ABSTRACT
High hydrostatic pressure is an alternative to thermal processing to inactivate spoilage and pathogenic microorganisms. Cashew apple juice has a pleasant flavor and is rich in vitamin C. Studies to determine the effect of high pressure on microorganisms in cashew apple juice are still lacking. In this study, the inactivation of natural micropopulation and inoculated Escherichia coli by high pressure was evaluated in fresh cashew apple juice. The microbiological stability of pressure-treated juice was also evaluated. The applied high pressure levels ranged from 250 to 400 MPa for periods of 3 to 7 min. Treatments with 350 MPa for 7 min and 400 MPa for either 3 or 7 min reduced the aerobic mesophilic bacteria count to a level below the detection limit. Pressure treatments were also efficient in inactivating yeast and filamentous fungi. The inoculated E. coli (10(6) CFU/mL) was reduced to below 10 CFU/mL after a pressure treatment of 400 MPa for 3 min. The inactivation of this microorganism followed a 1st-order reaction kinetics. The decimal reduction time (D-value) ranged from 1.21 to 16.43 min, while pressure resistance value (z-value) was 123.46 MPa. Neither natural micropopulation growth nor E. coli repair was observed in postprocessed (400 MPa for 3 min) cashew apple juice kept under refrigerated storage (at 4 degrees C) during 8 wk. The results of this study demonstrated the efficacy of high-pressure treatment for preserving cashew apple juice.
Subject(s)
Anacardium/microbiology , Bacteria/growth & development , Beverages/microbiology , Food Preservation/methods , Fungi/growth & development , Hydrostatic Pressure , Colony Count, Microbial , Food Contamination/analysis , Food Handling/methods , Food Microbiology , Kinetics , Time FactorsABSTRACT
The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.
Subject(s)
Lactoglobulins/chemistry , Animals , Cattle , Crystallography, X-Ray/methods , Genetic Variation , Lactoglobulins/genetics , Models, Molecular , Protein Conformation , Reproducibility of ResultsABSTRACT
Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.
Subject(s)
Lactoglobulins/chemistry , Animals , Cattle , Guanidine/pharmacology , Hydrogen-Ion Concentration , Lactoglobulins/genetics , Models, Molecular , Pressure , Protein Conformation , Protein Denaturation , Protein Folding , Protein Isoforms/chemistry , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.
