of the BD3526 TLP may be attributable to a partly degenerated calcium-binding site, Ca1-2, and a mutant calcium-binding site, Ca3.