Effects of pH and light on the storage stability of the purple pigment, hordeumin, from uncooked barley bran fermented broth

The pigment retention rate of hordeumin was higher than that of two standard anthocyanidins, cyanidin and delphinidin, when hordeumin and anthocyanidins were dissolved in Walpole buffer (pH 1.0) and stored. Moreover, when pigment solutions were stored at 15 degrees C under light irradiation, the pigment retention rate of the hordeumin solution became higher than those of standard anthocyanidins (2 to 10 times) as the storage period increased. Comparing various pH buffers (MacIlvaine buffer, pH 2.2 to 7.0), the pigment retention rate of hordeumin at pH 5.0 was highest. Furthermore, the half-life of hordeumin at pH 5.0 was increased from 9 days to 17.5 days when nitrogen gas was bubbled into the hordeumin solution. We considered that the storage stability of hordeumin is higher than standard anthocyanidins because hordeumin is a complex with anthocyanin, tannin, and protein.     

Storage Stability and Thermal Stability of Hordeumin,an Anthocyanin Pigment from Barley

Hordeumin stored at –40 to –80^oC in 1% HCI–methanol suffered neither from color reduction nor discoloration. After heating at 80°C for 60 min, hordeumin showed a pigment retention rate of 100%. This characteristic is because the pigment is a composite high-molecular weight compound consisting of anthocyanins and polyphenols, It was determined, however, that discoloration and browning occurred more rapidly than color reduction during storage and heating of the pigment.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Antimutagenicity of the purple pigment, hordeumin, from uncooked barley bran-fermented broth

The novel purple pigment hordeumin, an anthocyanin-tannin pigment, was produced from barley bran-fermented broth. The mutagenicity or antimutagenicity of hordeumin was investigated according to the Ames method, an indication of the safety of food, using Salmonella typhimurium TA98. Despite the presence of S-9 mix, hordeumin was not mutagenic. On the other hand, hordeumin effectively decreased a reverse mutation from Trp-P-1, Trp-P-2, IQ, and B[a]P. Furthermore, hordeumin also decreased the reverse mutation from dimethyl sulfoxide extracts of grilled beef.     

Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite

The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Immobilization of lipase on woolen fabrics: Enhanced effectiveness in stain removal

The aim of this research was to examine the effectiveness of an enzyme in enhancing the cleaning effectiveness of woolen fabric without addition of any detergent. As a model enzyme, lipase from Pseudomonas fluoresces was immobilized onto a woolen cloth using a unique protocol that involved: chlorination of the wool, adsorbing a polyethyleneimine (PEI) spacer, adsorbing, and cross‐linking with glutaraldehyde (GA) followed by adsorption of the lipase. It was determined that for this protocol, the immobilized activity was dependent on the GA solution pH and not on its concentration. The cloth exhibited excellent oily stain removal ability: after being stained with olive oil and stored for 1 day in air at room temperature, the oily stain could be easily removed by 0.05 M pH 8.5 Tris buffer without any detergent addition. This enhanced cleaning was stable also over a period of one month. The activity of the cloth (based on activity assay) dropped considerably over just 15 days storage in air. This therefore likely indicates that the enhanced cleaning seen over an extended storage period may not require as high an enzyme activity. The activity of the immobilized lipase was also very stable when stored under near ideal conditions: when the immobilized cloth was stored in 0.05 M Tris buffer (pH 8.5) for more than 80 days in a refrigerator, more than 80% of the lipase activity remained. Overall, results indicate that this immobilization protocol is a promising step towards producing a woolen fabric with enhanced cleaning properties. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:806–817, 2014     

The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae)

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4′-O-glucopyranoside as the main flavonol pigment.On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H₂O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] ⁺ at 2102 m/z (C₉₃H₁₀₅O₅₅ calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.     

Evaluation of the stability of creatine in solution prepared from effervescent creatine formulations

The objectives of this study were to determine the cause of the crystallization in a large volume creatine supplement solution made from effervescent powders containing di-creatine citrate, and to characterize these crystals using thermal analyses and x-ray diffractometry. Creatine effervescent powders were dissolved in deionized water (pH 6.2) and stored both at room temperature (RT) (25°C) and refrigerated condition (4°C) over a period of 45 days. Creatine concentration was determined using high-performance liquid chromatography (HPLC). Intrinsic dissolution and saturated solubility of creatine, creatine monohydrate, and di-creatine citrate in water were determined and compared. Crystal growth was detected only in the refrigerated samples on the seventh day of storage. Differential Scanning Calorimetry (DSC) and x-ray diffraction (XRD) studies revealed that the crystals formed were of creatine monohydrate. Ninety percent creatine degradation was observed within 45 days for RT samples. However, at refrigerated condition this degradation was 80% within the same time period. The pH of the RT samples also increased from 3.6 to 4.5 during storage. No such increase was observed in the case of refrigerated samples. The intrinsic dissolution rate constants of the compounds decreased in the following order: dicreatine citrate>creatine>creatine monohydrate. In conclusion, di-creatine citrate used in effervescent formulation dissociates to creatine in aqueous solution and eventually crystallizes out as creatine monohydrate. Significant decrease in solubility and effect of pH contribute to this crystallization process.     

Influence of pH on Cd and Cu uptake, distribution and their effect on nitrate reductase activity in cucumber (Cucumis sativus L.) seedling roots

Influence of different pH solutions (5.0 and 7.0) on Cu²⁺ and Cd²⁺ absorption and distribution in root cells as well as effects of these metals on nitrate reductase activity (NR) in roots of cucumber seedlings were estimated. The absorption of Cu and Cd by roots measured as metal depletion in uptake solution was similar, both metal absorption was independent of the pH of solution. However, after rinsing of roots in distilled water (30 minutes), more Cu than Cd was found in protoplasts of root cells. More Cu was measured in all cell fractions when Cu was uptaken from pH 5.0 than from 7.0. The nitrate reductase activity after one hour of metal treatments was drastically decreased by Cu. The strongest reduction of enzyme activity was observed in roots treated with Cu in buffer with pH 5.0. Influence of Cd on the enzyme activity was weaker and was independent of the pH of solution. Lower concentration of Cd in solution (20 μM) increased NR activity. The data obtained prove the higher mobility of Cu than Cd into the cells of root. The mobility of Cu depends on pH of solution. Cu ions, but not Cd, influenced membrane permeability (K leakage). Cu acted more drasticly than Cd on NR activity.     

不同干燥方式、存储温度对羊栖菜中岩藻黄素稳定性的影响

为探究不同干燥方式、不同存储温度和存储时间对羊栖菜中岩藻黄素稳定性的影响,本研究考察了三种不同干燥方式,包括自然阴干、低温烘干和冷冻干燥,对羊栖菜中岩藻黄素稳定性的影响。结果表明采用冷冻干燥所得羊栖菜,含水量低,岩藻黄素含量高,达到699.2μg/g,岩藻黄素保留率为94.7%,远高于自然阴干及低温烘干。因此,冷冻干燥是最佳干燥方法。考察了四个不同温度(-20、4、25、30℃)存储对冻干羊栖菜中岩藻黄素稳定性的影响,结果表明随着存储温度的升高,岩藻黄素的降解率明显增高,因此,-20℃为最佳储存温度。     

Changes in Resident Bacteria, pH, Sucrose, and Invert Sugar Levels in Sugarbeet Roots During Storage

Stored sugarbeet roots began fermenting within 24 h after oxygen was depleted at 26 C when the resident bacterial populations increased dramatically. Most of the bacteria present after anaerobic storage for 7 days at 26 C could hydrolyze sucrose in vitro. Although pH and sucrose levels decreased and invert sugar levels increased with time in aerobic storage at 26 C, these processes were significantly accelerated in beets stored anaerobically at 26 C. Under oxygen-depleted storage conditions at 26 C, the sucrose content was almost completely lost after 21 days.     

The extraction and assay of soil trehalase

Summary Trehalase is a specific enzyme for the hydrolysis of trehalose, a storage carbohydrate of insect and microbial species. The enzyme is of rare occurrence among higher plants. In cultivated soil, trehalase activity (Ta) was linearly related to both the amount of soil and assay incubation time. Ta increased sharply in response to substrate concentration over the range of 0 to 2% (w/v); higher substrate levels, however, showed a reduced rate of increase. Soil trehalase activity increased proportionally with increased incubation temperature over the range of 20 to 50°C but declined sharply at temperatures above 50°C. Ta was maximal at pH 5.0 when 0.5M acetate or propionate buffers were used; however, activity diminished with increased ionic strength of the buffer. Based on these findings, a standard assay method for Ta was developed. The enzyme was extracted from soil and eluted from a Sephadex G 200 column as high molecular weight organic matter. Treatment of the extract with beta-glucosidase reduced the molecular weights of the elution fractions exhibiting Ta.     

Cryopreservation of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase from bovine cardiac muscle

A method for the cryogenic storage of the cAMP-dependent protein kinase from bovine cardiac muscle is described. The catalytic parameters, kcat, KM, and kcat/KM are used to assess the activity of the enzyme both prior and subsequent to the freeze-thaw cycle. The enzyme is stored in cryogenic vials at -196 degrees C in liquid nitrogen. Complete retention of catalytic activity is dependent upon a rapid and efficient freeze-thaw cycle and the use of morpholinepropanesulfonic acid as the buffer. In addition, this buffer appears to eliminate the KCl- or NaCl-induced damage typically observed for enzymes stored at low temperature in phosphate buffer. As a result, morpholinepropanesulfonic acid may prove to be a more appropriate cryopreservation buffer than phosphate when the presence of salt is required for enzyme solubility or stability.     

Screening of novel excipients for improving the stability of retroviral and adenoviral vectors

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.     

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 degrees C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9 h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 degrees C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.     

Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection

A rapid method for the determination of olanzapine in plasma using high-performance liquid chromatography with ultra violet detection is described. Olanzapine was extracted from plasma with a mixture of hexane/dichloromethane (85:15), and then back extracted into phosphate buffer pH 2.8. Separation was achieved on a RP Select B C(18) column and commonly administered drugs did not interfere with the assay. The limit of quantitation was 1.5 microg/l and the inter-day and intra-day relative standard deviations were less than 10%. Olanzapine was shown to be stable in plasma for up to 7 days when stored at 4 degrees C. Moreover, the addition of ascorbic acid was not necessary for the achievement of chemical stability during storage, or during the assay procedure. The method has been used to measure olanzapine concentrations in patients treated with various doses of the drug varying from 5 to 40 mg/day.     

The solution and solid state stability and excipient compatibility of parthenolide in feverfew

The objectives of this research were to evaluate the stability of parthenolide in feverfew solution state and powdered feverfew (solid state), and explore the compatibility between commonly used excipients and parthenolide in feverfew. Feverfew extract solution was diluted with different pH buffers to study the solution stability of parthenolide in feverfew. Powdered feverfew extract was stored under 40 degrees C/0% approximately 75% relative humidities (RH) or 31% RH/5~50 degrees C to study the influence of temperature and relative humidity on the stability of parthenolide in feverfew solid state. Binary mixtures of feverfew powered extract and different excipients were stored at 50 degrees C/ 75% RH for excipient compatibility evaluation. The degradation of parthenolide in feverfew solution appears to fit a typical first-order reaction. Parthenolide is comparatively stable when the environmental pH is in the range of 5 to 7, becoming unstable when pH is less than 3 or more than 7. Parthenolide degradation in feverfew in the solid state does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. After 6 months of storage, parthenolide in feverfew remains constant at 5 degrees C/31% RH. However, approximately 40% parthenolide in feverfew can be degraded if stored at 50 degrees C/31% RH. When the moisture changed from 0% to 75% RH, the degradation of parthenolide in feverfew increased from 18% to 32% after 6-month storage under 40 degrees C. Parthenolide in feverfew exhibits good compatibility with commonly used excipients under stressed conditions in a 3-week screening study.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).