Thermal inactivation of the frozen thawed traditional meat starter culture, Pediococcus pentosaceus, in a meat model system

The equation, y_(t) = y₍₀₎e^kt, was fitted (R = 0.9281, 0.9220 and 0.9117, respectively) to thermal inactivation data (55, 60 and 65 °C) of the traditional meat starter culture Pediococcus pentosaceus (10⁷ cfu/ml) in a meat model system. The population reduction constant (‘k’) increased (about 2.5- and 3-fold) with an increase in the treatment temperature (from 55 to 60 °C and from 60 to 65 °C, respectively). The Q₁₀ (55–65 °C) for ‘k’ was 7.63. Thermal treatments of 19.1, 9.0 and 3.1 min (55, 60 and 65 °C, respectively) reduced the population of P. pentosaceus by 2.0 logs. The value of ‘k’ and the duration of the thermal treatment played an important role in the extent of the inactivation of the culture. The “zero inactivation” temperature (T₀) for P. pentosaceus was 49.9 °C. About 5 logs of the culture would be destroyed at 63 and 68 °C within about 15.5 and 6.5 min, respectively.     

Effect of season on color of Hanwoo (Korean native cattle) beef

A total of 1278 head of Hanwoo (Korean native cattle) slaughtered over four seasons were used to evaluate the effect of season on color characteristics of beef longissimus dorsi (LD) muscle. CIE L^*, a^*, b^*, C^* values and hue angle were significantly lower (P<0.05) in cattle slaughtered in the winter season. Meat color was darker in the winter than in the spring and autumn seasons. The L^* values among three average daily temperature (T_a) categories were different (P<0.05) in order of: [5 °CT_a<25 °C] > [T_a25 °C] > [T_a<5 °C], indicating that the meat color of cattle slaughtered at T_a<5 °C was darker. The a^*, b^*, C^* values and hue angle were significantly lower (P<0.05) in cattle slaughtered at T_a<5 °C. Season at slaughter is of great importance for meat color. Namely, meat color of Hanwoo beef was influenced by environmental temperature. Overall, cattle slaughtered in the winter season of T_a<5 °C produced beef with more undesirable meat color properties.     

Ochratoxin A in dried vine fruits on the Canadian retail market

Between 1998 and 2000, 151 samples of raisins and sultanas and two samples of currants were collected from retail outlets across Canada and analysed for ochratoxin A. Samples were extracted with methanol-sodium bicarbonate, and the extracts were cleaned-up by immunoaffinity column chromatography. Ochratoxin A was quantified by liquid chromatography with fluorescence detection. The minimum quantifiable level was 0.1 ng g^-1. Ochratoxin A was present, above the minimum quantifiable level, in 67 (79%) of 85 samples of raisins, in 39 (59%) of 66 samples of sultanas, and in both samples of currants. The overall mean level of ochratoxin A was 1.8 ng g^-1 in both the raisins and sultanas, and 2.8 ng g^-1 in the currants.     

Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis)

Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of N-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60 °C, respectively. Trypsins were stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed K_m of 0.11–0.29 mM and K_cat of 57.1–114 s⁻¹. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.     

Impact of sorbitol on the thermostability and heat-induced gelation of bovine serum albumin

The influence of sorbitol (0–40 wt.%) on the thermal denaturation and gelation of bovine serum albumin (BSA, pH 7.0) in aqueous solution has been studied. The effect of sorbitol on heat denaturation of 0.5 wt.% BSA solutions was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and was characterized by the thermal denaturation temperature (T_m). As the sorbitol concentration increased from 0 to 40 wt.%, T_m increased from 73.0 to 80.9 °C. The rise in T_m was attributed to the increased thermal stability of the globular state of BSA relative to its native state. The dynamic shear rheology of 4 wt.% BSA solutions containing 200 mM NaCl was monitored as they were heated from 30 to 90 °C at 1.5 °C min⁻¹, held at 90 °C for 120 min, and then cooled back to 30 °C at −1.5 °C min⁻¹. Sorbitol increased the protein gelation temperature (ΔT_gel +10 °C for 40 wt.% sorbitol), decreased the isothermal gelation rate at 90 °C, but increased the final shear modulus of the gels cooled to 30 °C. The impact of sorbitol on gel characteristics was attributed to its ability to increase protein thermal stability, increase the attractive force between proteins and decrease the protein–protein collision frequency.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.

The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

Identification of halothane genotypes by calcium accumulation and their meat quality using live pigs

The three halothane genotypes (NN, Nn, and nm) were identified by measuring the capacity for Ca²⁺ accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of M. longissimus dorsi with a Ca²⁺ specific electrode at 35°C. Significant differences (P < 0·001) in deterioration (%) of Ca²⁺ accumulation, 12% for NN, 35% for Nn, and 81% for nn pigs, were observed after ageing the whole muscle homogenate preparations for 24 h in ice.

Predictions of meat quality in live pigs (n = 34) based on the values for water-holding capacity, assessed as fluid (g/0·5 g wet wt LD), and pH (fluid) by using small biopsy LD samples (Cheah et al. 1993) were performed on all the halothane genotypes. The halothane genotype NN (n = 11) showed a fluid value of 0·37 ± 0·01 and a pH (fluid) value of 6·62 ± 0·03 as compared with 0·61 ± 0·02 and 5·84 ± 0·04, respectively, for the halothane genotype nn (n = 13). The Nn pigs (n = 10) showed fluid (0·49 ± 0·03) and pH (fluid) (6·19 ± 0·11) values between those values observed for the two homozygotes (NN and nn). Predictions of meat quality in live pigs from biopsy LD muscles were confirmed from assessments on post-mortem LD muscles based on pH₁ and fibre optic probe (FOP) measurements.

The extent of deterioration (%) in Ca²⁺ accumulation showed high correlations with fluid (r = −0·861) and pH (fluid) (r = −0·831) in the biopsy LD samples, and with pH₁ (r = 0·663), FOP (r = −0·812), and drip (%) loss (r = −0·777) in the post-mortem LD samples.     

Consumers' ability to discriminate aflatoxin-contaminated Brazil nuts

The objectives of the study were to investigate the extent to which consumers can separate nuts with a high content of aflatoxin from sound nuts, and whether sorting results can be improved by information or whether they are affected by certain factors. A test panel consisting of 100 subjects was asked to crack 300 g Brazil nuts and to sort the nuts into those they considered edible and inedible. The test showed that consumers can, on current behaviour, discriminate aflatoxin-contaminated Brazil nuts to a significant extent. The median and the 95th percentile of the total concentrations of aflatoxins (B₁, B₂, G₁, G₂) in the samples before sorting were 1.4 and 557 µg kg^-1, respectively, and in the edible fractions after sorting 0.4 and 56 µg kg^-1, respectively. Given that levels of aflatoxins before sorting exceed either 2 µg aflatoxin B₁ kg^-1 or totally 4 µg aflatoxins kg^-1, there was no effect of aflatoxin concentrations before sorting on the probability of exceeding these thresholds in the edible fraction. This means that similar sorting results were obtained for samples with aflatoxin levels exceeding either of the two thresholds, irrespective of if the thresholds were exceeded with a few µg kg^-1 or up to more than 1000 µg kg^-1. None of the tested factors (such as sex, age, level of education, ethnic background or knowledge of mycotoxins) had any effects on the probability of exceeding either of the two aflatoxin thresholds.     

Interaction between flaxseed gum and meat protein

Thermal properties, dynamic rheological properties, texture and microstructure of salt-soluble meat protein and flaxseed gum (SSMP-FG) mixtures were investigated. Two transitions, 57.0 °C (T_SSMP1) and 63.2 °C (T_SSMP2), were observed for SSMP without FG with differential scanning calorimetry (DSC). Addition of 2% FG to SSMP increased T_SSMP1 and T_SSMP2 by 1.9 °C and 5.9 °C, respectively. Two transitions, 53 °C (T_SSMP1′) and 75 °C (T_SSMP2′), were also observed for SSMP without FG with dynamic rheological measurement. Addition of 2% FG to SSMP increased T_SSMP1^′ and T_SSMP2^′ by 9 °C and 14 °C. These results indicated that addition of FG increased thermal stability of SSMP. Addition of FG also increased the storage modulus G′, gel strength, decreased syneresis, and changed the microstructure of SSMP gels with texture analyser and scanning electron microscope (SEM), respectively, suggesting that an interaction between FG and SSMP may have occurred. The results of addition of destabilizer to SSMP gels indicated that electrostatic forces seemed to be the main force involved in the formation and stability of protein–polysaccharide gel.     

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes

The aim of this work is to study and model the effect of a temperature shift on h₀, the product of the growth rate by the lag phase duration (μλ). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T_prior) and the growth temperature (T_growth) on λ values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h₀ as a function of T_prior and T_growth, and compare it to Whiting and Bagi's published polynomial model that describes the influence of T_prior and T_growth on λ independently of μ. For exponential as well as stationary phase cells, h₀ increases almost linearly with the magnitude of the temperature shift. A simple linear model of h₀ turns out to be more suitable to predict λ values than a polynomial model of λ.     

The effects of residual oxygen concentration and temperature on the degradation of the colour of beef packaged under oxygen-depleted atmospheres

Samples of beef longissimus dorsi (LD), approximately 5 × 5 × 1 cm, were packaged in pairs under 10 litre volumes of N₂ or CO₂ containing O₂ at concentrations between 100 and 1000 ppm. The packaged samples were stored at temperatures of 5, 1, 0 or −1·5°C, for times between 4 and 48 h. Samples of beef psoas major (PM) were packaged under N₂ or CO₂ containing O₂ at between 100 and 600 ppm, and stored at −1·5°C for 24 or 48 h. After storage, each sample was assessed for colour deterioration and discoloration, and for the fraction of metmyoglobin in the surface pigment.

The results obtained with N₂ and CO₂ atmospheres were similar. The colours of all LD samples had deteriorated after 4 h storage at 5 or 1°C, although the degree of deterioration increased with increasing O₂ concentration. All LD samples stored for 12 h at 5 or 1°C were extensively discoloured, with metmyoglobin fractions generally exceeding 60%, but those stored at −1·5°C for 48 h or less, under O₂ concentrations ≤ 400 ppm had undergraded colours. The colours of some LD samples stored at −1·5°C under about 600 ppm of O₂ were also undergraded, but the colours of samples stored under 800 or 1000 ppm had deteriorated by 24 h. The colours of LD samples stored at 0°C under > 200 ppm had deteriorated after 24 h storage, and the colours of samples stored under 100 ppm O₂ had deteriorated after 48 h storage. All PM samples were wholly discoloured after storage at −1·5°C. Evidently, the colour of beef muscle of high colour stability is resistant to degradation by atmospheres containing < 600 ppm of O₂ when the meat is stored at sub-zero temperatures, but not when the storage temperature is at or above 0°C. Beef muscle of low colour stability, such as the PM, will discolour at all low concentrations of O₂ irrespective of the storage temperature.     

NMR relaxometry and differential scanning calorimetry during meat cooking

By combining simultaneous nuclear magnetic resonance (NMR) T₂ relaxometry and differential scanning calorimetry (DSC) on pork samples heated to nine temperature levels between 25 and 75 °C, the present study investigates the relationship between thermal denaturation of meat proteins and heat-induced changes in water characteristics. Principal component analysis (PCA) on the distributed ¹H NMR T₂ relaxation data revealed that the major changes in water characteristics during heating occur between 40 and 50 °C. This is probably initiated by denaturation of myosin heads, which however, could not be detected in the DSC thermograms obtained directly on the meat. In contrast, the DSC thermograms revealed endothermic transitions at 54, 65 and 77 °C, probably reflecting the denaturation of myosin (rods and light chain), sarcoplasmic proteins together with collagen and actin, respectively. Simultaneous modelling of DSC and NMR data by partial least squares regression (PLSR) revealed a correlation between denaturation of myosin rods and light chains at 53–58 °C and heat-induced changes in myofibrillar water (T₂ relaxation time 10–60 ms) as well as between actin denaturation at 80–82 °C and expulsion of water from the meat. Accordingly, the present study demonstrates a direct relationship between thermal denaturation of specific proteins/protein structures and heat-induced changes in water mobility during heating of pork.     

Partial characterization of starches from Dioscorea opposita Thunb. cultivars

The physicochemical, morphological, thermal and crystal properties of the starches separated from four different Dioscorea opposita Thunb. cultivars (Yongji, Anguo, Jinpingyao and Shandongniutuimi) were studied. Amylose content of D. opposita Thunb. starches from different cultivars were in the range of 19.38–22.02%. Moisture content, swelling power, solubility and water-binding capacity of starches differed significantly. Scanning electron micrographs revealed the presence of smooth or rough surface, oval to spherical shaped granules, with mean particle size in the range of 29.2–36.96 μm. The gelatinization transition temperatures (T_o, T_p and T_c) and enthalpy of gelatinization (ΔH_gel), peak height index (PHI) and gelatinization temperature range (R) were determined. T_o, T_p, T_c of D. opposita Thunb. starches ranged from 70.2 to 75.8, 77.5 to 81.1, and 82.8 to 86.9 °C, respectively. D. opposita cv. Anguo starch showed the highest ΔH_gel values (12.47 J/g) while D. opposita cv. Shandongniutuimi starch showed the lowest values (10.54 J/g). The crystal type of starches separated from different D. opposita Thunb. cultivars was a typical C-type X-ray diffraction pattern. The relative crystallinity of the starches ranged from 34.3% to 43.1%.     

Purification and characterization of a dipeptidase from Lactobacillus delbrueckii subsp. bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu²⁺, and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50°C. Reducing agents, like dithiothreitol and β-mercaptoethanol, increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co²⁺ and Mn²⁺. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K_m-values for both substrates were about 0·56 and 1·23 mM, with turnover numbers of 870 and 480 s⁻¹, respectively.     

The storage life of chilled pork packaged under carbon dioxide

Pork cuts of longissimus dorsi muscle with overlaying fat and skin were packed under vacuum in film of low oxygen transmission rate, or under CO₂ in gas impermeable aluminium foil laminate. Cuts were stored at +3 or −1·5°C. Vacuum packaged cuts were grossly spoiled by Brochothrix thermosphacta after 2 weeks' storage at 3°C and after 5 weeks at −1·5°C. Cuts packaged under CO₂ were grossly spoiled by B. thermosphacta after 5·5 weeks' storage at 3°C. Growth of B. thermosphacta was suppressed when CO₂ packaged cuts were stored at −1·5°C. At that temperature, slow growth of enterobacteria was detected after a lag of about 18 weeks. The enterobacteria caused gross spoilage of an increasing proportion of cuts between 18 and 26 weeks. Muscle tissue with pale, soft, exudative (PSE) characteristics tended to lose colour after long storage periods, apparently because of loss of myogglobin with exudate. Until spoilage, the eating qualities of pork appeared little affected by prolonged storage.     

Simulation studies on the effects of solar heat on egg-laying, development and survival of Callosobruchus maculatus (F.) and Callosobruchus subinnotatus (Pic) in stored bambara groundnut Vigna subterranea (L.) Verdcourt

The effects of simulated solar heat on oviposition, development and survival of Callosobruchus maculatus (F.) and Callosobruchus subinnotatus (Pic) in stored bambara groundnut, Vigna subterranea (L.) Verdcourt, were evaluated at three high temperatures (40°C, 45°C and 50°C) at a constant, low humidity (30% relative humidity). Exposure to these temperatures for 6 h significantly reduced oviposition in C. maculatus and C. subinnotatus females. Females of both species that were exposed to 50°C laid significantly fewer eggs than those exposed to 40°C; and in the case of C. maculatus, females exposed to 45°C also laid significantly fewer eggs than those exposed to 40°C. The percentage of eggs laid by females of both species that reached adulthood after exposure to 50°C for 2–6 h was significantly lower than the percentage that developed from eggs laid by females that were exposed to 40°C. No adult developed from eggs of C. maculatus exposed for 6 h at 50°C or from eggs of C. subinnotatus exposed for 2 h at this temperature. For both species, no adult progeny subsequently emerged from seeds harbouring first instar larvae when exposed at 50°C for 2, 4 or 6 h. Older larvae of C. maculatus were more tolerant of exposure at 50°C: 26.8, 10.2 and 0.9% of late instar larvae exposed for 2, 4 and 6 h, respectively, developed to the adult stage. In contrast, no adults of C. subinnotatus emerged from seeds harbouring late instar larvae when exposed at 45°C for 6 h nor in seeds exposed to the temperature of 50°C for 4 or 6 h. On average, immature stages of C. subinnotatus were more susceptible to heat treatment than those of C. maculatus.     

Aflatoxin production by Aspergillus flavus in Brazil nuts

Experiments were conducted to evaluate the effects of relative humidity (r.h.; 75%, 80%, 85%, 97%) and temperature (10, 13, 15, 25, 30 °C) on aflatoxin production in previously dried (3.5% moisture content; m.c.) Brazil nuts. Initially Aspergillus spp. were isolated from the surfaces of whole in-shell (WIS) Brazil nuts imported from Peru using A. flavus and A. parasiticus agar (AFPA). Isolates were subsequently screened for aflatoxin production using yeast extract sucrose medium. Total aflatoxin (B₁+B₂+G₁+G₂+M₁) was analyzed using an immunoassay technique while the presence of aflatoxin was confirmed using thin-layer chromatography. The surface of shelled half-nuts (simulating damaged or trimmed nuts), shelled whole (SW) nuts, and WIS nuts following a chlorine wash and water rinse, served as sites for inoculation (10 μl; 10⁵/ml) using an aflatoxigenic isolate. Maximum concentrations of total aflatoxin and B₁ were detected in nuts stored at 97% r.h. and at temperatures of 25–30 °C. Shelled half-nuts contained the highest total (6817 ng/g) and B₁ (4483 ng/g) aflatoxin. WIS nuts contained the least total and B₁ toxin with maximum concentrations of 93 and 49 ng/g, respectively. Aflatoxin was not detected (detection limit of 1.75 ng/g) in nuts maintained at either 10 °C (97% r.h.) or at 30 °C (75% r.h.) for up to 60 d. Maximal moisture contents (%) and water activity values (a_W) for nuts stored at these conditions were 4.50 and 0.78, and 9.14 and 0.92, respectively. Results of this study indicate that the limiting moisture content and a_W values required to control aflatoxin production () in SW and WIS stored at 30 °C for up to 60 d are 4.5, 0.68, 5.0, and 0.75, respectively. Overall, increasing the relative humidity and temperature during storage resulted in an increase in aflatoxin and these were shown to be the most significant variables influencing toxin production in Brazil nuts.     

Microbiological and sensory characteristics of beef loin steaks: Role of subcutaneous fat

Beef loin steaks with the subcutaneous fat attached, without subcutaneous fat and the subcutaneous fat that was removed from steaks were packaged and stored at 4°C ± 1°C in polyvinyl chloride (PVC) film for 0–6 days and in high-oxygen barrier (HOB) film for 0–28 days. Aerobic plate counts (APCs) of subcutaneous fat of intact steaks and of subcutaneous fat that was packaged and stored separately in PVC and HOB films were greater (P < 0·05) than those of comparable lean samples. The APCs of lean of steaks without subcutaneous fat that were packaged and stored in HOB film were lower (P < 0·05) than those of the lean of intact steaks. APCs of the lean of these two types of steaks packaged and stored in PVC film did not differ (P > 0·05). Mean surface discoloration and mean overall appearance scores of intact steaks packaged and stored in HOB film were greater than those of steaks packaged and stored without subcutaneous fat; differences were significant (P < 0·05) after 21 and 14 days, respectively. This difference in surface discoloration was attributed to metmyoglobin formation due to possibly higher levels of oxygen remaining in the packages of steaks without subcutaneous fat than in packages containing steaks with the fat attached.     

Thermal inactivation of lectins (PHA) isolated from Phaseolus vulgaris

The influence of the thermal process on the loss of ability to bind a carbohydrate target was studied on lectins (PHA) purified from Phaseolus vulgaris seeds. Thermal inactivation of aqueous solutions of pure PHA occurred according to a biphasic first-order mechanism, the thermodynamic parameters, at pH 7·3, being as follows: ΔH*₁ = ΔH*₂ = 86·2 kcal mole⁻¹, ΔS*₁ = − 54·04 cal deg⁻¹ and ΔS*₂ = − 56·71 cal deg⁻¹. The first-order rate constants appeared to be dependent on pH (minimal around 7) and divalent cations. All different subunits constituting the whole PHA were inactivated at the same rate. The biphasic nature of this process is independent of the presence of 10 m Ca⁺⁺ or Mg⁺⁺ and appeared to indicate a discrete aggregation of PHA molecules.