Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

AIM: To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine. METHODS: Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro. RESULTS: TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut. CONCLUSION: Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

Model for human milk fat substitute evaluation based on triacylglycerol composition profile.

Being the dominant components in human milk fat (HMF), triacylglycerol (TAG) composition might be the best approximation index to represent the composing characteristics of HMF. In this study, TAG composition of HMF from different lactation stages was analyzed by RP-HPLC-APCI-MS, and the establishment of a model for the precise evaluation of human milk fat substitutes (HMFSs) based on TAG composition was indirectly realized by employment of fatty acid composition and distribution and polyunsaturated fatty acid (PUFA) and TAG compositions. The model was verified by the selected fats and oils with specific chemical compositions, and the results revealed the degrees of similarity of these fats and oils in different evaluation aspects reflected their differences in corresponding chemical composition with HMF. The newly established evaluation model with TAG composition as a comparison base could provide a more accurate method to evaluate HMFSs and might have some inspirations for HMFS production in the future.

Preparation of human milk fat substitutes from palm stearin with arachidonic and docosahexaenoic acid: combination of enzymatic and physical methods.

Human milk fat substitutes (HMFSs) were prepared by a two-step process, namely, Lipozyme RM IM-catalyzed acidolysis of interesterified high-melting palm stearin with fatty acids from rapeseed oil and blending of the enzymatic product with the selected oils on the basis of the calculation model. The optimum conditions for the enzymatic reaction were a mole ratio of palm stearin/fatty acids 1:10, 60 °C, 8% enzyme load (wt % of substrates), 4 h, and 3.5% water content (wt % of enzyme); the enzymatic product contained 39.6% palmitic acid (PA), 83.7% of the fatty acids at sn-2 position were PA (sn-2 PA), and the distribution probability of PA at the sn-2 position among total PA (% sn-2 PA) was 70.5%. With the fatty acid profiles of human milk fat (HMF) as a preferable goal, a physical blending model was established for the second step to guarantee the maximum addition of selected oils. Based on the model prediction, a desirable formula constituted enzymatic product/rapeseed oil/sunflower oil/palm kernel oil/algal oil/microbial oil at a mole ratio of 1:0.28:0.40:0.36:0.015:0.017, and the final product had PA content, sn-2 PA, and %sn-2 PA at 23.5, 43.1, and 61.1%, respectively. The contents of arachidonic and docosahexaenoic acids were 0.4 and 0.3%, respectively. Relying on the total and sn-2 fatty acid compositions of HMF and "deducting score" principle, the score for the similarity between the final product and HMF was scaled as 89.2, indicating the potential as a fat substitute in infant formulas.

Human milk fat globules from different stages of lactation: a lipid composition analysis and microstructure characterization.

The physicochemical properties of human milk fat globules (MFG) at different lactation stages from Danish mothers and the microstructure changes of MFG membrane (MFGM) at varied temperatures were investigated, and the relationship between chemical composition and the microstructure of MFGM was elucidated. The fat content in MFG was found to be significantly increased as lactation progressed, and colostrum MFG had the largest mean diameter of 5.75 ± 0.81 µm and the lowest ζ potential of -5.60 ± 0.12 mV. Chemical composition analyses of MFG revealed the following: (i) Colostrum milk fat constituted higher content in PUFAs (ω-6, and long-chain ω-6 and ω-3) than transitional and mature milk fats, with the corresponding lower content of SFA in its sn-2 position. (ii) The content of polar lipids among total lipids varied during lactation course (maximized at transitional stage); however, in terms of subclasses of polar lipids, no significant change of the relative content of sphingomyelin was observed, while the content of phosphatidycholine in mature milk was higher than that in colostrum and transitional milk. (iii) Inspection of fatty acid composition in phospholipids from different lactation milk revealed no remarkable and regular changes could be generalized; and no obvious difference of the morphologies of MFGM at different lactation stages can be visualized. An investigation of the microstructure change of MFGM vs temperature demonstrated that the segregated domains became larger as temperature decreased to 4 °C, while it became smaller when increased to 37 °C. This phenomenon indicated that, in addition to sphingimyelin and cholesterol, phospholipids might also contribute to increasing the segregated domains at lower temperature, while, at elevated temperature, these domains could be diminished, most likely due to a restructuring or distributing of sphingimyelin and cholesterol.

Preparation and characterization of catalase-loaded solid lipid nanoparticles based on soybean phosphatidylcholine.

BACKGROUND: High-purity soybean phosphatidylcholine (SPC) (94%) were prepared using macroporous resin adsorption chromatography previously. Catalase is a food enzyme for promoting health and protecting against many age-related disease. Solid lipid nanoparticles (SLN) are safe immobilizing systems for efficient protein transportation to biomembranes while avoiding adverse degradation of protein. This study was aimed at developing and characterizing catalase-loaded SLN using SPC as solubilizers and stabilizing agents, to protect catalase from proteolysis. RESULTS: Catalase-loaded SLN were prepared by the double emulsification method and solvent evaporation techniques, using acetone-methylene chloride (1:1, v/v) as an organic solvent, SPC-tripalmitin as oil phase and Poloxamer 188 as a surfactant. The optimized SLN were prepared using an SPC:tripalmitin ratio of 5% (w/w), 20 s plus 30 s sonication, 20 g L⁻¹ Poloxamer 188 and 1:2 (v/v) of oily phase:outer aqueous phase ratio. The mean particle size of SLN was 296.0 ± 7.0 nm, polydispersity index range and zeta potential were 0.322-0.354 and -36.4 ± 0.6, respectively, and encapsulation efficiency reached its maximum of 77.9 ± 1.56%. Catalase, which was found to distribute between the solid lipid and inner aqueous phase, was gradually released from SLN up to 20% within 20 h. Catalase-loaded SLN had stably retained 30% of H2O2-degrading activity for at least 24 h in a proteolytic environment, while free catalase lost its activity within 1 h. CONCLUSION: Catalase can indeed be loaded in tripalmitin-based SLN using SPC as solubilizers and stabilizing agents, which protected it against proteolysis, suggesting the potential application of SPC in delivery and protection of functional food enzyme.

Lipase-catalyzed preparation of human milk fat substitutes from palm stearin in a solvent-free system.

Human milk fat substitutes (HMFSs) were synthesized by lipozyme RM IM-catalyzed acidolysis of chemically interesterified palm stearin (mp = 58 °C) with mixed FAs from rapeseed oil, sunflower oil, palm kernel oil, stearic acid, and myristic acid in a solvent-free system. Response surface methodology (RSM) was used to model and optimize the reactions, and the factors chosen were reaction time, temperature, substrate molar ratio, and enzyme load. The optimal conditions generated from the models were as follows: reaction time, 3.4 h; temperature, 57 °C; substrate molar ratio, 14.6 mol/mol; and enzyme load, 10.7 wt % (by the weight of total substrates). Under these conditions, the contents of palmitic acid (PA) and PA at sn-2 position (sn-2 PA) were 29.7 and 62.8%, respectively, and other observed FAs were all within the range of FAs of HMF. The product was evaluated by the cited model, and a high score (85.8) was obtained, which indicated a high degree of similarity of the product to HMF.

Comparative analysis of lipid composition and thermal, polymorphic, and crystallization behaviors of granular crystals formed in beef tallow and palm oil.

Six rectangular block all beef tallow (BT)-based and all palm oil (PO)-based model shortenings prepared on a laboratory scale, respectively denoted BTMS and POMS, were stored under temperature fluctuation cycles of 5-20 °C until granular crystals were observed. The lipid composition and thermal, polymorphic, and isothermal crystallization behaviors of the granular crystals and their surrounding materials separated from BTMS and POMS, respectively, were evaluated. The changes of nanostructure including the aggregation of high-melting triacylglycerols (TAGs) and polymorphic transformation from ß' form of double chain length structures to complicated crystal structures, in which the ß and ß' form crystals of triple and double chain length structures simultaneously coexist, had occurred in granular crystals compared with surrounding materials, whether in BTMS or in POMS. Consequently, a slower crystallization rate appeared in granular crystal parts of both model shortenings noted above, which would yield larger and fewer crystals indicated by the Avrami model analysis that would further aggregate to form large granular crystals.

Characterization of graininess formed in all beef tallow-based shortening.

A batch of all beef tallow (BT)-based model shortening (divided into six rectangular block samples) was stored under temperature fluctuation cycles of 5-20 °C until granular crystals were observed. The lipid composition, thermal properties, and polymorphism of the granular crystals and their surrounding materials were evaluated. Furthermore, the isothermal crystallization behavior of two parts noted above was also examined by pulsed nuclear magnetic resonance (pNMR), rheology, and polarized light microscopy (PLM). The changes of nanostructure including the aggregation of high-melting triacylglycerols (TAGs) and transformation into the most stable ß polymorph occurred in granular crystals compared with surrounding materials. Concomitantly, a slower crystallization rate with a simultaneous increase in crystal growth led to the formation of large crystals and further aggregated to larger granular crystals when the size ultimately exceeded the sensory threshold.

Beta-D-glucosidase-catalyzed deglucosidation of phenylpropanoid amides of 5-hydroxytryptamine glucoside in safflower seed extracts optimized by response surface methodology.

Beta-D-glucosidase-catalyzed deglucosidation of phenylpropanoid amides of 5-hydroxytryptamine (PAHAs) glucoside in safflower (Carthamus tinctorius L.) seed extracts, including N-(p-coumaroyl)serotonin glucoside (CSG) and N-feruloylserotonin glucoside (FSG), was optimized by response surface methodology (RSM). The Box-Behken design (BBD) was employed to evaluate the interactive effects of independent variables on the deglucosidation rates of CSG and FSG. The variables involved were pH (5.6-6.2), temperature (45-55 degrees C), and enzyme load (2.0-3.0%, relative to the weight of the total substrate). The substrate concentration was fixed at 3.3 g/L on the basis of factorial experiments. The optimum conditions obtained via RSM at a fixed time of 2 h were as follows: pH, 5.9; temperature, 48 degrees C; and enzyme load, 3.0%. Under these conditions, the actual deglucosidation rates of CSG and FSG were 75.5 and 42.2%, respectively, which agree well with the predicted values (75.3 and 41.9%) by RSM. The final incubation time (10 h) was determined by the time course of the deglucosidation under the above-mentioned optimum conditions, which gave the deglucosidation rates of both CSG and FSG above 90%. Simultaneously, 2-hydroxyarctiin, a typical cathartic beta-glucoside, was also removed by 80.3%.

Solvent-free enzymatic preparation of feruloylated monoacylglycerols optimized by response surface methodology.

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of glyceryl ferulate (FG) and oleic acid for feruloylated monoacylglycerols (FMAG) preparation in a solvent-free system was investigated. Enzyme screening and the effect of glycerol on the initial reaction rate of esterification were also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 degrees C), the enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of FG conversion and FMAG yield. The optimum preparation conditions were as follows: temperature, 60 degrees C; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAG are 96.7 +/- 1.0% and 87.6 +/- 1.2%, respectively.

[Spectra study on the influence of drying process on palygorskite structure].

There are four different types of molecules of hydroxyl groups of the natural attapulgite. The band at 3614 cm(-1) was attributed to the stretching modes of hydroxyls coordinated with the magnesium. The band at 3415 cm(-1) is associated with the hydroxyl stretching vibrations of absorbed water. The bands at 3581 and 3552 cm(-1) were attributed to the symmetric and antisymmetric stretching modes of molecular water coordinated with the magnesium at the edges of the channels. The band at 1653 cm(-1) is associated with the hydroxyl stretching vibrations of zeolitic water. The structures of the natural palygorskite and its products dried at different temperatures for 30 min were analysed by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The absorbed, zeolitic and co-ordinated water decreased during the drying process at the same time. The absorbed water was completely-dehydrated firstly, then the zeolitic water, and lastly the co-ordinated water. And the hydroxyl groups remained until about 600 degrees C. When the co-ordinated water was dehydrated completely at 450 degrees C, the crystalloid was destroyed. The mechanism of the palygorskite structure change was also discussed in detail.