Fabrication of 1,4-alpha-D-glucan glucanohydrolase holding Gel-Scaffolds using Agar-Agar, a natural polysaccharide and Polyacrylamide, a synthetic organic polymer for continuous liquefaction of starch

1,4-alpha-D-glucan glucanohydrolase is among the most widely used commercial hydrolytic enzymes acting randomly on the glycosidic linkages of starch resulting in its saccharification and liquefaction. Its applicability in different industries can be improved by enhancing its stability and reusability. Therefore, in the present study attempts have been made to enhance the industrial applicability of 1,4-alpha-D-glucan glucanohydrolase from Bacillus subtilis KIBGE-HAR by adapting immobilization technology. The study developed mechanically stable, enzyme containing gel-frameworks using two support matrices including agar-agar, a natural polysaccharide and polyacrylamide gel, a synthetic organic polymer. These catalytic gel-scaffolds were compared with each other in terms of kinetics and stability of entrapped 1,4-α-D-glucan glucanohydrolase. In case of polyacrylamide gel, Km value for immobilized enzyme increased to 7.95 mg/mL, while immobilization in agar-agar resulted in decreased Km value i.e 0.277 mg/mL as compared to free enzyme. It was found that immobilized enzyme showed maximum activity at 70 °C in both the supports as compared to free enzyme having maximum activity at 60 °C. Immobilized 1,4-α-D-glucan glucanohydrolase exhibited no change in optimal pH 7.0 before and after entrapment in polyacrylamide gel and agar-agar. The enzyme containing gel-scaffold was found suitable for repeated batches of starch liquefaction in industrial processes. Agar-agar entrapped 1,4-α-D-glucanglucanohydrolase was capable to degrade starch up to seven repeated operational cycles whereas polyacrylamide entrapped enzyme conserved its activity up to sixth operational cycle.

Thermodynamics, kinetics and optimization of catalytic behavior of polyacrylamide-entrapped carboxymethyl cellulase (CMCase) for prospective industrial use.

In the current study, kinetic and thermodynamic parameters of free and polyacrylamide-immobilized CMCase were analyzed. The maximum immobilization yield of 34 ± 1.7% was achieved at 11% acrylamide. The enthalpy of activation (ΔH) of free and immobilized enzyme was found to be 13.61 and 0.29 kJ mol-1, respectively. Irreversible inactivation energy of free and immobilized CMCase was 96.43 and 99.01 kJ mol-1, respectively. Similarly, the enthalpy of deactivation (ΔHd) values for free and immobilized enzyme were found to be in the range of 93.51-93.76 kJ mol-1 and 96.08-96.33 kJ mol-1, respectively. Michaelis-Menten constant (Km) increased from 1.267 ± 0.06 to 1.5891 ± 0.07 mg ml-1 and the maximum reaction rate (Vmax) value decreased (8319.47 ± 416 to 5643.34 ± 282 U ml-1 min-1) after immobilization. Due to wide pH and temperature stability profile with sufficient reusing efficiency up to three successive cycles, the immobilized CMCase might be useful for various industrial processes.

Single step immobilization of CMCase within agarose gel matrix: Kinetics and thermodynamic studies.

In the current study, CMCase from Bacillus licheniformis KIBGE-IB2 was immobilized within the matrix of agarose gel through entrapment technique. Maximum immobilization yield (%) of the enzyme was obtained when 2.0 % agarose was used. The activation energy (Ea) of the enzyme increased from 16.38 to 44.08 kJ mol-1 after immobilization. Thermodynamic parameters such as activation energy of deactivation (ΔGd), enthalpy (ΔHd) and entropy (ΔSd) of deactivation, deactivation rate constant (Kd), half-life (t1/2), D-value and z-value were calculated for native/free and immobilized CMCase. The maximum reaction rate (Vmax) of the native enzyme was found to be 8319.47 U ml-1 min-1, which reduced to 7218.1 U ml-1 min-1after immobilization process. However, the Michaelis-Menten constant (Km) value of the enzyme increased from 1.236 to 2.769 mg ml-1 min-1 after immobilization. Immobilized enzyme within agarose gel matrix support can be reuse up to eight reaction cycles. Broad stability profile and improved catalytic properties of the immobilized CMCase indicated that this enzyme can be a plausible candidate to be used in various industrial processes.

Utilization of different polymers for the improvement of catalytic properties and recycling efficiency of bacterial maltase.

Current study deals with the comparative study related to immobilization of maltase using synthetic (polyacrylamide) and non-synthetic (calcium alginate, agar-agar and agarose) polymers via entrapment technique. Polyacrylamide beads were formed by cross-linking of monomers, agar-agar and agarose through solidification while alginate beads were prepared by simple gelation. Results showed that the efficiency of enzyme significantly improved after immobilization and among all tested supports agar-agar was found to be the most promising and biocompatible for maltase in terms of immobilization yield (82.77%). The catalytic behavior of maltase was slightly shifted in terms of reaction time (free enzyme, agarose and polyacrylamide: 5.0 min; agar-agar and alginate: 10.0 min), pH (free enzyme, alginate and polyacrylamide: 6.5; agar-agar, agarose: 7.0) and temperature (free enzyme: 45 °C; alginate: 50 °C; polyacrylamide: 55 °C; agarose: 60 °C; agar-agar: 65 °C). Stability profile of immobilized maltase also revealed that all the supports utilized have significantly enhanced the activity of maltase at higher temperatures then its free counterpart. However, recycling data showed that agar-agar entrapped maltase retained 20.0% of its initial activity even after 10 cycles followed by agarose (10.0%) while polyacrylamide and alginate showed no activity after 8 and 6 cycles respectively.

A comparative study on degradation of complex malathion organophosphate using of Escherichia coli IES-02 and a novel carboxylesterase.

Malathion organophosphates considered as the major constituent of herbicides, pesticides and insecticides. Extensively used in agricultural, horticultures and for numerous household applications contributes to precedence organic pollutants leading antagonistic effects on human health and environment. Therefore detoxification of malathion from contaminated site is of general interest. Simultaneously it is very emerging to isolated novel indigenous microbial strains from contaminated site with a record of pesticide application. In this study Escherichia coli IES-02 isolated from malathion contaminant effluent and the strain showed maximum efficiency in malathion degradation that utilized it as the sole source of carbon. Carboxylesterase (33.0, 30.0, 28.0 kDa) were purified (1685.71 U/mg) from Escherichia coli IES-02 showed significant results in malathion degradation approximately 81% within 20 min as compared with Escherichia coli IES-02 cells within 4 h (99.0 to 95.0%) into monocarboxylic acid and diacid derivatives. The generation time of Escherichia coli was also observed at 60 min with 0.1 ppm, 68 min with 0.5 ppm, 74.5 min with 2.0 ppm and 91.37 min with 50 ppm of malathion. The degradation rate and transformation metabolites were estimated by Gas Chromatography-Mass Spectrometry respectively. Malathion metabolites pathway proposed in this study which revealed the potential application against lethal environmental pollution.

Maltose deterioration approach: Catalytic behavior optimization and stability profile of maltase from Bacillus licheniformis KIBGE-IB4.

Maltase is an economically valuable enzyme that is used to catalyze the hydrolytic process of maltose and yields d-glucose as a product. In this study, the catalytic behavior of maltase was optimized under various physicochemical condition. Results indicated that bacterial maltase exhibited maximum catalytic activity at 45 °C and pH-6.5 after 5.0 min. It presented greater stability within 0.1 M K2HPO4 buffer having pH-6.5 and showed 100 % activity even after 1.0 h. It retained 83.6 % and 45.0 % activity at 40 °C after 1.0 and 3.0 h, respectively. The enzyme retained 90.0 % activity at -20 °C even after 60 days. The molecular weight of enzyme was deduced to be 157.2 kDa as calculated using polyacrylamide gel electrophoresis (PAGE) and zymography. It was concluded that the characterized maltase has notable stability profile with reference to temperature, pH and other reaction conditions which anticipates its utilization in various starch and maltose hydrolyzing processes for the synthesis of glucose.

Characterization of cross-linked amyloglucosidase aggregates from Aspergillus fumigatus KIBGE-IB33 for continuous production of glucose.

Current research deals with immobilization of amyloglucosidase through carrier-free approach using cross-linking strategy. Cross-linked amyloglucosidase aggregates (CLAAs) with aggregation yield of 94% were prepared in 04 h by incorporating 40% ammonium sulfate and 1.5% glutaraldehyde in enzyme solution. CLAAs were characterized by optimizing various conditions including reaction time, pH, temperature and substrate concentration. It was noticed that after cross-linking no change in optimum reaction time and substrate concentration was observed however, a 5-degree shift in optimum temperature from 60 °C to 65 °C was obtained as compared to soluble amyloglucosidase. Activation energy (Ea) of amyloglucosidase as calculated from Arrhenius plot was 5.5 kcal mol-1 and 5.2 kcal mol-1 for soluble and cross-linked aggregates, respectively. Stability studies revealed that CLAAs can be used at higher temperatures for longer time period than soluble amyloglucosidase. Furthermore, data of recycling studies showed that CLAAs can be efficiently reused for 20 cycles with the retention of 63% of its initial activity. Due to the continuous reusability of CLAAs, the product formation is also increased 8 times from 5.71 mg ml-1 (soluble enzyme) to 46.548 mg ml-1 (CLAAs). Findings of this research show that carrier-free strategy is more effective for continuous hydrolysis of starch and production of glucose.

Screening, purification and characterization of thermostable, protease resistant Bacteriocin active against methicillin resistant Staphylococcus aureus (MRSA).

BACKGROUND: The emergence of serious issues of multidrug resistance in the past few years have enforced the use of bacteriocins for combating infections. Threat posed to public health by various multidrug resistant (MDR) organisms can be resolved by discovering new antimicrobial proteins with broad spectrum of inhibition. RESULTS: In the current study, Bacteriocin (BAC-IB17) produced by Bacillus subtilis KIBGE-IB17 is found to be effective against different strains of methicillin resistant Staphylococcus aureus (MRSA). The approximate molecular mass of BAC-IB17 is 10.7 kDa. This unique bacteriocin is found to be highly thermostable and pH stable in nature. It also showed its stability against various heavy metals, organic solvents, surfactants and proteolytic enzymes. Amino acid profile of BAC-IB17 clearly showed that this protein mainly consists of non-polar and basic amino acids whereas; some acidic amino acids were also detected. Sequence of first 15 amino acid residues obtained from N-terminal sequencing of BAC-IB17 were NKPEALVDYTGVXNS. CONCLUSIONS: The anti-MRSA property of purified bacteriocin may be used to prevent the spread of MRSA infections. Remarkable features of BAC-IB17 suggests its applications in various pharmaceutical and food industries as it can function under a variety of harsh environmental conditions.

Enhanced biosynthesis of dextransucrase: A multivariate approach to produce a glucosyltransferase for biocatalysis of sucrose into dextran.

The current study reported the statistically designed experimental method to enhance the biocatalytic efficacy of dextransucrase from Weissella confusa. Various environmental and nutritional parameters were optimized using multiple responses under submerged fermentation environment. Statistical models were constructed to screen the influence of nine factors on the biocatalysis of dextransucrase. Among them, fermentation time, pH, sucrose and peptone exhibited significant probability (P < 0.05) and are considered as substantial constituents in accordance with Plackett-Burman design. Central composite design was further implemented to optimize the levels of selected variables for maximum enzyme yield. The predicted optimum conditions were pH of 7.5 under fermentation time of 8 h with 30.0 g l-1 sucrose and 1.0 g l-1 peptone. The overall enzyme yield increased from 11.4 DSU ml-1 to 52.75 DSU ml-1 with 4.62-fold upsurge after the implementation of the statistical models. Furthermore, SEM analysis showed the biocatalytic conversion of sucrose into highly porous dextran when utilizing dextransucrase. The biopolymer produced under the current optimized model could be utilized as an emulsifying, gelling, stabilizing and thickening agent in food industry.

Characterization and interplay of bacteriocin and exopolysaccharide-mediated silver nanoparticles as an antibacterial agent.

Metallic nanoparticles have a substantial scientific interest because of their distinctive physicochemical and antimicrobial properties and the emergence of multidrug resistant pathogens could unlock the potential of nanoparticles to combat infectious diseases. The aim of the current study is to enhance the antibacterial potential of purified bacteriocin by combining bacteriocin and antibacterial silver nanoparticles (AgNPs). Hence, the interaction of natural antimicrobial compounds and antibacterial nanoparticles can be used as a potential tool for combating infectious diseases. In this study, a green, simple and effective approach is used to synthesize antibacterial AgNPs using fungal exopolysaccharide as both a reducing and stabilizing agent. The AgNPs were characterized by spectroscopic analysis, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and Dynamic Light Scattering (DLS). Furthermore, the synergistic effect of bacteriocin-AgNPs was determined against pathogenic strains. The histogram of AgNPs indicated well-dispersed, stabilized and negatively charged particles with variable size distribution. The combination of bacteriocin with nanoparticles found to be more effective due to broad antibacterial potential with possibly lower doses. The current study is imperative to provide an alternative for the chemical synthesis of silver nanoparticles. It showed environmental friendly and cost effective green synthesis of antibacterial nanoparticles.

Agar-agar immobilization: An alternative approach for the entrapment of protease to improve the catalytic efficiency, thermal stability and recycling efficiency.

The catalytic performance of an immobilized enzyme could be enhanced by using entrapment technique. In this contemporary study agar-agar, a natural polysaccharide, is subjected to entrap serine-protease produced by Aspergillus niger KIBGE-IB36. The results revealed that maximum enzymatic activity was attained when 3.0% agar-agar was used. It was observed that in case of both free and entrapped forms the enzyme was stable at pH-5.0. While, an increment in reaction temperature and time was noticed from 50 to 55 °C and 15.0 to 20.0 min, respectively. Km value increased from 1.883 mM to 2.399 mM and Vmax value decreased from 1753 U mg-1 to 1372 U mg-1 after agar-agar entrapment of protease as compared to soluble enzyme. Additionally, entrapped protease within the polymer exhibited significant increase in the thermal stability at various temperatures and retained approximately 68.0% of its residual activity at 60 °C. However, at this extreme temperature the soluble protease lost its catalytic performance. Storage stability considerably improved as entrapped protease revealed enzymatic activity up to 30 days as compared to soluble enzyme. Recycling efficiency was calculated up to eight cycles which is an exceptional characteristic for economic feasibility and continuous reusability of protease.

Bioprospecting of indigenous resources for the exploration of exopolysaccharide producing lactic acid bacteria.

Exploration of biodiversity lead towards the discovery of novel exopolysaccharide (EPS) producing microbes that have multiple applications. The safety compatibility status of lactic acid bacteria (LAB) makes it an attractive candidate for the production of EPS in industries. Therefore, new bacterial isolates are continuously being identified from different habitats. Current research was conducted to explore indigenous biodiversity for the production of dextransucrase, which is involved in the synthesis of dextran. Dextran is an EPS which is used in different industries. In this study, thirty-nine LAB were isolated from different food samples. The isolates were identified as genus Leuconostoc, Weissella and Streptococcus based on genotypic and phenotypic characteristics. Screening revealed that only eight isolates can produce dextransucrase in high titres. Fermentation conditions of dextran producing LAB was optimized. The results indicated that Weissella confusa exhibited maximum specific activity (1.50 DSU mg-1) in 8 h at 25 °C with pH 7.5. Dextran produced from Weissella proved to be a useful alternative to commercially used dextran produced by Leuconostoc mesenteroides in industries for various applications.

Utilization of agro waste pectin for the production of industrially important polygalacturonase.

In the present study, a variety of agro-industrial wastes have been utilized for meaningful purpose to produce valuable biocatalyst. All wastes used were low cost and easily accessible while, some available throughout the year. A number of bacterial strains isolated from rotten fruits and vegetables were screened for the production of industrially important polygalacturonase (PGase) using pectin present in these agro-industrial wastes. The strain producing maximum PGase was identified as Bacillus licheniformis KIBE-IB3 on the basis of taxonomic studies and 16S rDNA analysis. Among different agro-industrial wastes studied, high yield of PGase was achieved from fermentation broth having wheat bran (1.0%) as a substrate in to the medium supplemented with nitrogen sources in combination of NaNO3 and yeast extract while KH2PO4 was selected as suitable micronutrient. After optimizing fermentation parameters it was noticed that Bacillus licheniformis KIBE-IB3 was capable of producing maximum PGase at 37 °C, pH 7.0 and after 48 h of incubation time. From the current research, wheat bran was proven as a cheap and easily available source throughout the year for hyper production of pectinase. The utilization of the waste will also help to minimize the concerned environmental issues.

Enzymatic and acidic degradation of high molecular weight dextran into low molecular weight and its characterizations using novel Diffusion-ordered NMR spectroscopy.

Low molecular weight fractions were derived from native high molecular weight dextran produced by Leuconostoc mesenteroides KIBGE-IB26. Structural characterization of native and low molecular weight fractions obtained after acidic and enzymatic hydrolysis was done using FTIR and NMR spectroscopy. The molecular weight was estimated using Diffusion Ordered NMR spectroscopy. Native dextran (892kDa) is composed of α-(1→6) glycosidic linkage along with α-(1→3) branching. Major proportion of 528kDa dextran was obtained after prolong enzymatic hydrolysis however, an effective acidic treatment at pH-1.4 up to 02 and 04h of exposure resulted in the formation of 77kDa and 57kDa, respectively. The increment in pH from 1.4 to 1.8 lowered the hydrolysis efficiency and resulted in the formation of 270kDa dextran fraction. The results suggest that derived low molecular weight water soluble fractions can be utilized as a drug delivery carrier along with multiple application relating pharmaceutical industries.

Production of α-1,4-glucosidase from Bacillus licheniformis KIBGE-IB4 by utilizing sweet potato peel.

In the current study, sweet potato peel (Ipomoea batatas) was observed as the most favorable substrate for the maximum synthesis of α-1,4-glucosidase among various agro-industrial residues. Bacillus licheniformis KIBGE-IB4 produced 6533.0 U ml-1 of α-1,4-glucosidase when growth medium was supplemented with 1% dried and crushed sweet potato peel. It was evident from the results that bacterial isolate secreted 6539.0 U ml-1 of α-1,4-glucosidase in the presence of 0.4% peptone and meat extract with 0.1% yeast extract. B. licheniformis KIBGE-IB4 released 6739.0 and 7190.0 U ml-1 of enzyme at 40 °C and pH 7.0, respectively. An improved and cost-effective growth medium design resulted 8590.0 U ml-1 of α-1,4-glucosidase with 1.3-fold increase as compared to initial amount from B. licheniformis KIBGE-IB4. This enzyme can be used to fulfill the accelerating demand of food and pharmaceutical industries. Further purification and immobilization of this enzyme can also enhance its utility for various commercial applications. Graphical abstract Pictorial representation of maltase production from sweet potato peel.

Polyacrylamide Gel-Entrapped Maltase: An Excellent Design of Using Maltase in Continuous Industrial Processes.

Bacterial maltase catalyzes the hydrolysis of maltose and is known as one of the most significant hydrolases. It has several applications in different industrial processes but widely used in food fermentation technology and alcohol production. In the current study, entrapment technique was comprehensively examined using polyacrylamide gel as a matrix support to improve the stability and catalytic efficiency of maltase for continuous use. Maximum entrapment yield of maltase was achieved at 10 % polyacrylamide concentration with 3.0-mm bead size. Optimized conditions indicated an increase in the reaction temperature from 45 to 55 °C after maltase entrapment while no change was observed in the reaction time and pH. An increase in the K m value of entrapped maltase was attained whereas V max value decreased from 8411.0 to 6813.0 U ml(-1) min(-1) with reference to its free counterpart. Entrapped maltase showed remarkable thermal stability and retained 16 % activity at 70 °C even after 120.0 min. Entrapped maltase also exhibited excellent recycling efficiency up to eight consecutive reaction cycles. With respect to economic feasibility, entrapped maltase indicates its high potential to be used in various biotechnological applications.

Calcium alginate matrix increases the stability and recycling capability of immobilized endo-ß-1,4-xylanase from Geobacillus stearothermophilus KIBGE-IB29.

Exploration of microbial pool from extremely diversified ecosystem is significantly important for various industrial applications. Bacterial communities from extreme habitats including volcanic vents, hot springs, and industrial sectors are eagerly explored for the isolation of thermophiles. Geobacillus stearothermophilus KIBGE-IB29, isolated from blast furnace site of a steel processing industry, is capable of producing thermostable endo-ß-1,4-xylanase. In the current study, this enzyme was immobilized within calcium alginate beads using entrapment technique. Amalgamation of sodium alginate (40.0 gL(-1)) and calcium chloride (0.4 M) was used for the formation of immobilized beads. It was observed that temperature (50 °C) and pH (7.0) optima of immobilized enzyme remained same, but enzyme-substrate reaction time increased from 5.0 to 30.0 min as compared to free enzyme. Diffusion limit of high molecular weight xylan (corncob) caused a decline in V max of immobilized enzyme from 4773 to 203.7 U min(-1), whereas K m value increased from 0.5074 to 0.5722 mg ml(-1) with reference to free enzyme. Immobilized endo-ß-1,4-xylanase showed its stability even at high temperatures as compared to free enzyme and retained 18 and 9 % residual activity at 70 and 80 °C, respectively. Immobilized enzyme also exhibited sufficient recycling efficiency up to five reaction cycles which indicated that this enzyme can be a plausible candidate in paper and pulp industry.

Purification, characterization and end product analysis of dextran degrading endodextranase from Bacillus licheniformis KIBGE-IB25.

Degradation of high molecular weight dextran for obtaining low molecular weight dextran is based on the hydrolysis using chemical and enzymatic methods. Current research study focused on production, purification and characterization of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 36 folds with specific activity of 1405 U/mg and molecular weight of 158 kDa. It was found that enzyme performs optimum cleavage of dextran (5000 Da, 0.5%) at 35 °C in 15 min at pH 4.5 with a Km and Vmax of 0.374 mg/ml and 182 µmol/min, respectively. Relative amino acid composition analysis of purified enzyme suggested the presence of higher number of hydrophobic, acidic and glycosylation promoting amino acids. The N-terminal sequence of dextranase KIBGE-IB25 was AYTVTLYLQG. It exhibited distinct amino acid sequence yet shared some inherent characteristics with glycosyl hydrolases (GH) family 49 and also testified the presence of O-glycosylation at N-terminal end.

Morphological and molecular based identification of pectinase producing Bacillus licheniformis from rotten vegetable.

Pectinase catalyzed the degradation of pectin substances and has been used in various biotechnological industries. In the current study, 23 bacterial strains were isolated from rotten vegetables, soil and air. The isolated bacterial strains were qualitatively screened for pectinase production on pectin agar medium and only three strains HR 4, HR 21 and HR 23 were observed to produce extracellular pectinase. These strains were further screened quantitatively for pectinase production through submerged fermentation technology in pectin containing fermentation medium. Strain HR 4 from rotten brinjal (Solanum melongena) was found to produce higher pectinase as compared to others. The maximum pectinase producing bacterial strain was identified as Bacillus licheniformis on the basis of morphological, physiological and biochemical characteristics. For further confirmation of identification, 16S rDNA sequence analysis was performed. The 16S rDNA sequences were aligned and the phylogenetic tree was constructed. The phylogenetic tree confirmed that the strain was belonging to B. licheniformis. The 16S rDNA sequences of this new strain were submitted to GenBank and designated as B. licheniformis KIBGE-IB21 with the GenBank accession number JQ 411812. The newly isolated pectinase producing B. licheniformis used apple pectin as carbon and yeast extract as nitrogen source for maximum pectinase production.

Continuous degradation of maltose: improvement in stability and catalytic properties of maltase (α-glucosidase) through immobilization using agar-agar gel as a support.

Maltose degrading enzyme was immobilized within agar-agar support via entrapment method due to its industrial utilization. The maximum immobilization efficiency (82.77%) was achieved using 4.0% agar-agar keeping the diameter of bead up to 3.0 mm. The matrix entrapment showed maximum catalytic activity at pH 7.0 and temperature 65 °C. Substrate saturation kinetics showed that the K m of immobilized enzyme increased from 1.717 to 2.117 mM ml(-1) where as Vmax decreased from 8,411 to 7,450 U ml(-1 )min(-1) as compared to free enzyme. The immobilization significantly increased the stability of maltase against various temperatures and immobilized maltase retain 100% of its original activity after 2 h at 50 °C, whereas the free maltase only showed 60% residual activity under same condition. The reusability of entrapped maltase showed activity up to 12 cycles and retained 50% of activity even after 5th cycle. Storage stability of agar entrapped maltase retain 73% of its initial activity even after 2 months when stored at 30 °C while free enzyme showed only 37% activity at same storage conditions.