Chemical and Physical Stability of Astaxanthin-Enriched Emulsion-Based Delivery Systems

Astaxanthin (AST) is carotenoid that is considered to have many potential benefits for human health. However, its poor water-solubility and chemical instability hamper its use as a functional food ingredient. The present study evaluated the effects of storage temperature, pH, ionic strength, and light exposure on the physical and chemical stability of AST-enriched emulsions prepared using caseinate as emulsifier. The chemical degradation of AST increased with increasing temperature, but the emulsions remained physically stable (droplet diameter = 230 nm; ζ-potential = ?40 mV) at all incubation temperatures (5–70 °C). Solution pH, ionic strength and light exposure had little impact on the chemical stability of AST, except at pH 4 and 5. Conversely, the emulsions were highly unstable to droplet aggregation at pH values near the isoelectric point of the caseinate. This work highlights the benefits and drawbacks of using caseinate-stabilized emulsions as delivery systems for AST in functional foods and beverages.     

Effect of Temperature,pH, Ionic Strength,and Sodium Nitrate on Activity of LiPs: Implications for Bioremediation

The present work is aimed to show the effects of environmental parameters such as temperature, pH, ionic strength, and sodium nitrate on enzyme activity of a LiP Isoenzymes Mixture (LIM) obtained from an immobilized culture of Phanerochaete chrysosporium. LIM enzyme stability was also evaluated. The results are discussed in detail and a comparison with literature data is carried out. LIM showed high activity at pH 3.0 in the temperature range 30 to 40°C, it is able to catalyze oxidation reactions at acid pH (2.5<pH<6) and over a wide range of temperatures (25 to 60°C). Ionic strength below 0.2?M had no effect on enzyme activity at pH 4.7 and 39°C. An evaluation of the time decay constant of LIM activity under a specific combination of parameters was also conducted. Finally, an LIM activity and durability map shows the optimal working conditions that might be suitable for its practical application in waste bioremediation processes.     

Biophysical analysis of phaseolin denaturation induced by urea,guanidinium chloride,pH, and temperature

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Characterization of a tannase from Emericela nidulans immobilized on ionic and covalent supports for propyl gallate synthesis

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t₅₀) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.     

Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding

Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60°C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na₂SO₄ strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmCl denaturation and had a transition midpoint at 1.2 M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture; 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase. Received: June 22, 1998 / Accepted: December 11, 1998     

Molecular basis of the structural stability of hemochromatosis factor E: A combined molecular dynamic simulation and GdmCl‐induced denaturation study

Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidinium chloride (GdmCl) was measured by monitoring changes in [θ]₂₂₂ (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F₃₄₆) and the difference absorption coefficient at 287 nm (Δε₂₈₇) at pH 8.0 and 25°C. Coincidence of denaturation curves of these optical properties suggests that GdmCl‐induced denaturation (native (N) state ? denatured (D) state) is a two‐state process. The GdmCl‐induced denaturation was found reversible in the entire concentration range of the denaturant. All denaturation curves were analyzed for , Gibbs free energy change associated with the denaturation equilibrium (N state ? D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 133–142, 2016.     

Structural Changes in Halophilic and Non-halophilic Proteases in Response to Chaotropic Reagents

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein–urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca²⁺ and Na⁺ ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.     

Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae

An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind withK _d=80 and 130 μM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae

An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind withK _d=80 and 130 μM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Thermal stability and chain conformational studies of xanthan at different ionic strengths

The aim of the present study was to determine the influence of the ionic strength on the thermal stability of xanthan, i.e. xanthan resistance to chain breaking at high temperatures. Xanthan solutions of various ionic strengths were kept at 80, 90 and 95°C for periods up to 95 h. The thermal stability was determined by measuring the intrinsic viscosity after the heating periods. The experiments showed a critical ionic strength for the thermal stability of xanthan between 10 and 100 mm NaCl or KCl in this temperature range. Below the critical ionic strength the intrinsic viscosity was rapidly reduced, whereas above the critical ionic strength the intrinsic viscosity was virtually unaffected by heating.We then looked for a possible correlation between thermal stability and secondary structure of xanthan. The transition ionic strength (I_m) of xanthan solutions, i.e. where xanthan is midway between an ordered and a disordered structure, was determined by NMR at constant temperatures. I_m was found to be in the range of 24 mm at 80°C to 60 mm NaCl at 95°C, thus lying in the range of the critical ionic strength of the thermal stability. This suggests a close relationship between thermal stability and secondary structure of xanthan, indicated by the enhanced thermal stability in the ordered state. We believe this enhanced thermostability arises from a double-stranded conformation in the ordered state, as in DNA. The presence of double-stranded xanthan is also indicated by electron micrographs taken at both high and low ionic strengths.The transition temperature (T_m) of xanthan was determined by NMR and optical rotation measurements. At the ionic strength of 7·5 mm the two methods resulted in T_m values of 67 and 52°C respectively. This difference in T_m can possibly be due to the fact that the observed NMR and optical rotation (OR) effects are caused by different molecular phenomena.     

Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis

An esterase gene, est10, was identified from the genomic library of a deep-sea psychrotrophic bacterium Psychrobacter pacificensis. The esterase exhibited the optimal activity around 25 °C and pH 7.5, and maintained as high as 55.0 % of its maximum activity at 0 °C, indicating its cold adaptation. Est10 was fairly stable under room temperatures, retaining more than 80 % of its original activity after incubation at 40 °C for 2 h. The highest activity was observed against the short-chain substrate p-nitrophenyl butyrate (C4) among the tested p-nitrophenyl esters (C2–C16). It was slightly activated at a low concentration (1 mM) of Zn²⁺, Mg²⁺, Ba²⁺, Ca²⁺, Cu²⁺, Fe³⁺, urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10 activity (up to 143.2 % of original activity at 2 M NaCl) and stability (up to 126.4 % after incubation with 5 M NaCl for 6.5 h), proving its salt tolerance. 0.05 and 0.1 % Tween 20, Tween 80, Triton X-100 and CHAPS increased the activity and stability of Est10 while SDS, CTAB had the opposite effect. Est10 was quite active after incubation with several 30 % organic solvents (methanol, DMSO, ethanediol) but exhibited little activity with 30 % isopropanol, ethanol, n-butanol and acetonitrile.     

Kinetics of chromium nucleotide isomer interconversion

The rates of chromium nucleotide isomer interconversion were studied as a function of pH, ionic strength, and temperature. Nucleotide isomers were separated using high voltage electrophoresis and gel filtration chromatography. The rate of conversion of monodentate adenosine 5°-monophosphate-chromium salt (CrADP) to the bidentate complex increased with increasing pH, temperature, and ionic strength. Optimal stability for CrADP complexes was found to be at pH 3.5 with a temperature of 4°C. It was found that at pH values above 7.0, the chromium complexes rapidly decomposed even at 4°C. It was found that the conversion of monodentate CrADP to binentate CrADP required the removal of one proton by the solvent. The activation energy for the conversion was found to be 7.3 kcal/mol at pH 6.5. The kinetics of the isomer interconversion are described in terms of possible conversion mechanisms.     

Stability of Emulsions Using a New Natural Emulsifier: Sugar Beet Extract <Emphasis Type="Italic">(Beta vulgaris L.)</Emphasis>

This study describes the influence of environmental stresses on the stability of emulsions prepared by a natural sugar beet extract (Beta vulgaris L.). The emulsion stabilizing performance was compared to that of Quillaja extract, which is widely used within the food and beverage industry as natural surfactant. We investigated the influence of pH, ionic strength, heating and freeze-thawing on the mean particle size, ζ-potential and microstructure of oil-in-water emulsions (10% w/w oil, 0.75% w/w emulsifier). The emulsions stabilized by the anionic sugar beet extract were stable at pH 5–8 and against thermal treatments up to 60 °C. However, the prepared emulsions were unstable at acidic (pH 2–4) and basic pH conditions (pH 9), at high temperature (>60 °C), and at salt additions (> 0.1 M NaCl / CaCl₂). Moreover, they also phase separated upon freeze-thawing. Our results show that sugar beet extract is capable of stabilizing emulsions and may therefore be suitable as natural emulsifier for selected applications in the food and beverage industry.     

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ～400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.     

Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis

A protease producing marine bacterium, Bacillus halodurans CAS6 isolated from marine sediments, was found to produce higher enzyme by utilizing shrimp shell powder. Optimum culture conditions for protease production were 50 °C, pH 9.0, 30 % NaCl and 1 % shrimp shell powder (SSP) and the protease purified with a specific activity of 509.84 U/mg. The enzyme retained 100 % of its original activity even at 70 °C, pH 10.0 and 30 % NaCl for 1 h. The purified protease exhibited higher stability when treated with ionic, non-ionic (72–94 %) and commercial detergents (76–88 %), and organic solvents (88–126 %). Significant blood stain removal activity was found with the enzyme in washing experiments. The culture supernatant supplemented with 1 % SSP showed 93.67 ± 2.52 % scavenging activity and FT-IR analysis of the reaction mixture confirmed the presence of antioxidants such as cyclohexane and cyclic depsipeptide with aliphatic amino groups. These remarkable qualities found with this enzyme produced by Bacillus halodurans CAS6 could make this as an ideal candidate to develop the industrial process for bioconversion of marine wastes and antioxidant synthesis.     

Serum opacity factor, a streptococcal virulence factor that binds to apolipoproteins A-I and A-II and disrupts high density lipoprotein structure

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.     

GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA₃) followed by incubation at dark, and (iv) immersion in 400 ppm GA₃ followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

Effects of different cold-air exposure intensities on the risk of cardiovascular disease in healthy and hypertensive rats

Ten-week-old male Wistar rats (systolic blood pressure, 106–116 mmHg; body weight, 300–320 g) and spontaneously hypertensive rats (systolic blood pressure, 160–176 mmHg; body weight, 210.9–244.9 g) were used as healthy and hypertensive subjects to determine the effects of varying degrees of cold-air exposure in a climate chamber box. The three cold-air ranks were cold air I [minimum temperature (TMIN) 6.4 °C, ↓?T₄₈ 8.6 °C], cold air II (TMIN 3.8 °C, ↓?T₄₈ 11.2 °C), and cold air III (TMIN ?0.3 °C, ↓?T₄₈ 15.3 °C), as established from the cold-air data of Zhangye City, China. Each cold-air rank consisted of a temperature drop and a temperature increase with the same initial and terminal temperatures (15 °C). After cold-air exposure, the risk factors for cardiovascular disease (CVD) such as systolic blood pressure, whole blood viscosity (10/s and 150/s), plasma fibrinogen, and blood lipids of the rats were determined. The results indicated that the CVD risk factors of the healthy and hypertensive rats increased significantly with cold-air exposure intensities. The increase in systolic blood pressure was greater during temperature drops, whereas the increases in whole blood viscosity and plasma fibrinogen were greater after cold-air exposure. The effects of cold-air exposure on the CVD risk factors of healthy rats, particularly the systolic blood pressure, whole blood viscosity (150/s), and LDL/HDL, were greater than those in hypertensive rats. In conclusion, CVD risk may increase with cold-air ranks. Blood pressure-induced CVD risk may be greater during cold-air temperature drop, whereas atherosclerosis-induced CVD risk may be greater after cold-air exposure. The effect of cold air on the CVD risk factors in healthy subjects may be more significant than those in hypertensive subjects.     

Proteomic Studies of Saliva: A Proposal for a Standardized Handling of Clinical Samples

Background

In recent years, differential analysis of proteins from human saliva, i.e., proteomic analysis, has received much attention mainly due to its unstressful sampling and its great potential for biomarker research. It is widely considered that saliva is a highly stable medium for proteins thanks to a large amount of antiprotease agents, even at ambient and physiological temperatures.

Objective

To find the best protocol for the handling of samples, we have investigated the stability of saliva proteins stored at different temperatures (from ?80 to 20°C) by one- and two-dimensional electrophoresis.

Results

At 20°C, no major changes were observed on protein one-dimensional profiles following 1 day of storage; however, between 7 days and 30 days, the native alpha-amylase band decreased slightly to give several bands with molecular weight between 35 and 25 kDa. The same phenomenon appeared after 30 days of storage at 4°C. Two-dimensional analysis of salivary maps revealed degradation from day 7 of several protein groups for samples stored at 20°C.

Conclusion

All these findings have to be carefully considered when saliva is collected for clinical proteomic analysis. We can conclude that, to maintain the optimum stability of saliva proteins, saliva samples should be collected on ice followed by the addition of protease inhibitor cocktail, centrifuged to remove insoluble material, and stored at ?20 or ?80°C.