Decreased Heat Stability Research Articles

Relationship between whey protein nitrogen index of skim milk powder and the heat stability of recombined filled evaporated milk

In this study, it was evaluated whether the whey protein nitrogen index (WPNI) of a range of skim milk powder (SMP) samples was correlated with the heat stability of the corresponding recombined filled evaporated milk (RFEM), as well as with the extent of lactosylation and changes in whey proteins (WPs). Pulsed field gradient NMR results showed that the diffusion coefficients of lactose in all samples varied only to a small extent, while the diffusion coefficients of the WP fraction ranged more widely. A lower heat stability of RFEM produced from SMP with a lower WPNI was found as compared with those with a higher WPNI: principal component analysis (PCA) and correlation analysis revealed that the decreased heat stability could be at least partly explained by the increased calcium solubilization induced by the lower pH of dispersions of SMP with a lower WPNI. Although it was claimed over the years that high-heat SMP (with a low WPNI) is beneficial to ensure a better heat stability of RFEM, it is clear from our results that a low WPNI of SMP alone was not sufficient to guarantee a better RFEM-heat stabilizing functionality.

The heat stability of Rhamnolipid containing egg-protein stabilised oil-in-water emulsions

The heat stability of egg white stabilised emulsions was studied in the presence of monoglycerides, lecithin and rhamnolipid biosurfactants. Rhamnolipids have a greater effect on the reduction of emulsion droplet particle size than either lecithin or monoglycerides, with both the latter having virtually no effect even at high concentration. The effect of all surfactants on heat stability is concentration dependent. There is trend towards a minimum in heat stability (represented by the first order rate constant, k) for emulsions stabilised by all surfactants at a molar ratio (R) of egg protein to surfactant of R = 50 for monoglycerides, R = 34 for lecithin and R = 61 for rhamnolipids. However, this trend is only statistically significant for the rhamnolipid emulsions. The largest decrease in heat stability is observed for the rhamnolipid stabilised emulsion. The rhamnolipid biosurfactant is also the only one of the three surfactants that is able to provide heat stability to the emulsion at high molar ratios, presumably due to competitive displacement of the protein from the emulsion droplet surface. Analysis of the thermodynamics of the heat-induced destabilization of the emulsions suggests differing mechanisms for the three surfactants, probably related to the way in which the different surfactants interact with the adsorbed protein layer. Molecular docking and molecular dynamics simulations confirm different binding affinity to egg proteins for the rhamnolipid and lecithin surfactants.

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.

Comparison of heat stability of cow's milk subjected to ultra‐high temperature and in‐container sterilisation

Cow's milk of different fat contents with or without stabilising salts was sterilised by in‐container and UHT sterilisation. Heat stability decreased with increasing fat content following both UHT and in‐container sterilisation. Adding small amounts of stabilising salts (6.4 mM) increased heat stability in UHT‐treated milk, while excessive addition (12.8 mM) decreased heat stability. In contrast, increasing stabilising salts from 6.4 to 12.8 mM caused a decrease in heat stability after in‐container sterilisation. Increasing in‐container sterilisation time from 10 to 30 min caused a decrease in heat stability arising from an increase in particle size. This study confirmed discrepancies in heat stability for two sterilisation procedures.

Negative charge at aspartate 151 is important for human lens αA-crystallin stability and chaperone function

Aggregation of lens protein is a major cause of senile cataract. Lens crystallins contain many kinds of modification that accumulate over lifespan. In particular, isomerization of Asp 151 in αA-crystallin has been found in aged lenses; however, its significance is unknown. The purpose of this study was to determine the effects of isomerization of Asp 151 in αA-crystallin. Trypsin digestion followed by liquid chromatography–mass spectrometry analysis of the water-soluble high molecular weight (HMW) fraction from human lens samples showed that isomerization of Asp 151 in αA-crystallin is age-independent, and that 50% of isomerization occurs shortly after birth. However, the extent of Asp 151 isomerization varied with the size of αA-crystallin oligomer species separated from the HMW fraction from aged lens. To evaluate the effects of modification, Asp 151 of αA-crystallin was replaced by glycine, alanine, isoleucine, asparagine, glutamate, or lysine by site-directed mutagenesis. All substitutions except for glutamate decreased heat stability and chaperone function as compared with wild-type αA-crystallin. In particular, abnormal hydrophobicity and alteration of the charge state at Asp 151 caused loss of stability and chaperone activity of αA-crystallin; these properties were recovered to some extent when the mutant protein was mixed 1:1 with wild-type αA-crystallin. The results suggest that, by itself, age-independent isomerization of Asp 151 in αA-crystallin may not contribute to cataract formation. However, the long-term deleterious effect of Asp 151 isomerization on the structure and function of αA-crystallin might cooperatively contribute to the loss of transparency of aged human lens.

Synthesis of 2-Hydroxypropyl Trimethylammonium Chloride Chitosan and its Application in Bamboo Fiber Paper

To obtain a functional composite paper with antibacterial activity, 2-hydroxypropyl trimethylammonium chloride chitosan (HACC) was prepared and sprayed onto the surface of paper made of bamboo fiber. HACC was synthesized from chitosan with 2,3-epoxypropyl trimethylammonium chloride (GTAMC), and the optimal preparation conditions were selected by single-factor tests. The highest degree of substitution (DS) of HACC reached 0.868, when the ratio of chitosan to GTAMC was 1:4, and the water solubility was noticeably improved. The structural characterization demonstrated the successful modification on the original chitosan with a decrease in heat stability and the peak correlated with hydroxypropyl trimethylammonium chloride groups in FTIR. The addition of HACC in the bamboo fiber paper greatly increased the antibacterial activity, and water absorption was higher as well. These results may serve as a basis for the modification and the preparation of chitosan antibacterial agents.

Aggregated whey proteins and trace of caseins synergistically improve the heat stability of whey protein-rich emulsions

Heat treatments are used to extend the shelf life of manufactured food emulsions, which in turn require excellent heat stability. Whey protein aggregation prior to homogenization is a means to modify emulsion heat stability but the underlying mechanism of heat stabilization has hardly been studied in an industrial context where whey protein ingredients contain caseins. Emulsions were prepared with 30% anhydrous milk fat and 70% whey protein/casein solutions with protein concentrations ranging from 3 to 6%. The proteins were either unheated (WP/Cas samples) or heat-aggregated (A-WP/Cas samples). After homogenization, the fat droplet interface was characterized and emulsion stability was analyzed visually and at microscopic level. WP/Cas emulsions were heat stable at low protein concentrations but exhibited a gradual decrease in heat stability when the protein concentration increased (>3%). This instability was due to the co-gelation of the protein-coated fat droplets and the proteins in the dispersing phase. In contrast, A-WP/Cas emulsions were rapidly heat destabilized at low protein concentrations (<4%) but were more heat stable than WP/Cas emulsions at higher protein concentrations. The stability of the A-WP/Cas emulsions at protein concentrations higher than 4% was correlated with the heat stability of the whey protein aggregates in the dispersing phase and the decrease in the proportion of whey protein aggregates at the oil/water interface due to increasing competition with caseins present in the whey protein ingredient. This study contributes to greater understanding of the functional role of aggregated whey proteins and residual caseins in emulsions stabilized by industrial WP ingredients.

Effect of peppermint and citronella essential oils on properties of fish skin gelatin edible films

Fish skin gelatin films incorporated with peppermint and citronella essential oils at difference concentrations (10, 20 and 30% w/w) were prepared by solution casting. Addition of peppermint oil contributed to a significant decrease of tensile strength and Young's modulus, while the percent elongation at break showed an obvious increase except at 30% w/w. On the other hand, addition of citronella oils promoted a great increase of tensile strength and young's modulus, but an intense decrease of the percent elongation at break. At the predetermined content, the film incorporated with citronella oils outperformed the one with peppermint oils in term of water vapor transmission and solubility in water. Thermal properties of gelatin films with citronella oils exhibited an enhancement in heat stability, while the one with peppermint oils showed slight decrease in heat stability. The additions with both of essential oils exhibited excellent antibacterial properties against both Staphylococcus aureus and Escherichia coli.

Perbandingan Karakteristik Kimia Trypsin Inhbibitor Biji dan Kecambah Kedelai

This study investigated the changes in trypsin inhibitor (TI) activity, amino acid composition and protein patterns after 36 hours germination of soybeans. TI was extracted by a combination of pH precipitation and centrifugation. TI activity of seeds and germinated soybean were 1.64 and 1.68 IU/mg seeds (db) respectively. Heating of crude TI decreased heat stability of TI. The decrease of TI activity of germinated soybeans due to heating was lower than seeds. The amino acid composition and protein patterns of soybeans changed after 36 hours germination. Thr, Ala, Trp, Met and Lys decreased significantly, while Asp, Glu, Val, Phe, and Ile increased significantly. The SDS-PAGE analysis showed that the seeds contain high-molecular weight of proteins which were degraded after 36 hours germination result in smaller molecular weight of proteins.

Fortification of reconstituted skim milk powder with different calcium salts: Impact of physicochemical changes on stability to processing

Reconstituted skim milk powder (RSMP) was fortified with 12.5 mM/L calcium (Ca) using soluble [Ca chloride (CChlor), Ca gluconate (CGluc) or Ca hydroxide (CHyd)] or insoluble [Ca carbonate (CCarb), Ca phosphate (CPhos) or Ca citrate (CCit)] salts. CPhos and CCit decreased heat stability moderately at 140 °C, while CCarb had no effect. Soluble salts had a pronounced destabilising effect at 140 °C due to increased ionic Ca levels. After a laboratory‐scale high‐temperature short‐time heating process, CHyd‐fortified RSMP had a lower viscosity than all other samples. CChlor and CGluc increased sedimentation during accelerated physical stability testing, with CHyd causing greater sedimentation than CChlor or CGluc.

Heat stability of reconstituted, protein-standardized skim milk powders

We determined the effects of standardization material, protein content, and pH on the heat stability of reconstituted milk made from low-heat (LH) and medium-heat (MH) nonfat dry milk (NDM). Low-heat and MH NDM were standardized downward from 35.5% to 34, 32, and 30% protein by adding either edible lactose powder (ELP) or permeate powder (PP) from skim milk ultrafiltration. These powders were called standardized skim milk powders (SSMP). The LH and MH NDM and SSMP were reconstituted to 9% total solids. Furthermore, subsamples of reconstituted NDM and SSMP samples were set aside to measure heat stability at native (unadjusted) pH, and the rest were adjusted to pH 6.3 to 7.0. Heat stability is defined as heat coagulation time at 140°C of the reconstituted LH or MH NDM and SSMP samples. The entire experiment was replicated 3 times at unadjusted pH values and 2 times at adjusted pH values. At an unadjusted pH, powder type, standardization material, and protein content influenced the heat stability of the samples. Heat stability for reconstituted LH NDM and SSMP was higher than reconstituted MH NDM and SSMP. Generally, decreased heat stability was observed in reconstituted LH or MH SSMP as protein content was decreased by standardization. However, adding ELP to MH SSMP did not significantly change its heat stability. When pH was adjusted to values between 6.3 and 7.0, powder type, standardization material, and pH had a significant effect on heat stability, whereas protein content did not. Maximum heat stability was noted at pH 6.7 for both reconstituted LH NDM and SSMP samples, and at pH 6.6 for both reconstituted MH NDM and SSMP samples. Furthermore, for samples with adjusted pH, higher heat stability was observed for reconstituted LH SSMP containing PP compared with reconstituted milk from LH SSMP containing ELP. However, no statistical difference was observed in the heat stability of reconstituted milk from MH NDM and MH SSMP samples. We conclude that powder type (LH or MH) and effect of standardization material (ELP or PP) can help explain differences in heat stability. The difference in the heat stability of powder type may be associated with the difference in the pH of maximum heat stability and compositional differences in the standardization material (ELP or PP).

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

Biochemical and physiological analyses of a hemolytic toxin isolated from a sea anemone Actineria villosa

A species of venomous sea anemone Actineria villosa was recently found inhabiting the coastal areas of Okinawa, Japan. This marine animal produces various proteinous toxins, so that a local health organization was called for medical treatment for those who had accidental contact with this animal. In this study we analyzed the biochemical and physiological properties of hemolytic protein from A. villosa. The toxin purified from the tentacles of the animals was found to be a protein with a molecular weight of approximately 19 kDa. We named this newly found hemolytic toxin of A. villosa, Avt-I. Incubation of the toxin with sphingomyelin inhibited hemolytic activity by up to 85%, showing that Avt-I may target sphingomyelin on the erythrocyte membrane. The hemolytic activity was stably maintained at temperatures below 45 °C, however, a sharp linear decrease in heat stability was observed within the range of 45–55 °C. Our results provide the first evidence that A. villosa produces a toxin with strong hemolytic activity similar in biochemical and physiological properties to other members of actinoporin family previously isolated from related species of sea anemones.

The maize Sh2r6hs ADP-glucose pyrophosphorylase (AGP) large subunit confers enhanced AGP properties in transgenic wheat ( Triticum aestivum)

The rate of cereal seed starch biosynthesis is determined largely by the activity of starch biosynthetic enzymes within the endosperm. The heterotetrameric allosteric enzyme ADP-glucose pyrophosphorylase (AGP) controls the first committed step in starch synthesis, and is inhibited by phosphate (P i) and activated by 3-PGA. An altered AGP large subunit ( Sh2r6hs) from maize containing two alterations relative to the wild-type maize Sh2 sequence was introduced into wheat. The rev6 alteration confers reduced P i inhibition and decreased heat stability in maize and the hs alteration has been shown to increase AGP subunit interactions and enzyme stability in Escherichia coli. We have previously shown that low-level expression of Sh2r6hs under control of the maize Sh2 promoter increases yield in wheat and rice. Our objective here was to examine how Sh2r6hs affects AGP activity and yield in wheat and whether further yield increases were possible with greater Sh2r6hs expression. We therefore developed an additional population of transgenic wheat expressing SH2R6HS under control of the endosperm specific wheat Glutenin ( Glu) promoter. Yield increases in high-expressing Glu promoter Sh2r6hs lines were comparable to those with the Sh2 promoter despite the fact that the Glu promoter confers ∼20 times higher levels of Sh2r6hs expression than the Sh2 promoter and five times more SH2R6HS accumulation versus native wheat large AGP subunits. However, the high expression allowed endosperm AGP kinetic analysis. The I 0.5 of wheat AGP was increased from 2.0 to 4.1 mM P i by SH2R6HS expression and the A 0.5 was decreased from 0.82 to 0.31 mM 3-PGA. Two properties of Sh2r6hs AGP were identified that indicate greater subunit stability: wheat AGP small subunit levels were higher without variation in small subunit transcript levels, and in vitro activity was twice as great as untransformed AGP activity after heat treatment. Both r6 and hs alterations act independently and synergistically to modify AGP allosteric-regulation, increase AGP subunit stability, and increase yield in plants expressing Sh2r6hs.

Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring.

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

Deletion of leucine 61 in glucose-6-phosphate dehydrogenase leads to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections

AbstractIn this study the blood cells of 4 male patients from 2 unrelated families with chronic nonspherocytic anemia and recurrent bacterial infections were investigated. The activity of glucose-6- phosphate dehydrogenase (G6PD) in the red blood cells and in the granulocytes of these patients was below detection level. Moreover, their granulocytes displayed a decreased respiratory burst upon activation. Sequencing of genomic DNA revealed a novel 3–base pair (TCT) deletion in the G6PD gene, predicting the deletion of a leucine at position 61. The mutant G6PD protein was undetectable by Western blotting in the red blood cells and granulocytes of these patients. In phytohemagglutinin-stimulated lymphocytes the G6PD protein was present, but the amount of G6PD protein was strongly diminished in the patients' cells. Purified mutant protein from an Escherichia coli expression system showed decreased heat stability and decreased specific activity. Furthermore, we found that the messenger RNA of G6PD180-182delTCT is unstable, which may contribute to the severe G6PD deficiency observed in these patients. We propose the name “G6PD Amsterdam” for this new variant.

Function Assignment to Conserved Residues in Mammalian Alkaline Phosphatases

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.

Kinetics of droplet aggregation in heated whey protein-stabilized emulsions: effect of polysaccharides

The kinetics of heat-induced destabilization of whey protein emulsions was studied in the presence of varied concentrations of the polysacchride stabilizer xanthan, carrageenan or propylene glycol alginate (PGA). Both xanthan and carrageenan increased the rate of emulsion droplet aggregation in heated whey protein emulsions, whilst PGA had no observable effect. The maximum effect for xanthan was a 3.5-fold increase in the rate of destabilization for concentrations of 0.05 wt% and above. For carrageenan the increase in aggregation rate was smaller, with a 2.7 times increase observed at a carrageenan concentration of 0.1 wt%. For xanthan gum the decrease in heat stability was explained in terms of the known tendency for xanthan to cause depletion flocculation in emulsions. For carrageenan the effect was related to evidence that the polysaccharide can interact with denatured whey protein, but not with the native molecule.

Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degrees C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.

Tenderization of beef by lactic acid injected at different times post mortem

The potential to tenderize beef muscles by the injection of lactic acid (0.5 M, 10% w/w) was studied using the pectoralis profundus muscle from cull cows. The injection was performed either 1 h (pre rigor) or 24 h (post rigor) post mortem, and the meat was stored for 2 or 14 days post mortem. Both treatments caused a rapid pH drop to around 5.0 within 4 h of injection. Other effects were: (1) an accelerated release of lysosomal enzymes into the cytosol; (2) a greater degradation of myosin heavy chains; (3) ultrastructural alterations of the myofibrils which included a general weakening or rupture in the M-lines and, to a lesser extent, in the I-bands; (4) a decreased heat stability of perimysial collagen indicated by a lower insoluble collagen content, lower differential scanning calorimetry transition temperature, and lower transition temperatures in isometric tension tests on muscle strips. The lactic acid injections improved significantly the textural traits of the meat (shear value, tensile strength, sensory scores) at 2 days post mortem with little further improvement when storage was extended to 14 days post mortem. Changes in texture were of similar amplitude at both post mortem injection times. The tenderization mechanisms of lactic acid injection are discussed.