Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Characterization of Bostrycoidin: An Analytical Analog of Zearalenone

: Twenty-nine strains from Fusarium section Liseola were screened for a zearalenone-like compound using analytical thin-layer chromatography (TLC). One metabolite had the same retention time on TLC and a similar multi-peak ultraviolet (UV) spectrum to zearalenone. Mass spectral and nuclear magnetic resonance (1H, 13C, 1H-13C heteronuclear multiple-quantum coherence) analysis gave information that helped identify the compound as the Fusarium pigment 8-O methyl bostrycoidin. The pigment was readily resolved from zearalenone with reversed-phase high-performance liquid chromatography analysis. Because this compound has a similar retention time, care should be exercised when using acid charring to visualize spots during zearalenone TLC analysis.

Experimental design in reversed-phase high-performance liquid chromatographic analysis of imatinib mesylate and its impurity

For the determination of the optimal RP-HPLC chromatographic conditions for the separation of imatinib mesylate and its impurity STI 509-00 experimental design 2 4 was applied. All the factors that affect imatinib mesylate/STI 509-00 separation, as well as their mutial interations were investigated. Methanol and triethylamine content in the mobile phase, pH of the mobile phase and column temperature were independent variables or factors to be investigated in two levels: “low” and “high”. Capacity factor was chosen as a dependent variable. From the experimentally determined capacity factor values, it was defined the factors that affect to chromatographic system the most. Applying response surface methodology the appropriate graphs were constructed from experimental points and optimal chromatographic conditions for the separation were defined. Optimal conditions for the separation of imatinib mesylate and STI 509-00 were obtained using X Terra 150 mm×4.6 mm, particle size 5 μm column at 25 °C. Mobile phase consisted of 250 ml of methanol, 740 ml of water and 10 ml of triehylamine. pH of water phase was adjusted to 2.4 with 85% orthophosphoric acid and than methanol was added.

Ontogeny of a novel decapeptide derived from POMC-A in the brain and pituitary of the rainbow trout

Trout POMC-A exhibits a unique C-terminal extension of 25-amino acids which is processed in the pituitary and hypothalamus to generate two novel decapeptides, EQWGREEGEE and ALGERKYHFQ-NH 2. The fibers containing these two decapeptides are widely distributed in the brain, suggesting that they may exert neurotransmitter or neuromodulator activities. In the present study, we have investigated the ontogeny of the decapeptide EQWGREEGEE in the trout pituitary and brain. In the pituitary of 29-day embryos and 33-day alevins, EQWGREEGEE-immunoreactive material was observed in a cluster of cells located in the central and rostral region of the gland, respectively. In 47-day alevins, a second group of cells exhibiting EQWGREEGEE-like immunoreactivity was detected in the caudal region of the pituitary and the intensity of labeling in these cells increased in 61-day fry. In the brain, EQWGREEGEE immunoreactivity was detected in 47-day alevins. In 47- and 61-day larvae, immunoreactive elements were mainly detected in the diencephalon. Characterization of the immunoreactive material by reversed-phase high-performance liquid chromatographic analysis combined with radioimmunoassay detection revealed the existence of two major forms which exhibited different retention times than synthetic EQWGREEGEE. The present study indicates that EQWGREEGEE-related peptides are present in the trout pituitary early during ontogeny and appear in the brain only later, and that processing of the C-terminal extension of POMC-A generates distinct molecular species at different developmental stages. These data suggest that alternative processing of the C-terminal domain of POMC-A gives rise to various peptide products that may exert specific activities during trout development.

Development and validation of an automated extraction method (accelerated solvent extraction®) and a reverse-phase HPLC analysis method for assay of ivermectin in a meat-based chewable formulation

A new method for monitoring ivermectin content in HEARTGARD CHEWABLES® has been developed and validated. The method consists of the automated extraction of ivermectin from the meat-based formulation under conditions of elevated temperature and pressure (accelerated solvent extraction, ASE®), and determination of the active by reverse-phase high performance liquid chromatography (HPLC). The method resolves both active species of ivermectin (components H 2B 1a and H 2B 1b) from the formulation matrix.

Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin

Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein’s affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.

Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol–water partition coefficients

The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol–water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5–210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb’s Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol–water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation ( r=0.87) between drug lipophilicity, expressed as octanol–water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument. Index Descriptors and Abbreviations: Transtegumental diffusion; Benzimidazole anthelmintics; Cestodes; Moniezia benedeni; Lipophilicity; Octanol–water partition coefficient

Dietary toxicity of decomposed arborescent leaf litter against larval mosquito: involvement of a lignin-polypeptidic complex.

To characterize the toxic compounds involved in the dietary toxicity of decomposed arborescent leaf litter against larval mosquito, a toxic fraction was extracted from crude leaf litter by using hot water. Preliminary characterization of this fraction, called the insoluble fraction (IF) because it progressively precipitates after extraction, has suggested the involvement of lignin-like compounds in the toxicity. Further analyzes are currently being performed by using additional phytotoxicity-based methods. The involvement of lignin-like compounds in the toxicity was indicated by both the comparative effects of different enzymatic oxidative treatments and reversed-phase high-performance liquid chromatography analysis of the phenolic aldehydes and acids obtained after alkaline nitrobenzene oxidation. However, these lignin-like compounds may not be involved alone in the toxicity, as no specific feature of those components was associated with the toxicity. Among the possible compounds associated with lignin-like compounds in the toxicity, peptidic compounds were suggested by comparative determination of the C/N ratio and then revealed by denaturation experiments, use of specific binding protein molecules, and thin-layer chromatography analysis. A possible role of these peptidic compounds associated with lignin-like compounds in the dietary toxicity of the leaf litter against the larval mosquito midgut is discussed.

Simple method for reversed-phase high-performance liquid chromatographic analysis of fungal pigments in fruit-bodies of Boletales (Fungi)

A reversed-phase HPLC method has been developed for the analysis of hydroxylated pulvinic acid derivatives which are responsible for the pigmentation of fruit-bodies belonging to the order Boletales (Fungi). Variegatic, xerocomic and atromentic acid as well as variegatorubin were detected and separated in methanolic extracts of Boletus permagnificus and Xerocomus parasiticus, and the pigment profile of these species was compared. The identity of the pigments was confirmed by means of LC–atmospheric pressure chemical ionization (APCI) mass spectrometry.

Aldosterone augments adrenomedullin production without stimulating pro-adrenomedullin N-terminal 20 peptide secretion in vascular smooth muscle cells.

Both adrenomedullin (AM) and pro-adrenomedullin N-terminal 20 peptide (PAMP), processed from the same precursor of prepro-adrenomedullin (preproAM), have differential biological properties; AM dilates blood vessels and presumably affects the vascular remodeling, while PAMP inhibits catecholamine secretion. Since aldosterone has been shown to be involved in vascular remodeling, we examined the effects of aldosterone on AM and PAMP secretion and preproAM gene expression in human aortic vascular smooth muscle cells (VSMC). AM and PAMP secreted from human VSMC incubated with aldosterone were measured by radioimmunoassay, and preproAM gene expression was evaluated by quantitative polymerase chain reaction. Cultured human VSMC secreted both AM and PAMP into the media, while the secretion rate of AM was much higher than that of PAMP. Aldosterone increased preproAM gene expression in the cultured VSMC in a dose-dependent fashion following incubation for 48 h, with a concomitant increase in AM secretion from the cells, but PAMP secretion remained unchanged. Aldosterone-stimulated AM secretion was significantly reduced by spironolactone. Reverse-phase high-performance liquid chromatography analyses showed that immunoreactive AM secreted from the VSMC untreated or treated with aldosterone emerged at the point of human AM(1-52)-NH2. AM production was stimulated by aldosterone in cultured human VSMC without an increase in PAMP secretion, suggesting a possible role of AM in modulating vascular remodeling by aldosterone.

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C 18 stationary phases. A small sorbent particle size (2.5 μm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA–HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Partition behavior of virgin olive oil phenolic compounds in oil-brine mixtures during thermal processing for fish canning.

The chemical modifications and partitioning toward the brine phase (5% salt) of major phenol compounds of extra virgin olive oil (EVOO) were studied in a model system formed by sealed cans filled with oil-brine mixtures (5:1, v/v) simulating canned-in-oil food systems. Filled cans were processed in an industrial plant using two sterilization conditions commonly used during fish canning. The partitioning of phenolic compounds toward brine induced by thermal processing was studied by reversed-phase high-performance liquid chromatographic analysis of the phenol fraction extracted from oils and brine. Hydroxytyrosol (1), tyrosol (2), and the complex phenolic compounds containing 1 and 2 (i.e., the dialdehydic form of decarboxymethyl oleuropein aglycon 3, the dialdehydic form of decarboxymethyl ligstroside aglycon 4, and the oleuropein aglycon 6) decreased in the oily phase after sterilization with a marked partitioning toward the brine phase. The increase of the total amount of 1 and 2 after processing, as well as the presence of elenolic acid 7 released in brine, revealed the hydrolysis of the ester bond of hydrolyzable phenolic compounds 3, 4, and 6 during thermal processing. Both phenomena (partitioning toward the water phase and hydrolysis) contribute to explain the loss of phenolic compounds exhibited by EVOO used as filling medium in canned foods, as well as the protection of n-3 polyunsaturated fatty acids in canned-in-EVOO fish products.

Profiling solid-phase synthesis products by free-solution conjugate capillary electrophoresis.

Solid-phase synthesis of oligomers, both natural and nonnatural, has proved to be invaluable for the development of many areas of biotechnology. A critical step in the solid-phase synthesis of any oligomer is determining the number and concentration of different constituents present in the product mixture resulting from the synthesis, both before and after purification. Most typically, this analysis is performed by reversed-phase high performance liquid chromatography (RP-HPLC), with the separated components detected by UV absorbance. Recently, we described a novel technique, free-solution conjugate electrophoresis (FSCE), for the high-resolution separation and sensitive laser-induced fluorescence (LIF) detection of uncharged, synthetic polymers, PEG in particular. In this report, we apply this bioconjugate capillary electrophoresis technique to analyze products of the solid-phase synthesis of oligomeric polyamides, namely poly(N-substituted glycines), or polypeptoids. When compared to more traditional RP-HPLC analysis, FSCE analysis of oligomeric peptoids results in separation resolutions that are approximately five times higher and separation efficiencies that are increased by 150%. Moreover, when FSCE with LIF detection is applied to the analysis of oligomeric polyamides after HPLC purification, impurities that are not detectable in RP-HPLC analysis are readily separated and detected. With the advent of capillary array electrophoresis (CAE), which allows for automated, parallel analysis of many different samples, we believe that FSCE will be especially applicable to the analysis of combinatorial synthesis products, by allowing researchers to evaluate many different samples in a single, highly parallel, fully automated analysis. This is in contrast to RP-HPLC analysis, in which samples must be analyzed in series.

Phosphorylation of beta-D-ribosylbenzimidazoles is not required for activity against human cytomegalovirus.

We have previously reported that 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(beta-D-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5'-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [(3)H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5'-monophosphate (BDCRB-5'-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5'-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5'-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5'-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5'-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5'-deoxy analog (5'-dTCRB) did not affect viral DNA synthesis, but 5'-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5'-dTCRB and the 5'-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5'-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5' position for antiviral activity.

HPLC ANALYSIS OF GINSENOSIDES IN THE ROOTS OF ASIAN GINSENG (PANAX GINSENG) AND NORTH AMERICAN GINSENG (PANAX QUINQUEFOLIUS) WITH IN-LINE PHOTODIODE ARRAY AND EVAPORATIVE LIGHT SCATTERING DETECTION

In-line connected ultraviolet (UV) and evaporative light scattering (ELS) detection were compared in the reversed-phase high-performance liquid chromatographic (HPLC) analysis of ginsenosides in ginseng roots. Similar to all the QA/QC literature on ginseng and ginseng products, employing HPLC-UV, the UV detection showed a good linearity in the range of 100–2000 ng of ginsenosides, with minimum detectable concentration of 10 ng of ginsenosides on the column. In contrast, the sensitivity of the ELSD was found to be 5 times lower than that obtained with the UV, with minimum detectable concentration of 50 ng of ginsenosides on the column. Further, the calibration curve obtained with the ELSD was exponential linear in the range of 200–2000 ng of ginsenosides. Quantitative evaluation in terms of ginsenosides using both methods of detection showed similar results. But, in view of the better sensitivity and easier handling, the UV is still recommended for the routine analysis of ginseng samples.

Biochemical and molecular characterisation of Glu-1 loci in Argentinean wheat cultivars.

The high molecular weight glutenin subunit (HMW-GS) composition of a collection of 107 Argentinean bread wheat cultivars was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Allelic variation at the Glu-1 loci was identified and its frequency calculated. Eleven alleles were detected, three encoded at the Glu-A1 locus, six at the Glu-B1 locus and two at the Glu-D1 locus. A low frequency of the null allele at the Glu-A1 locus and a high frequency of subunits 5+10 at the Glu-D1 locus were observed. Reversed phase-high performance liquid chromatography (RP-HPLC) analysis was used to further characterise HMW-GS. Two different types of Bx subunit 8 (named subunits 8 and 8*) were detected, the latter having shorter elution time. Subunit 8* was not identifiable by SDS-PAGE. According to quantification by RP-HPLC analysis, two groups of subunit 7 were observed. One group, with a relatively high proportion of subunit 7 (approximately 39% of the total amount of HMW-GS) appeared in cultivars with allele 7+8* at the Glu-B1 locus; a second group of subunit 7 (around 24% of the total amount of HMW-GS), was found in alleles 7+8, 7+8* and 7+9. Restriction fragment length polymorphisms (RFLP) analyses of HMW-GS genes were also carried out after digestion of genomic DNA with HindIII and TaqI restriction enzymes. The relationship between DNA fragment size and glutenin subunits, as estimated by electrophoretic mobility in SDS-PAGE, was also examined. The restriction enzyme TaqI demonstrated to be a useful tool to detect homozygous plants in selection programs against the Glu-A1 null allele.

Phytoplankton pigments designation: an application of RP-HPLC in qualitative and quantitative analysis

An account is given of the use of RP-HPLC (reverse-phase high performance liquid chromatography) analysis of high purity chlorophylls and carotenoids standards originating from reference phytoplankton monocultures. All important chromatography parameters were established: retention time, detection limit, spectrum, wavelength of maximum absorption, calibration curve, response factors. Data were also obtained for pigment mixtures in naturalphytoplankton populations in Spitsbergen and the Gulf of Gdansk. The identity and purity of all pigments were confirmed by co-elution with standards, including canthaxanthin as an internal standard. The data presented enable the qualitative and quantitative analysis of naturally existing phytoplankton pigments at concentrations down to 0.1 μg L-1 to be carried out without the need for standards from commercial sources.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M+H] + ion at m/ z 1141.58 which co-eluted with activity that displaced [ 3H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean.

Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to analyze various fatty acids in recombinant mouse L cells. These fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C18RP-HPLC column using a gradient of acetonitrile (ACN)-H2O. The eluted fatty acids were monitored by ultraviolet (UV) absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolved various fatty acids and provided a tool for the elucidation of the fatty acid metabolic pathway in the cells.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY INTERCOMPARATIVE STUDY FOR AMINO ACID ANALYSIS IN TWO TISSUES BY PITC- AND OPA-DERIVATIZATIONS

In this paper, we have extensively investigated the pre-column derivatization method with o-phthaldialdehyde (OPA) to analyze cysteine, cystine, among with other 18 amino acids, using reversed-phase high-performance liquid chromatography analysis (RP-HPLC), and after we have compared it with an other method involving phenyl-isothiocyanate (PITC) in order to have a two sensitive methods to use accordingly the matrix. We have studied the analytical conditions for the OPA-reaction together with precision, linearity range, correlation coefficient (>0.998) and limit of detection (between 14 fmol and 1 pmol) for each amino acid. Then, we have applied the two methods to the analysis of larynx and kidney tissues: between the two methods, the amino acid recoveries (between 80% and 100%) and the correspondence of the OPA and PTC results are satisfactory considering the different involved derivatization procedures. PTC- and OPA-chromatograms obtained in the analysis of actual samples are reported together with the amino acid level, with particular regard to the cysteine/cystine determination.

Proteolysis in reduced sodium Kefalograviera cheese made by partial replacement of NaCl with KCl

Abstract Kefalograviera cheeses (five trials) of different sodium content were made from split lots of curd by varying the salting processes, i.e. brine — and dry — salting, with NaCl (control) or a mixture of NaCl/KCl (3:1 or 1:1, w/w basis). The extent and characteristics of proteolysis in the cheeses were monitored during aging by Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen [WSN], trichloroacetic acid [TCA]-SN, phosphotungstic acid [PTA]-SN), the cadmium-ninhydrin method for the determination of total free amino acids (FAA), urea-polyacrylamide gel electrophoresis of cheese proteins, followed by densitometric analysis of the αs1- and β-casein fractions, reverse-phase high-performance liquid chromatography (HPLC) analysis of the water-soluble extracts of cheeses, and ion-exchange HPLC analysis of FAA. The results showed that proteolysis was similar in control and experimental cheeses at all sampling ages, indicating that the partial substitution of NaCl by KCl in the manufacture of Kefalograviera cheese did not significantly influence the extent and characteristics of proteolysis during cheese aging.