Fractional Ethanol Precipitation Research Articles

Effects of Ethanol Concentrations on Primary Structural and Bioactive Characteristics of Dendrobium officinale Polysaccharides.

Ethanol fractional precipitation can initially separate polysaccharides according to the structure, which exhibits strong correlation with the biological activities. This study aimed to investigate the impact of varying ethanol concentrations on the structural characteristics, and the antitumor and antioxidant activities of polysaccharides derived from Dendrobium officinale through ethanol fractional precipitation, as well as their internal relationships. The polysaccharides acquired by absolute alcohol additions at a final liquor-ethanol volume ratio of 1:1, 1:2, and 1:4 were named DOP-1, DOP-2, and DOP-4, and the supernatant was named DOP-S. The results of the structural analysis revealed that the increase in ethanol concentrations resulted in a reduction in the molecular weights and the acetylation degree of the polysaccharides, as well as a decrease in mannose content and an increase in glucose content. In vitro experiments demonstrated that DOP-S exhibited optimal antitumor and antioxidant activities. Animal experiments further confirmed that DOP-S suppressed the growth of solid tumors significantly, enhanced lymphocytes, mediated immune ability, and improved the activity of antioxidant enzymes. These findings would establish a theoretical foundation and provide technical support for further advances and applications of polysaccharides derived from D. officinale in the fields of food and medicine.

Preparation of a new resource food-arabinogalactan and its protective effect against enterotoxicity in IEC-6 cells by inhibiting endoplasmic reticulum stress

Plant polysaccharides can be used as bioactive natural polymers that provide health benefits, however high molecular weight neutral polysaccharides have not shown good bioactivity. In this study, high molecular weight neutral arabinogalactan was isolated and structurally characterized to investigate it antioxidant activity against IEC-6 cells. In this study, a neutral polysaccharide (AG-40-I-II) was obtained from the roots of Larix gmelinii (Rupr.) Kuzen. and purified using ethanol fractional precipitation and purification on a DEAE-52 cellulose column and a Superose 12 gel filtration column. The structural characteristics of AG-40-I-II was detected by chemical and spectroscopic methods. The results showed that the average molecular weight of AG-40-I-II was 18.6 kDa, the main chain was composed of →4)-β-D-Gal-(1, → 4, 6)-β-D-Gal-(1 and →4)-β- D-Glc-(1, the side chain is composed of T-β-L-Araf(1 → 6). The effect of AG-40-I-II on H2O2-induced IEC-6 cell injury was determined by MTT method. Besides, AG-40-I-II could reduce the level of MDA and increase SOD activity on IEC-6 cells, which could significantly inhibit the production of ROS. Importantly, AG-40-I-II inhibited the splicing of XBP1 by IRE1α through the ERS pathway and reduced the cell apoptosis induced by H2O2. In summary, the results of this study indicate that AG-40-I-II, as a natural source of plant polysaccharides, has good antioxidant activity, and is expected to become a safe plant source of natural antioxidants, which has great potential in biomedicine potential.

Efficient isolation of immunostimulatory polysaccharides from Lentinula edodes by autoclaving-ultrasonication extraction and fractional precipitation

A hyphenated process, autoclaving followed by ultrasonication (AU), was evaluated for efficient extraction of polysaccharides (PS) from Lentinula edodes (Shiitake) mushroom. The PS yield (w/w) was 8.44 % from hot-water extraction (HWE), 11.01 % by autoclaving extraction (AE), and 16.3 % by AUE. The AUE water extract was subject to fractional precipitation in four-steps with increasing ethanol concentration of 40 %, 50 %, 70 % and 80 % (v/v), yielding four PS fractions in descending molecular weight (MW), PS40 > PS50 > PS70 > PS80. All the four PS fractions were composed of four monosaccharide residues, mannose (Man), glucose (Glc) and galactose (Gal) but in different mole ratios. The PS40 fraction with the highest average MW (4.98 × 106) was the most abundant fraction, accounting for 64.4 % of the total PS mass and also had the highest glucose molar ratio (~80 %). PS40 also most significantly enhanced the NO, and ROS generation and phagocytic activity in RAW 264.7 cells. The results proved that AUE followed by fractional ethanol precipitation is an efficient strategy with reduced solvent expenditure for isolation of the major immunostimulatory PS from L. edodes mushroom.

Characterization of hawthorn pectin gained via different ethanol concentrations.

Pectin is identified as an effective delivery material due to its excellent gel-forming ability, low immunogenic properties, biocompatibility, and biodegradability. These excellent properties depend on the preparation method of pectin. In the study, four pectin fractions (named: CAHP30, CAHP40, CAHP50, and CAHP60, respectively) were obtained by different ethanol precipitations (30%, 40%, 50%, and 60%). Physicochemical properties, antioxidant activity, and emulsifying ability of HP were investigated and analyzed. Results showed that the surface structure of pectin was changed by ethanol fractional precipitation, and four fractions were low methoxy pectin. They had different monosaccharide compositions, but all rich in GalA. The Mw/Mn of CAHP30, CAHP40, CAHP50, and CAHP60 were 3.29, 2.57, 2.66, and 2.77, respectively. CAHP30 and CAHP60 had excellent emulsifying ability; moreover, CAHP60 was endowed with additional lipid antioxidant capacity and had the best thermal stability. E-CAHP40 exhibited a property between the entangled network structure. Overall, pectin with specific properties could be obtained by different ethanol concentrations.

Rheological behaviors of ethanol-fractional polysaccharides from Dendrobium officinale in aqueous solution: Effects of concentration, temperature, pH, and metal ions

Ethanol fractional precipitation is an important technology to classify polysaccharides at different molecular weights. In this study, polysaccharides of Dendrobium officinale were step-precipitated by final ethanol concentrations of 40%, 60% and 80%, named as EPDO-40, EPDO-60 and EPDO-80, respectively. Chemical composition, molecule weights and monosaccharides were identified. Effects of concentration, temperature, pH and metal ions on rheological properties were investigated. Results showed that all EPDOs behaved as non-Newtonian fluid in a certain shear rate range of 0.1–1000 s−1 which is affected by their different chemical characteristics. Viscosities of all polysaccharides showed a concentration dependent manner, and they decreased with the increased temperatures from 10 to 90 °C. Viscosities of EPDO-40 and EPDO-80 increased at a range of lower pH significantly, while that of EPDO-60 decreased. Na+, K+, Mg2+ and Ca2+ could all affect the viscosity of EPDOs but in different patterns. Accordingly, it demonstrated that EPDOs with different chemical characteristics had different rheological properties, which could be applied more accurately in foods industry.

Purification, structural characterization and immunoregulatory mechanism of PSPW-3-a isolated from wine-processed Polygonatum sibiricum

A novel polysaccharide (PSPW-3-a) with a molecular weight of 1.29 × 104 Da was extracted and isolated from wine-processed Polygonatum sibiricum (PS) using fractional ethanol precipitation and column chromatography. Structural analysis illustrated that the backbone of PSPW-3-a was composed of →4)-β-D-Galp-(1→, and the terminal branch was present as the β-D-Galp connected to the O-6 glycosidic bond of the main chain. Additionally, PSPW-3-a promoted the secretion of IL-1β, TNF-α, and IFN-γ cytokines and induced the activity of T-SOD, GSH, and CAT oxidases in RAW264.7 cells. Immunoregulatory mechanism studies showed that PSPW-3-a significantly up-regulated the mRNA expression levels of NF-κB, iNOS, IL-6, Nrf2, HO-1, and NQO1 and activated MyD88/TRAF6/NF-κB and MyD88/ TRAF6/ (p38-MAPK, ERK-MAPK)/Keap1-Nrf2 signaling pathways in RAW264.7 cells. Our results provide a specific experimental basis for research of the immunomodulatory properties of wine-processed PS. PSPW-3-a shows potential as a natural immunomodulator.

Optimization of alkali extraction, structure, and antioxidant activity of protein-bound polysaccharide from seeds of Plantago ovata Forssk

In this study, the effects of different solvents on the viscosity of the seeds of Plantago ovata Forssk (POF) were compared to select the extraction solvent; then, a three-level and four-variable Box–Behnken design (BBD) was applied to determine the optimum extraction conditions for the maximum yields of polysaccharides from POF. Two novel protein-bound heteropolysaccharides (POFG-1a and POFG-2a) with molecular weights of 118 kDa and 113 kDa were isolated and purified by ethanol fractionation precipitation, DEAE-650 M anion exchange column chromatography, and Sephadex G-100 column chromatography. POFG-1a and POFG-2a contain 49.6% and 17.2% of polysaccharide, 26.9% and 56.4% of protein, 9.1% and 5.0% of uronic acid, respectively; they are rich in monosaccharides, Ara, Gal, and Rha, and amino acids, Ser, Pro, Ile, and Phe. Based on FT-IR, β-elimination, methylation analysis, NMR spectroscopy, and some literature data, it was found that POFG-1a is a homogeneous acidic protein-bound heteropolysaccharide with O-glycosidic linkage. The main chain of POFG-1a is connected by 1→4 Araf and 1→5 Araf with a high degree of branching, connected to the branch at the O-2 and O-3 positions of 1→5 Araf. The POFGs exhibited strong scavenging activity on DPPH, ABTS, and NO free radicals, which is rarely observed in other plant polysaccharides. These results indicate that POFGs might have potential applications in the medical and food industries as novel natural antioxidants.

Assessment of biological activities of exopolysaccharides with different relative molecular masses extracted from Rhizopus nigricans

To evaluate the antioxidant, anti-tumor and immunomodulatory activities of exopolysaccharides with different molecular masses isolated from Rhizopus nigricans. Three polysaccharides with different molecular masses, namely RPS-1, RPS-2 and RPS-3, were separated from the fermentation broth of Rhizopus nigricans by fractional ethanol precipitation, and their capacity for scavenging DPPH, ABTS, and hydroxyl radicals was assessed. Cell counting kit-8 was used to analyze the changes in the viability of MFC, A549 and RAW 264.7 cells following treatments with the 3 polysaccharides; The level of nitric oxide in the supernatant of RAW 264.7 cells was detected using a nitric oxide detection kit, and the apoptosis rate of A549 cells was analyzed with flow cytometry. All the 3 polysaccharides had good antioxidant activities, and among them RPS-1 with a medium molecular mass exhibited the strongest scavenging capacity for DPPH and ABTS radicals (P < 0.05) while RPS-3 with the lowest molecular mass had the best scavenging activity for hydroxyl radicals (P < 0.01). All the 3 polysaccharides were capable of inhibiting the proliferation of MFC cells and A549 cells, activating the macrophages RAW 264.7 cells, and inducing apoptosis of A549 cells. RPS-2 with the highest molecular mass showed the strongest inhibitory effects against MFC and A549 cells (P > 0.05), and RPS-2 had the strongest activity for inducing apoptosis in A549 cells (P < 0.05). Compared with the other two polysaccharides, RPS-2 more strongly promoted the proliferation of RAW 264.7 cells and enhanced NO release from the cells (P < 0.05). The 3 polysaccharides all have antioxidant, anti-tumor and immunomodulatory activities, and among them RPS-1 and RPS-3 have better antioxidant activities, and RPS-2 has stronger anti-tumor and immunomodulatory activities.

A method for quantitative characterization of incomplete degradation products of polygalacturonic acid

Biological activity of incomplete degradation products of polygalacturonic acid (IDPP) is closely related to its molecular weight and molecular weight distribution. Therefore, it is necessary to provide a reliable quantitative characterization method for evaluating these types of bioproducts. A novel method was established in this work for the quantitative characterization of IDPP based upon ethanol fractional precipitation. IDPP was fractionated into several fractions with high recovery (>95%), and the average molecular weights of each fraction was in descending order with the increase of ethanol concentration. Oligosaccharides (polymerization degree: 2–20) could be effectively harvested from the polygalacturonic acid enzymatic hydrolysate by ethanol precipitation. Moreover, the developed method had good repeatability and could also be applied to quantify enzymatic hydrolysis products of citrus-derived pectin polysaccharides. In conclusion, this paper provides a simple, accurate method for the quantitative characterization of IDPP and a strategy for the extraction of oligosaccharides.

A facile quantitative characterization method of incomplete degradation products of galactomannan by ethanol fractional precipitation

A novel method for the quantitative characterization of incomplete degradation products of galactomannan (IDPG) was developed by ethanol fractional precipitation. In this work, IDPG in the enzymatic hydrolyzate of Sesbania cannabina galactomannan was fractionated into several fractions with high recovery (>96 %) by ethanol fractional precipitation. The average molecular weights of obtained fractions were in descending order with varied corresponding contents of galactomannan degradation products. Thus, the quantitative characterization of IDPG can be achieved. Moreover, the results of statistical validation and method robustness showed that the method has good reproducibility. Surprisingly, when this method was applied to the guar gum or locust bean gum enzymatic hydrolyzates, it was found that the lower the mannose to galactose ratio of un-hydrolyzed galactomannan, the higher the molecular weight of their degradation product obtained under same ethanol concentration. This method is effectively applied to the quantitative characterization of IDPG from other sources.

Bioactivities and Molecular Characterizations of the Polysaccharide(s) from Ultrasonic-Circulating Extracts of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Total polysaccharide (i.e., GLP-UCE) was effectively isolated from the crude polysaccharide extract powder of Ganoderma lucidum by ultrasonic-circulating extraction, which was separated into three fractions (i.e., GLP-U40, GLP-U60, and GLP-U80) by ethanol fractional precipitation. The detection of antioxidant enzymes (GSH-Px, SOD) and oxidation metabolites (MDA, LF); liverfunctiontestof ALT, AST, and LDH; and western blot for antioxidant proteins of GSH-Px and MMP-2 showed that the GLP-UCE displayed a favorable in vivo antioxidant and hepatoprotective activities for reducing the oxidative damage in CCl4-hepatopathy SD rats. The GLP-UCE and its fractions were analyzed and compared by Fourier transform infrared (FT-IR), high-performance liquid chromatography, high-performance gel permeation chromatography, and antioxidant activity in vitro assay. These studies revealed that the fraction GLP-U80 exhibited stronger antioxidant activities in vitro than that of GLP-UCE and other fractions (p<Mw) of 1.05 × 104 g·mol-1 and composed of D-mannose, D-ribose, D-glucose, D-galactose, and L-fucose in a molecular ratio of 1:0.26:8.26:0.64:0.08.

Valorization of Fish Viscera for Crude Proteases Production and Its Use in Bioactive Protein Hydrolysate Preparation

Increasing environmental pollution and limited biological resources has emphasized the need for better utilization of fish processing waste. In the present study, visceral proteases from little tuna (Euthynnus affinis), catla (Catla catla) and tilapia (Oreochromis mossambicus) belonging to different habitats using acetone, ethanol and ammonium sulfate fractional precipitation were isolated and characterized. Enzymes precipitated from little tuna and tilapia exhibited higher specific activity at 40% saturation for ammonium sulfate fractional precipitation and the specific activities were 18.19 and 13.67 U/mg for little tuna and tilapia enzymes, respectively. Enzymes precipitated from catla reported higher specific activity of 8.32 U/mg at 60% saturation for ammonium sulfate fractional precipitation. Acetone precipitated fraction exhibited higher recovery for all crude enzymes studied. The effect of different combinations of crude tuna visceral waste enzyme and papain enzyme on hydrolysate properties was optimized using response surface methodology. Second order response surface regression model was used to explain the total variability in terms of quadratic and interaction effects of combination enzyme concentration on the properties of croaker protein hydrolysate with a significant R2 value for protein content (0.92), tyrosine content (0.99), degree of hydrolysis (0.99), DPPH free radical scavenging activity (0.97), ferric reducing antioxidant power (0.93), metal chelating activity (0.88), foaming capacity (0.92) and foam stability (0.88). Based on the desirability score, papain at 0.81% concentration and crude tuna visceral protease at 4.36% was found to be optimal for achieving desired properties of croaker protein hydrolysate.

Structural characterization and immunostimulatory activity of a glucan from natural Cordyceps sinensis

A water-soluble polysaccharide, named NCSP-50, was obtained from natural Cordyceps sinensis by hot water extraction and ethanol fractionation precipitation. It was eluted as a single symmetrical peak and had an average molecular weight of 9.76 × 105 Da. The structure was determined by monosaccharide composition, methylation analysis, 1D/2D NMR spectroscopy, and enzymatic hydrolysis and characterization of the oligosaccharides by MALDI-TOF mass spectrometry. The repeating unit of this polysaccharide was proposed as follows: [Display omitted] This glucan showed potent immunostimulatory activity on the basis of its significant abilities to promote macrophage proliferation, enhance NO production, as well as and cytokines (IL-1β and TNF-α) secretion.

Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847.

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained Km and kcat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (Ki of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn2+-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.

Purification, characterization and antitumor activity of polysaccharides extracted from Phellinus igniarius mycelia

Water-soluble intracellular polysaccharides (IPS) were extracted from cultured mycelia of Phellinus igniarius. The IPS were purified by ethanol fractional precipitation, ion-exchange and size exclusion chromatography in that order. Homogeneous polysaccharide IPSW-1, IPSW-2, IPSW-3, and IPSW-4 were obtained, which molecular characteristics were examined using multiangle laser-light scattering and refractive index detector system. The average molecular weights of them were 34.1, 17.7, 15.1, 21.7kDa, respectively. GC analysis indicated that IPSW-1, IPSW-2 and IPSW-3 all only contained glucose, while IPSW-4 was composed of rhamnose, xylose, mannose, glucose and galactose in a molar ratio of 1.29:1.21:1:43.86:1.86. UV and IR analysis suggested they belonged to α-type of the pyran group and didn’t contain protein. These homogeneous polysaccharides could inhibit the growth of SW480 and HepG2 cells to a certain extent in a dose-dependent manner. So they could be beneficial for the further development of a natural carcinoma preventive agent and functional food.

Study on prebiotic effectiveness of neutral garlic fructan in vitro

Garlic is traditionally believed to have many health benefits including prevention of gastrointestinal diseases. One of the major components, garlic fructan (GF), was evaluated for its prebiotic effectiveness on human intestinal microflora. Garlic fructans A (DP 16) and B (DP 21) were prepared by ethanol fractionation precipitation. Then, they were added to an in vitro fermentation system as the sole carbon source inoculated with human fecal suspension. The total anaerobic bacteria, Bacteroides and Bifidobacteria, were enumerated by plate counting on selective media. Terminal restriction fragment length polymerization (tRFLP) was used to analyze DNA extracted from the in vitro cultures. The results indicated that the log CFUs of both Bacteroides (GF A 6.96, GF B 7.15) and Bifidobacteria (GF A 7.74, GF B 7.74) grown in the GF cultures at 24h were significantly higher than those at 0h (Bacteroides 4.93, Bifidobacteria 4.78) (P<0.05). Terminal restriction fragments (TRFs) 256–258bp (ascribed to Bifidobacterium) in the profiles were also observed higher in the TRFLP profiles from the garlic fructans’ cultures. In this study, GFs were found to selectively stimulate the growth of beneficial Bifidobacteria from human fecal microflora. The prebiotic effectiveness of GFs supports the use of garlic as a way to prevent some gastrointestinal diseases.

Purification and characterization of β-glucosidases and β-xylosidase of Aspergillus niger NCIM 1207

Background: The extracellular β-glucosidases (cellulose and xylan induced) and xylan-induced β-xylosidase from Aspergillus niger NCIM 1207 were purified to homogeneity. The protocols were based on fractional ethanol precipitation, pH and thermal stability, and separation of impurities by thermal denaturation. Results: The molecular weights of all the three enzymes were 122 and 336 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel perme\\ation chromatography. Mass spectrometric analysis revealed that cellulose and xylan-induced β-glucosidases showed 24 and 12% sequence coverage, respectively, with β-glucosidase A of A. niger CBS 513.88/FGSC A1513. Xylan-induced β-xylosidase exhibited 35% homology with probable exo-1,4-β-xylosidase of A. niger CBS 513.88/FGSC A1513 and 11% homology with β-glucosidase A of A. niger CBS 513.88/FGSC A1513. Conclusion: The enzymes are high-molecular weight proteins and are trimeric in nature. This is the first report on high-molecular weight trimeric β-xylosidase from fungal species.

Plasma-derived medicines: access and usage issues.

Background to plasma-derived medicines Although the first plasma-derived medicines were anti-toxins, raised in horses against pathogens (diphtheria, tetanus)1, the inception of plasma protein therapy occurred when Edwin Cohn developed his fractional ethanol precipitation scheme to isolate a stable albumin solution for the treatment of battle fi eld injury and blood loss2. The Cohn scheme yielded albumin as a fi nal product while producing, among others, therapeutically useful fractions of fi brinogen and immunoglobulins which could not be employed widely because of safety issues. Following initial safety problems, albumin gained widespread medical acceptance because of its dramatic effectiveness reported in victims of shock3, and was the plasma industry's staple product until the 1970s. Its position as a safe and effective plasma expander went unchallenged up to the 1990s, when the introduction of cheaper synthetic colloids and a Cochrane metaanalysis4 threw doubts on its use, which were subsequently dispelled through clinical trials5. While studies showed that careful plasma processing of Cohn's Fraction I could yield a product which was therapeutically useful in haemophilia A6, it took Judith Pool's widespread adoption of cryoprecipitate from blood bank plasma7 to result in the next milestone in the history of plasma protein therapies. Pool's technique was rapidly adapted for large-scale fractionation without affecting the Cohn method8 and resulted in the fi rst industrial scale production of haemophilia therapy. The capacity to treat a previously life-limiting disease effectively made the manufacture of factor VIII (FVIII) concentrate the driver for the plasma industry in the 1970s, usurping albumin's historical position. The revolution this produced in the life of haemophiliacs cannot be underestimated, just as the effects, on patients and industry alike, of viral transmission by the products cannot be underappreciated, although in the heady days of the 1970s this risk of this transmission was under recognised. While industry hastened to introduce enhanced safety measures, particularly viral inactivation which by the mid 1980s had made haemophilia products safe, an effect of this tragedy was the rapid development of recombinant FVIII concentrates, once the F8 gene had been cloned in 19849. The results of clinical trials, published in 198910, rapidly led to widespread acceptance of this therapy to its current position as the dominant haemophilia treatment modality in many countries of the developed world, including the United States, the United Kingdom, Canada and Australia, and greatly increased the market and the availability of FVIII, allowing interventions such as prophylaxis and tolerisation. In some other, mostly European countries, plasma-derived FVIII has retained a strong presence, due, primarily, to the continuing debate regarding the different capacity of different FVIII products to result in inhibitors to FVIII11. This development would have had a profound effect on the economic, and indeed, the viability of the industry, but other developments in the fi eld of immunotherapy obviated it. Cohn's original method allowed the harvesting of immunoglobulin (Ig) fractions which could be concentrated into solutions and used to treat patients with Ig defi ciencies12. In addition, Ig solutions from the plasma of donors immunised to specifi c antigens could be used for the treatment or prophylaxis of various diseases; the use of the Rh Ig fraction is the most famous of these applications13. However, early clinical observations that intravenous administration of Ig solutions led to severe reactions meant that Ig administration was limited to the intramuscular route, limiting dosage and patients' comfort. Efforts to address this problem led to several imperfect intravenously administrable Ig products, in which measures, such as enzymatic digestion of the entities

Strain Screening, Fermentation, Separation, and Encapsulation for Production of Nattokinase Functional Food

This study presents a novel and integrated preparation technology for nattokinase functional food, including strain screening, fermentation, separation, and encapsulation. To rapidly screen a nattokinase-productive strain, PCR-based screening method was combined with fibrinolytic activity-based method, and a high productive strain, Bacillus subtilis LSSE-22, was isolated from Chinese soybean paste. Reduction of poly-γ-glutamic acid (γ-PGA) concentration may contribute to separation of nattokinase and reduction of late-onset anaphylaxis risk. Chickpeas were confirmed as the favorable substrate for enhancement of nattokinase production and reduction of γ-PGA yield. Using cracked chickpeas, the nattokinase activity reached 356.25 ± 17.18 FU/g (dry weight), which is much higher than previous reports. To further reduce γ-PGA concentration, ethanol fractional extraction and precipitation were applied for separation of nattokinase. By extraction with 50 % and precipitation with 75 % ethanol solution, 4,000.58 ± 192.98 FU/g of nattokinase powders were obtained, and the activity recovery reached 89 ± 1 %, while γ-PGA recovery was reduced to 21 ± 2 %. To improve the nattokinase stability at acidic pH condition, the nattokinase powders were encapsulated, and then coated with methacrylic acid-ethyl acrylate copolymer. After encapsulation, the nattokinase was protected from being denatured under various acid conditions, and pH-responsible controlled release at simulated intestinal fluid was realized.

Monosaccharide composition and bioactivity of tea flower polysaccharides obtained by ethanol fractional precipitation and stepwise precipitation

Yields of tea flower polysaccharides (TFPS) were investigated by ethanol fractional precipitation and stepwise precipitation and the monosaccharide composition of TFPS was investigated by ion chromatography(IC). Furthermore, a preliminary study on bioactivities of TFPS was discussed. The result indicated that the molecular weight distribution of TFPS was wide. Results of ethanol fractional precipitation and stepwise precipitation suggested that most of polysaccharides were precipitated when the final concentration of ethanol was adjusted to 30% (v/v). TFPS contained acid polysaccharides and it was composed of rhamnose, arabinose, galactose, glucose, xylose, mannose, GalA, and GluA with a molar ratio of 4.95:11.57:11.89:4.26:1.31:1.98:8.41:1.0. Furthermore, TFPS exhibited strong inhibitory effect on α-glucosidase and proliferation effect on lymphocytes.