Buffer Concentration Research Articles

Amherstia Nobilis: an Approach to in Vitro Anti-inflammatory Activity

Herbal plant compounds were used in the inhibition of inflammation from traditional period and its main benefit is the less side effects. The aim of this study is to determine the anti-inflammatory potential of Amherstia nobilis by albumin denaturation method. The leaves of the plant were collected, dried and powdered. The egg albumin, phosphate buffer and different concentrations of the plant extracts were incubated at 37°C for 15 minutes and heated at 70°C for 5 minutes. The rate of inhibition of protein denaturation is calculated. The result verified the anti-inflammatory potential of Amherstia nobilis and its prospective use in alleviating inflammation as a natural remedy.

Effect of phosphate buffer concentration on the isomerization of galactose to rare sugars under subcritical water conditions

Galactose was treated in sodium phosphate buffer at various concentrations (0.1–500 mmol/L) under subcritical water conditions (140 °C), and the effects of the buffer concentration and reaction time (0–300 s) on the reaction behavior were evaluated. The reaction proceeded rapidly at higher buffer concentrations. Rare sugars (tagatose, talose, and sorbose) were formed from galactose by isomerization. The highest yield of the main product, tagatose, was approximately 14 % in 50 mmol/L buffer. However, the tagatose yield did not increase further with increasing buffer concentration. On the other hand, the formation of talose and sorbose was accelerated at higher buffer concentrations, with the highest yields of approximately 5 % and 12 %, respectively, in 500 mmol/L buffer. At the same time, the formation of byproducts (organic acids and colored substances) was also accelerated in high-concentration buffers. These results suggest that phosphate buffer promoted all reactions occurring under subcritical water conditions.

The effects of pH, temperature, and buffer concentration on the self-assembling behavior, secondary structure, and surface hydrophobicity of donkey and bovine β-casein

The self-assembling behavior, secondary structure, and surface hydrophobicity of purified donkey β-casein in terms of pH, temperature, and buffer concentration were investigated in comparison with commercial bovine β-casein. Critical micelle concentration of both β-caseins decreased with the lowering of pH (pH 8.0–6.0) and the increasing temperatures (25–50 °C). Critical micelle temperature of both β-caseins increased moving from pH 6.0 to 8.0 and aggregates larger than micelles formed at pH 6.0 that is close to their isoelectric point. Fluorescence spectroscopy analysis demonstrated that the maximum surface hydrophobicity was achieved at pH 6.0. The secondary structure was examined using circular dichroism spectroscopy, highlighting an increase of α-helix content and a decrease of unordered structures with the decrease of pH and increase of temperature. This work provides insights on parameters promoting molecular interactions involved in donkey β-CN self-association, useful to develop nanocarriers for encapsulating bioactive compounds in pharmaceutical and nutraceutical applications.

Application of a Quality by Design Approach to Develop a Simple, Fast, and Sensitive UPLC-MS/MS Method for Quantification of Safinamide, an Antiparkinson’s Drug, in Plasma

Safinamide is an orally active, selective monoamine oxidase-B inhibitor with dopaminergic and non-dopaminergic properties approved by the European Medicine Agency and US Food and Drug Administration for the treatment of mid- to late-stage fluctuating Parkinson’s disease (PD) used in combination with other PD medications such as levodopa. In this study, an analytical quality by design (AQbD) approach was applied to optimize an LC-MS/MS bioanalytical method to determine safinamide in human plasma. A full 33 factorial design was used to optimize safinamide separation conditions, with a method first screened and optimized using chromatographic responses, including peak area and retention time. The results showed that temperature had a significant indirect effect on retention time and peak area (p < 0.05), while ammonium acetate concentration had an insignificant indirect impact on peak area or retention time. However, the temperature was significantly agonistic to the effect of buffer concentration (p < 0.05). The resultant optimized chromatography conditions utilized 9.0 mM ammonium acetate buffer and acetonitrile (22.0:78.0) as mobile phases at a column temperature of 23.2 °C. The assay was linear from 0.1–1000 ng/mL, met acceptance criteria for inter- and intra-assay precision and accuracies across three quality controls, and was successfully applied to in vitro microsomal metabolic stability. The UPLC/MS/MS method was found to be adequately sensitive and suitable for routine safinamide pharmacokinetic studies.

A Novel Approach to Monitor the Concentration of Phosphate Buffers in the Range of 1 M to 0.1 M Using a Silicon-Based Impedance Sensor.

We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination of the post-dilution concentration of the buffers is necessary for ensuring optimal buffer capacity, pH stability, and to assess solution reproducibility. In this study, we focused on phosphate buffer as the test liquid to achieve precise post-dilution concentration determinations. The impedance chip consisting of a top gold ring electrode, where a test volume of 20 μL to 30 μL of phosphate buffer was introduced for impedance measurements within the frequency range of 40 Hz to 1 MHz. For impedance investigation, we used phosphate buffers with three different pH values, and the impedance was measured after diluting the phosphate buffers to a concentration of 1.00 M, 0.75 M, 0.50 M, 0.25 M, 0.10 M, 0.05 M, and 0.01 M. In order to analyze the distinctive changes in the measured impedance, an equivalent circuit was proposed and modeled. From the impedance modeling, we report that the circuit parameter RAu/Si showed exponential dependence on the concentration of phosphate buffer and no dependence on the pH values of the phosphate buffer and on the added volume inside the ring electrode. The proposed silicon-based impedance chip is quick and uses reduced liquid volume for post-dilution concentration measurements of buffers and has perspective applications in the pharmaceutical and biological domains for regulating, monitoring, and quality control of the buffers.

Optimization and in-house validation of the analytical procedure for official control of bentonites as aflatoxin inactivators

In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) above 90%. BCAfB1 is determined by an official adsorption assay using an aflatoxin solution (4 mg/L) in acetate buffer (pH 5.0) and a bentonite at 0.02% (w/v). To date, the robustness of this method has not been investigated.In this work, we addressed this challenge and performed a robustness study by analyzing six bentonite samples that met the mineralogical requirements for claim code 1 m558. Leading factors selected for robustness testing were (1) preparation mode of bentonite suspension, (2) residual amount of acetonitrile in the test trial, (3) acetate buffer concentration, (4) incubation time, and (5) centrifugation. It was statistically evinced that factors 2 and 5 affected the results. Due to its weakness, the method excluded 4 out of six bentonites to be marketed in EU, being BCAfB1 < 90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated. This protocol yielded BCAfB1 > 90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat < 2. Its validity was proven by the isotherm approach, comparing Langmuir adsorption parameters with BCAfB1 values. Application of the protocol to bentonites other than montmorillonite was demonstrated.

Tetraethylenepentamine-derived carbon dots and tetraethylenepentamine co-immobilized silica stationary phase for hydrophilic interaction chromatography

In this work, tetraethylenepentamine (TEPA) was used as precursor and reaction medium to prepare tetraethylenepentamine-functionalized carbon dots (TEPACDs), the resultant mixture was subsequently silanized and then grafted on the surface of bare silica. The obtained tetraethylenepentamine-functionalized carbon dots and tetraethylenepentamine co-modified silica stationary phase (Sil-TEPA/CDs) was characterized by multiple ways, such as Fourier transformed infrared spectroscopy (FTIR), elemental analysis and transmission electron microscope, which revealed the successful preparation of the mixed stationary phase and higher density of functional groups on co-modified stationary phase than precursor single-modified stationary phase. The synergistic effect of TEPACDs and TEPA was proved by comparing the separation performance of Sil-TEPA/CDs and Sil-TEPA toward amino acids, nucleosides, and nucleobases, which distinctly enhanced the selectivity of Sil-TEPA/CDs. Thus, 12 nucleosides and nucleobases and 11 amino acids was nicely separated on Sil-TEPA/CDs. By study the influences of the changes of mobile phase composition, mobile phase buffer concentration and buffer pH on the retention behaviors of Sil-TEPA and Sil-TEPA/CDs, it was found that both hydrophilic partitioning and adsorption of analytes on Sil-TEPA/CDs were enhanced benefit from the co-existence of TEPA and TEPACDs, which provided the analytes better separation performance. By comparing the column quality of Sil-TEPA/CDs with four commercially available columns, Sil-TEPA/CDs exhibited the best peak asymmetry of 0.98, and second best column efficiency of 43895 m−1 using guanosine as analyte. The RSD (n = 9) of the retention times of five selected analytes on Sil-TEPA/CDs were within 0.30–0.61% during 40 h of continuously elution, which implied excellent stability of prepared packing material.

QuEChERS-assisted ion pair chromatography/fluorescence detection method for determination of antimigraine combination therapy in rabbit plasma samples: Application to a pharmacokinetic study

Antimigraine combination therapy has shown significant effectiveness in relieving pain, as well as reducing the frequency, duration, and severity of migraine attacks if compared to a single migraine medication. This work represents the first analytical investigation for emphasizing the synergistic effect of combining ophthalmic beta blockers with triptans in migraine treatment. The presented study was conducted to investigate the pharmacokinetic profile of almotriptan (ALM), a serotonin (5-HT1B/1D) receptor agonist used to treat migraine, when coadministered with timolol (TIM) or verapamil (VER) which are considered as an adjuvant therapy in migraine prevention. Ion pair chromatography (IPC) with online fluorescence detection was applied to simultaneously detect and quantify the binary mixtures of ALM/TIM and ALM/VER in rabbit plasma samples. The separation was achieved using a Platinum C18 analytical column with a mobile phase composed of methanol: 35 mmol L-1 phosphate buffer solution containing 10 mmol L-1 SDS at pH = 6.8 (60:40 v/v). Several parameters were evaluated during the optimization of separation conditions including mobile phase composition, buffer concentration, buffer pH and concentration of ion pair reagent. A thorough investigation of the retention mechanism was performed, and the results showed that Coulomb forces were the main contributors to the overall retention mechanism, which may be hydrophobically assisted. QuEChERS extraction technique was utilized to extract the investigated drugs from plasma samples and a detailed study was carried out to optimize partition/extraction solvents, pH, extraction salts, sample volume and clean-up step. The method had a limit of detection and quantitation of 5.6 and 16.9 ng mL-1 for ALM in ALM/TIM mixture and 2.5 and 7.6 ng mL-1 for ALM in ALM/VER mixture, with an overall recovery not less than 95.22%. This newly proposed method offers a faster alternative to existing chromatographic methods for extraction and determination of ALM in binary mixtures with TIM or VER in rabbit plasma and provides a platform for studying pharmacokinetic parameters. The coadministration of either TIM or VER with ALM resulted in a notable rise in Cmax (maximum plasma concentration) and AUC (area under the plasma concentration-time curve) of ALM, implying possible alterations in the absorption and overall exposure of ALM.

Enhancing biotechnological applications of l-asparaginase: Immobilization on amino-epoxy-agarose for improved catalytic efficiency and stability

l-asparaginase has been shown to be effective in reducing acrylamide formation in food and in treating certain cancers, such as acute lymphoblastic leukemia. This study aimed to improve the application of l-asparaginase in these areas, through its immobilization in two distinct supports: epoxy-agarose and amino-epoxy-agarose. Different immobilization conditions were investigated, including immobilization pH and buffer concentration. The best activity results were obtained employing amino-epoxy-agarose, being the 0.1 M sodium carbonate buffer pH 10 the conditions that offered the highest immobilization yield (76%), immobilization efficiency (89.8%) and relative activity (68.2%). Immobilization of l-asparaginase in amino-epoxy-agarose improved its catalytic efficiency and stability, including at pH 4 and 60 °C. All immobilized biocatalysts preserved more than 70% of their activity after 7 cycles of reuse, and maintained about 82% of their activity after 60 days of storage at 4°C. These results show the efficacy of immobilized biocatalysts in the preservation of enzymatic activity and highlight their potential for biotechnological applications.

Controllable Synthesis of Hemoglobin-Metal Phosphate Organic-Inorganic Hybrid Nanoflowers and Their Applications in Biocatalysis.

In recent years, organic-inorganic hybrid nanoflower technology has become an effective method for enzyme immobilization. Here, seven hierarchical flower-like hemoglobin-phosphate organic-inorganic hybrid nanomaterials (Hb-M3(PO4)2·nH2O HNFs) were synthesized through an improved universal one-pot wet-chemical method, with Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+ as inorganic components. In this synthesis process, the metal cations are successively involved in the coordination reaction with Hb and the metathesis reaction to generate phosphate precipitation. The coordination ability of metal cations and the generation rate of phosphate precipitations were evaluated, then the progress of the two chemical reactions was controlled synchronously by adjusting the phosphate buffer (PB) concentration, and finally a flower-like structure conducive to substrate diffusion and transport was obtained. Due to the conformational transformation of hemoglobin and the abundant Cu2+/Fe3+ active sites, the hemoglobin-Cu3(PO4)2·3H2O nanoflowers have extremely high catalytic activity, which is ∼14 times that of Hb. Importantly, this method is suitable for the monometallic-ionic, polymetallic-ionic and polyvalent metal-ion nanoflowers, which broadens the chemical composition and structural diversity of nanoflowers.

Bovine serum albumin and myoglobin separation on HAp

The separation of the proteins Bovine Serum Albumin (BSA) and Myoglobin (Mb) was achieved experimentally by the simulated moving bed (SMB) technology using HAp as adsorbent and by performing a buffer step-elution in the FlexSMB-LSRE unit. Batch experiments determined the BSA and Mb adsorption equilibrium using pH 7 at different buffer concentrations. Then, fixed bed experiments were also performed for both proteins using the same buffer concentration. The validity of a phenomenological mathematical model, specifically the general rate model combined with steric mass action equilibrium, was confirmed through experimental validation using fixed bed dynamic adsorption tests at various buffer concentrations. Based on the results, a separation region was computed, and suitable operating conditions for an SMB experiment were chosen from within this region. Experimental verification was performed to validate the selected operating point, and purity, recovery, and productivity of Mb on the extract were 92%, 87% and 1.04×10-3 molprotein · kgads−1· day−1, and for BSA on the raffinate were 95%, 98%, and 1.24×10-3 molprotein · kgads−1· day−1 respectively.

Influence of Phosphate Buffer on the Reaction of 3‐Nitrophenylboronic Acid with Alizarin Red S

AbstractThe influence of phosphate buffer on the reaction of boronic acid with 1,2‐diol was studied using a reaction system suitable for kinetics and the reaction of 3‐nitrophenylboronic acid (3‐NO2PhB(OH)2) with alizarin red S. The reaction was accelerated with increasing concentrations of phosphate buffer. 1H and 11B NMR spectra were recorded for four pH‐controlled solutions, each containing different boronic acids, to determine the cause of the reaction rate acceleration. The results indicated that HPO42− interacted with both 3‐NO2PhB(OH)2 and 3‐NO2PhB(OH)3−, whereas H2PO4− hardly interacted with either 3‐NO2PhB(OH)2 or 3‐NO2PhB(OH)3−. The reaction rates were enhanced by the formation of outer‐sphere complexes between RB(OH)2 and HPO42− and RB(OH)3− and HPO42−.

Fasten the analysis of metal-binding proteins with GE-ICP-MS via increasing the electrolyte concentration of the running buffer

The coupled system of column gel electrophoresis and inductively coupled plasma mass spectrometry (GE-ICP-MS) is a highly effective technique for detecting metal-binding proteins. However, it takes a long time for this method to test a single sample, which greatly limits its application. In this study, GE-ICP-MS system was optimized by adjusting the analytical conditions, including the concentration and pH of running buffer and the proportion of polyacrylamide gel. The results of the experiment showed that the migration speed of proteins in GE was enhanced by increasing the electrolyte concentration in the running buffer solution. Additionally, the ICP-MS response, which was dramatically decreased because of the change in running buffer solution, can be stabilized by adjusting pH of running buffer. Meanwhile, the optimization of polyacrylamide gel ratio allows GE-ICP-MS to maintain high resolution for proteins of similar molecular weight with increased detection speed. After increasing the concentration of running buffer by 10 times, four iodine labeled proteins were successfully separated at baseline by the GE-ICP-MS system at pH 8.0 in 40 min using a resolving gel (8%, 7 cm) and a stacking gel (4%, 1 cm), which was three times faster than the original one. Finally, the optimized method was proved by detecting a silver-binding protein in rat plasma samples. The above method provided an effective and rapid detection for metal-binding proteins in organism.

Mesoporous Silica Particle as an RNA Adsorbent for Facile Purification of In Vitro-Transcribed RNA.

Messenger RNA vaccines against SARS-CoV-2 hold great promise for the treatment of a wide range of diseases by using mRNA as a tool for generating vaccination antigens as well as therapeutic proteins in vivo. Increasing interest in mRNA preparation warrants reliable methods for in vitro transcription (IVT) of mRNA, which must entail the elimination of surplus side products such as immunogenic double-stranded RNA (dsRNA). We developed a facile method for the removal of dsRNA from in vitro transcribed RNA with mesoporous silica particles as RNA adsorbents. Various polyamines were tested for the facilitation of RNA adsorption onto mesoporous silica particles in the chromatography. Among the polyamines tested for RNA adsorption, spermidine showed a superior capability of RNA binding to the silica matrix. Mesoporous silica-adsorbed RNA was readily desorbed with elution buffer containing either salt, EDTA, or urea, possibly by disrupting electrostatic interaction and hydrogen bonding between RNA and the silica matrix. Purification of IVT RNA was enabled with the adsorption of RNA to mesoporous silica in a spermidine-containing buffer and subsequent elution with EDTA. By differing EDTA concentration in the eluting buffer, we demonstrated that at least 80% of the dsRNA can be removed from the mesoporous silica-adsorbed RNA. When compared with the cellulose-based removal of dsRNA from IVT RNA, the mesoporous silica-based purification of IVT RNA using spermidine and EDTA in binding and elution, respectively, exhibited more effective removal of dsRNA contaminants from IVT RNA. Thus, mRNA purification with mesoporous silica particles as RNA adsorbents is applicable for the facile preparation of nonimmunogenic RNA suitable for in vivo uses.

Arabinoxylan in Water through SANS: Single-Chain Conformation, Chain Overlap, and Clustering.

Using small-angle neutron scattering (SANS), we examine the structure and conformational behavior of wheat arabinoxylan (AX) prepared at various concentrations in a sodium phosphate aqueous buffer. As for another major hemicellulose, xyloglucan, we observe a small number of large clusters surrounded by AX chains that behave exactly as a polymer in good solvent with a Flory exponent ν = 0.588. The fit of the data at high q-values to a standard worm-like chain model gives the persistence length lp = 45 Å and cross section of the chains 2Rc = 11-12 Å. In addition, using a dedicated modeling approach, we extract from the SANS data at the intermediate q-range the correlation length ξ of the solutions in the semidilute regime. The decay of ξ with concentration follows a scaling law that further confirms the self-avoiding statistical behavior of the AX chains. This first comprehensive study about the properties of water-soluble AX at different length scales may help in the development of products and processes involving AX as a substitute for fossil carbon molecules.

Determination of rhein and physcion in rhubarb by microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection.

Microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection was developed for the separation and detection of physcion and rhein in rhubarb. In contrast to the conventional capillary electrophoresis method, ammonium acetate-dimethyl sulfoxide was used as the basic buffer system in this method. The effects of background buffer, buffer apparent pH*, buffer concentration, water ratio, sample preparation method, and separation voltage on separation and detection were investigated. Optimized separation and detection conditions were obtained: the buffer consisted of 20mmol/L of ammonium acetate in hydro-organic solvent composed dimethyl sulfoxide, formamide, and water mixed at 60/20/20 (v/v/v) ratio. The separation voltage was 1.9kV. Under these conditions, the physcion, rhein, and other components of rhubarb can be completely separated within 150 s. Under the methodological verification, good linearity (R ≥ 0.9995) for physcion and rhein, and low limits of detection (0.085μg·mL-1 and 0.077μg·mL-1 , respectively), satisfactory peak area precisions, migration time precisions (1.74%-3.09%), and accuracy (recovery rate 97.8% and 101.4%) were achieved. It is shown that the proposed method is simple, efficient, fast, sensitive, simple instrument, consumes few samples, has low operating cost, and is linear.

Photo-induced site-specific oxidative fragmentation of IgG1 mediated by iron(III)-containing histidine buffer: Mechanistic studies and excipient effects

Fragmentation may compromise the clinical efficacy and safety profile of monoclonal antibodies (mAbs). We recently reported that Fe(III)-containing histidine (His) buffer mediates site-specific mAb fragmentation within the Fc domain when exposed to visible light (Y. Zhang and C. Schöneich, Mol. Pharm. 2023, 20, 650–662). Here, we show that this fragmentation proceeds even more efficiently under near-UV light. Several formulation strategies were applied in an attempt to reduce the photo-induced fragmentation. In solution formulations, the fragmentation can be mitigated by reducing the concentration of His buffer, adding Fe(III)-chelating agents, and replacing His with other amino acids. Fragmentation can be almost completely inhibited by formulating the protein in the lyophilized state. Mechanistically, His plays a critical role in the fragmentation process, likely due to its affinity for Fe(II), driving a photo-redox reaction towards product formation.

Synthesis of hybrid Cu nanoflowers by using Tornabea scutellifera lichen extract, and evaluation of their dye degredation, and antioxidant activities

In this study, it was aimed to obtain Cu based organic@inorganic hybrid nanoflowers (Cu-hNFs) cheaply and effectively by using Tornabea scutellifera lichen extract as the organic component instead of expensive biomolecules such as DNA and enzymes. The effects of lichen extract concentration and pH of PBS buffer on the morphology of Cu-hNFs synthesized by the coordination of T. scutellifera lichen extract and Cu+2 were examined. It has been determined that Cu-hNFs did not synthesized under the pH ≤5 and, pH> 9 conditions of the PBS. Different morphological structures of Cu-hNFs synthesized under the pH 7.4 and, 9 conditions of PBS have been identified. In addition, it has been observed that the extract concentration used as an organic component has a significant effect on the morphology of Cu-hNFs. It has been recorded that synthesized Cu-hNFs have antioxidant activity against DPPH (IC50 = 0.98 mg/ml, R² = 0.9834). In addition, catalytic activity was demonstrated depend on pH of PBS and, concentration of Cu-hNFs against methylene blue and brillant blue. It is thought that these research data will be a guide for the synthesis and application potentials of various nanomaterials with T. scutellifera extract.

Spatio temporal interdependent calcium and buffer dynamics regulating DAG in a hepatocyte cell due to obesity.

Calcium ions (Ca2+) serve as a crucial signaling mechanism in almost all cells. The buffers are proteins that bind free Ca2+ to reduce the cell's Ca2+ concentration. The most studies reported in the past on calcium signaling in various cells have considered the buffer concentration as constant in the cell. However, buffers also diffuse and their concentration varies dynamically in the cells. Almost no work has been reported on interdependent calcium and buffer dynamics in the cells. In the present study, a model is proposed for inter-dependent spatio-temporal dynamics of calcium and buffer by coupling reaction-diffusion equations of Ca2+ and buffer in a hepatocyte cell. Boundary and initial conditions are framed based on the physiological state of the cell. The effect of various parameters viz. inositol 1,4,5-triphosphate receptor (IP3R), diffusion coefficient, SERCA pump and ryanodine receptor (RyR) on spatio-temporal dynamics of calcium and buffer regulating diacylglycerol (DAG) in a normal and obese hepatocyte cell has been studied using finite element simulation. From the results, it is concluded that the dynamics of calcium and buffer impact each other significantly along the spatio-temporal dimensions, thereby affecting the regulation of all the processes including DAG in a hepatocyte cell. The proposed model is more realistic than the existing ones, as the interdependent system dynamics of calcium and buffer have different regulatory impacts as compared to the individual and independent dynamics of these signaling processes in a hepatocyte cell.

Investigating freezing-induced acidity changes in citrate buffers

Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.