The proteolytic activity of the crude enzyme extracts of Baltic cod (Gadus morhua) alimentary tract

The crude enzyme extract from Baltic cod alimentary tract had maximum activity of aspartic proteinases towards haemoglobin at pH 2.0 and 3.4, and of serine proteinases with casein as the substrate at pH 8.3 and 10.4. With bovine myofibrils as the substrate the proteolysis at pH 5.0-8.0 was maximum at the lowest and highest pH values. the optimum temperature for the proteolytic activity at pH 3.4 and 10.4 was 30–45°C and at pH 8.3 it was 40–50°C. Heating the crude extract for 10 min at pH 3.4,8.3, and 10.4 in the absence of the substrate had no effect on the activity of the acid and alkaline proteinases up to about 35–40°C. the stability at higher temperature decreased gradually and total inactivation occurred at 55–60°C. In the pH range 5.5–7.5 the proteolytic activity against bovine myofibrils was low at 0°C but brought about significant loss of myosin heavy chain at 20°C.     

Analysis of the kinetic patterns of horseradish peroxidase thermal inactivation in sodium phosphate buffer solutions of different ionic strength

The thermal inactivation of horseradish peroxidase was studied in sodium phosphate buffer solutions and in pure water at pH 7 in the temperature range of 70–95°C. The sodium phosphate ions concentration affected both the thermostability and the kinetic patterns and had a stabilizing effect. The gradual change observed at low concentrations made a series-type mechanism theoretically more coherent with the experimental observations than the conventionally applied two-fraction model. In water the kinetics is apparently First order at high temperatures, while the results obtained at 25°C support the occurrence of a series-type inactivation mechanism. The pH and enzyme concentration also affect the inactivation proFile, supporting the conclusion that the thermal inactivation is not a monomolecular process with respect to protein concentration.     

Effects of Dense Phase Carbon Dioxide Pasteurization on the Physical and Quality Attributes of a Red Grapefruit Juice

ABSTRACT:  Red grapefruit juice was treated with continuous dense phase carbon dioxide (DPCD) equipment to inactivate yeasts and molds and total aerobic microorganisms. A central composite design was used with pressure (13.8, 24.1, and 34.5 MPa) and residence time (5, 7, and 9 min) as variables at constant temperature (40 °C), and CO₂ level (5.7%) after experimentally measuring CO₂ solubility in the juice. Five log reduction for yeasts and molds and total aerobic microorganisms occurred at 34.5 MPa and 7 min of treatment. A storage study was performed on the fresh juice DPCD treated at these conditions. °Brix, pH, titratable acidity (TA), pectinesterase (PE) inactivation, cloud, color, hue tint and color density, total phenolics, antioxidant capacity, and ascorbic acid were measured after the treatment and during 6 wk storage at 4 °C. During storage, the DPCD-treated juice showed no growth of total aerobic microorganisms and yeasts and molds. Cloud increased (91%) while percent PE inactivation was partial (69.17%). No significant (α= 0.05) differences were detected between treated and untreated samples for °Brix, pH, and TA. Treated juice had higher lightness and redness and lower yellowness. No significant differences (α= 0.05) were detected for the hue tint values while the color density value was higher for the treated samples compared to the untreated. The treatment and the storage did not affect the total phenolic content of the juice. Slight differences were detected for the ascorbic acid content and the antioxidant capacity. The experimental results showed evidence that the treatment can maintain the physical and quality attributes of the juice, extending its shelf life and safety.     

Effect of pH on Inactivation of Escherichia coli K12 by Sonication, Manosonication, Thermosonication, and Manothermosonication

ABSTRACT:  The effect of pH on inactivation of Escherichia coli K12 by sonication at 100 kPa/40 °C, manosonication (MS) at 400 kPa/40 °C, thermosonication (TS) at 100 kPa/61 °C, and manothermosonication (MTS) at 400 kPa/61 °C at acoustic energy density of 3 W/mL and 6 W/mL was investigated. Five linear and nonlinear kinetic models were used to examine the inactivation kinetics. At all pH levels, the inactivation rates of E. coli K12 in a buffer by TS and MTS were significantly higher than those by sonication and MS. A 5 log reduction of E. coli K12 population by TS and MTS was achieved in 0.5 and 0.25 min, respectively. With an initial count of 10⁸ CFU/mL, no colonies were detected at pH 3 after a 0.25-min MTS treatment. The lethal effect of MTS was enhanced at low pH (pHs 3 and 4), whereas at nonlethal temperature of 40 °C, no increased killing was observed. Regardless of pH, the treatment by MTS, TS, and MS exhibited a rapid initial reduction followed by tailing-off on the inactivation curves. The biphasic linear, log-logistic, and modified Gompertz kinetic models allowed better fitting of the inactivation data for MTS, TS, and MS treatments than the 1st-order and Weibull models. The survival counts of sonication-treated E. coli K12 at all pH levels fitted well to a 1st-order kinetic model.     

EFFECT OF BREAK TEMPERATURE AND HOLDING TIME ON PECTIN AND PECTIC ENZYMES IN TOMATO PULP

SUMMARY –The effect of break temperature and holding time on chemical and physical properties of canned tomato pulp and on the activity of polygalacturonase (PG) and pectin esterase (PE) was investigated. Samples preheated above 200°F were slightly higher in pH value and lower in titratable acidity than those preheated at 160°F and 140°F. The gross viscosity and serum viscosity of the canned tomato pulp increased as break temperature increased. A slight increase in serum viscosity was also observed when holding time was lengthened. The effect of break temperature on serum viscosity was much greater than that of holding time. The total pectin content of the tomato pulp as well as that of the serum increased as the break temperature increased. The same observation holds true for the effect of holding time on the total pectin content of the tomato pulp and tomato serum. This was explained by the inactivation of pectic enzymes at higher break temperature and longer holding periods. The level of PE and PG activity in the frozen tomato pulp decreased as break temperature and holding time increased. No PE activity was detected in samples preheated at 180°F for 15 sec. Samples preheated at 220°F for 15 sec showed no PG activity, while those preheated at 200°F for 15–87 sec still show some PG activity with a retention of about 3.54–5.68%. PG in tomatoes may be inactivated by giving the macerated fruits a hot break temperature higher than 200°F. The critical conditions for inactivation of PE (180°F for 15 sec) and of PG (220°F for 15 sec) found in the course of this investigation are supported by the data obtained on pectin analysis and serum viscosity.     

Partial characterization of the in situ activity of pectinesterase in Bramley apple

The characteristics of pectinesterase (PE) have been examined in waste material (peel, cores and offcuts) obtained from Bramley seedling apples barn stored for 2, 4 and 12 weeks. A crude enzyme extract was prepared by suspending the dried and milled apple waste in 0.1 m NaCl at pH 8.5. The activity of PE under standard assay conditions of 0.5% apple pectin, 0.1 m NaCl, pH 8.5 and 30°C was low, between 4 and 8 units g⁻¹ dry matter, but activities up to 60 units g⁻¹ dry matter were obtained at higher temperatures. The optimum temperature was 60°C with the enzyme stable up to 40°C with 5 min heating. The mean activation energy for PE in the three samples was calculated at 39.2 kJ mol⁻¹ K⁻¹. The optimum pH was high at 10.0 probably due to the PE assay measuring the extraction/solubilization and stability of the enzyme in addition to its activity. Optimum activity was obtained in 0.15 m NaCl with optimum stability at 0.5 m.     

Pectin Methyl Esterase Activity in Southern Peas (Vigna sinensis)

SUMMARY: Pectin methyl esterase (PME) activity was investigated in vitro in Southern peas (Vigna sinensis). Experiments were conducted to determine the effect of NaCl concentration, pH, temperature, maturity, frozen storage and rinsing the peas on PME activity.
The optimum salt level for maximum PME activity in the Purple Hull Pink Eye (PHPE) and Princess Ann (PA) varieties was ascertained to be 0.25 M. The pH optima were: PHPE, 8.5; and PA, 7.5 to 8. The remaining studies were made using peas of the PHPE variety which were harvested at 3 stages of maturity. Maximum enzymatic activity occurred at 50°–60°C. Partial inactivation occurred at 65°C. The mean Q₁₀ was 1.35 over the range of 30°–50°C.
PME activity was dependent upon the maturity of the peas. The most immature peas had an activity level about 2.5 times that found in the most mature peas. Peas subjected to frozen storage had a higher activity than the fresh peas; the increased activity was more pronounced in the more mature peas.
Rinsing the peas removed significant amounts of the enzyme. A greater proportion of PME was removed from the more immature peas than from the more mature ones. With the immature, frozen peas PME activity was reduced 22.7% by rinsing; whereas, in the fresh counterpart the reduction was 11.1%. The rinse water from frozen peas contained more PME activity than did rinse water from fresh peas. Also, the activity in rinse water from the most immature peas was about 2.7 times that from the most mature ones. Upon standing, PME activity in the slurries prepared from frozen peas continued to increase up to 3–4 hr.     

Effects of pH and temperature on inactivation of Salmonella typhimurium DT104 in liquid whole egg by pulsed electric fields

Pulsed electric field (PEF) exposes a fluid or semi-fluid product to short pulses of high-energy electricity, which can inactivate microorganisms. The efficacy of PEF treatment for pasteurisation of liquid eggs may be a function of processing temperature. In this study, effects of PEF, temperature, pH and PEF with mild heat (PEF + heat) on the inactivation of Salmonella typhimurium DT104 cells in liquid whole egg (LWE) were investigated. Cells of S. typhimurium were inoculated into LWE pH adjusted to 6.6, 7.2 or 8.2 at 15, 25, 30 and 40 °C. The PEF field strength, pulse duration and total treatment time were 25 kV cm⁻¹, 2.1 μs and 250 μs respectively. Cells of S. typhimurium in LWE at pH 7.2 were reduced by 2.1 logs at 40 °C and 1.8 logs at 30 °C. The PEF inactivation of S. typhimurium cells at 15 or 25 °C was pH dependent. Heat treatment at 55 °C for 3.5 min or PEF treatment at 20 °C resulted in c. 1-log reduction of S. typhimurium cells. Combination of PEF + 55 °C achieved 3-log reduction of S. typhimurium cells and was comparable to the inactivation by the heat treatment at 60 °C for 3.5 min. With further development, PEF + heat treatment may have an advantage over high-temperature treatment for pasteurisation of liquid eggs.     

EFFECT OF pH, ACIDULANT, SODIUM CHLORIDE AND TEMPERATURE ON THE GROWTH OF LISTERIA MONOCYTOGENES

Growth of two Listeria monocytogenes strains in tryptic soy broth containing NaCI or combinations of NaCI and acidulants at different pHs and temperatures was investigated . L. monocytogenes was capable of growing in 10% NaCI at 35°C and 12% NaCI at 25°C and 10°C. The maximum NaCI for growth changed when NaCI and pH, in combination with different acidulants and temperature, were tested. The minimum pH/salt level for initiation of growth of L. monocytogenes ranged from 5.0–5.6/8–10% at 35°C and 25°C and 5.6/8% at 10°C, depending upon the acidulant and the strain. Greatest antimicrobial activity occurred at 35°C. Greatest survival occurred at 10°C. In this study L. monocytogenes appeared to persist and tolerate a combination of low pH, high salt and low temperatures .     

Combined effects of hydrostatic pressure, temperature, and pH on the inactivation of spores of Clostridium perfringens type A and Clostridium sporogenes in buffer solutions

ABSTRACT:  To develop a spore inactivation strategy, the effect of 15-min hydrostatic pressure treatments (550 and 650 MPa) at 55 and 75 °C in citric acid buffer (4.75 and 6.5 pH) on spores of 5 isolates of Clostridium perfringens type A carrying the gene that encodes the C. perfringens enterotoxin ( cpe ) on the chromosome (C- cpe ), 4 isolates carrying the cpe gene on a plasmid (P- cpe ), and 2 strains of C. sporogenes were investigated. Treatments at 650 MPa, 75 °C and pH 6.5 were moderately effective against spores of P- cpe (approximately 3.7 decimal reduction, DR) and C. sporogenes (approximately 2.1 DR) but not for C- cpe (approximately 1.0 DR) spores. Treatments at pH 4.75 were moderately effective against spores of P- cpe (approximately 3.2 DR) and C. sporogenes (approximately 2.5 DR) but not of C- cpe (approximately 1.2 DR) when combined with 550 MPa at 75 °C. However, when pressure was raised to 650 MPa under the same conditions, high inactivation of P- cpe (approximately 5.1 DR) and C. sporogenes (approximately 5.8 DR) spores and moderate inactivation of C- cpe (approximately 2.8 DR) spores were observed. Further advances in high-pressure treatment strategies to inactivate spores of cpe -positive C. perfringens type A and C. sporogenes more efficiently are needed.     

Technical Approach to Simplify the Purification Method and Characterization of Microbial Transglutaminase Produced from Streptoverticillium ladakanum

In order to fast and economically purify MTGase from Streptoverticillium ladakanum , a stepwise elution method was developed and compared with linear gradient elution method. MTGase was purified to electrophoretical homogeneity by using CM Sepharose CL-6B and Blue Sepharose Fast Flow chromatographies by linear gradient or stepwise methods. The recovery of MTGase by linear gradient and stepwise methods were 68.4% and 81.0%, respectively. The optimal temperature and pH were 40 °C and 5.5, respectively. It was stable at pH 5.0 to 7.0 and had a rate constant (KD) of 6.21 °o 10^-5 min^-1 for thermal inactivation at 45 °C. The purified MTGase was activated by K⁺ Na⁺, Ca²⁺, Mn²⁺, and Mg²⁺, not affected by Fe³⁺, EDTA, but inhibited by Cu²⁺, Zn²⁺, Hg²⁺, Ni²⁺, Co²⁺, Cd²⁺, PCMB, NEM, IAA, and PMSF. A simple stepwise method was developed for the purification of MTGase from S. ladakanum.     

EFFECTS OF pH ON QUALITY OF STORED TOMATO JUICE

Tomato juice was prepared by the hot-break method and filled hot into cans containing sufficient concentrated HCl to adjust pH to 4.0, 3.5, 3.0, 2.5 and 2.0. Controls were also prepared with no HCl added. Product was stored at ambient (18–22°C) and 40°C up to 3 months. As pH decreased below 4.0, consistency also showed a significant decline through pH 2.0. At the same time there was no apparent pH effect on serum viscosity. Further studies indicated that this effect was noticeable after 4 hr, at pH 3.0 to 5.0. Addition of as much as 1% NaCl to tomato juice had no effect on consistency of tomato juice. Lightness (Hunter color L ) decreased with pH after 3.5, redness (a_L) decreased after pH 4.0, and yellowness (b_L) decreased after pH 3.0. All storage effects were more profound at the higher temperature. The storage of tomato juices at pH less than 4.0 does not appear to be advantageous to the processor.     

IDENTIFICATION AND CHARACTERIZATION OF SOME OXIDIZING ENZYMES OF THE MC FARLIN CRANBERRY

SUMMARY– Enzyme extracts were prepared from acetone powders with and without phenol-binding agents such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) and buffered PVP. The acetone-PVP combination was found most effective in reducing the polyphenolic content of the enzyme extract. Highest specific activity was obtained by using a buffered PVP extract. The pH optimum of cranberry peroxidase activity was 6.0. Heat inactivation of cranberry peroxidase was determined to follow first order kinetics. There was 90% destruction at 70, 80, and 90°C requiring 9.40, 1.60, and 0.47 min of heat treatment, respectively. Activation energy for the thermal inactivation of cranberry peroxidase was observed to be 37.2 kcal/mole. Guaicol, o-phenylene diamine (OPDA), and pyrogallol were tested for their sensitivity to cranberry peroxidase with OPDA determined as most sensitive. The pH optimum for catalse activity was found to range from 7.5 to 9.2. Kinetics for the heat inactivation of cranberry catalase was observed not to be of the first order nor zero order. Approximately 50% of the catalase activity was inactivated after heating for 17, 1.8, and 0.6 min at temperatures of 50, 60, and 70°C, respectively. The pH optimum for cranberry polyphenolase activity was determined to be 7.0. Heat inactivation of cranberry poly-phenolase was found to follow first order kinetics. There was 90% destruction at 50, 60, and 70°C requiring 15.85, 7.05, and 1.37 min of heat treatment, respectively. The activation energy for the inactivation of cranberry polyphenolase was observed to be 27.7 kcal/mole.     

The Effect of pH-Temperature Treatments on the Calcium Accumulating Ability of Purified Sarcoplasmic Reticulum

SUMMARY: Pig sarcoplasmic reticulum fragments obtained from the longissimus dorsi muscle at 0- and 24-hours post-mortem were purified by salt extraction and density gradient centrifugation. The calcium uptake activity of 0-hour purified preparations was more than 20-fold higher than that from 24-hr old muscles, but there was no significant difference between fractions for calcium activated ATPase activities. When observed electron microscopically after negative staining, the ultrastructures of the 0. and 24-hour membrane fragments were found to be essentially identical. Incubation of isolated sacroplasmic reticulum fragments at pH 7.2 and 37°C or pH 5.6 and 0°C caused negligible inhibitoin of their calcium accumulating ability. However, treatment at pH 5.6 and 37°C for 1 hr almost completely abolished the sarcoplasmic reticulum calcium uptake. Thus it appears that low muscle pH and high temperature may be responsible for the inactivation of the calcium accumulating ability of the sarcoplasmic reticulum that occurs in situ.     

TEMPERATURE ACCLIMATION OF ATLANTIC COD (GADUS MORHUA) AND ITS INFLUENCE ON FREEZING POINT AND BIOCHEMICAL DAMAGE OF POSTMORTEM MUSCLE DURING STORAGE AT 0° and -3°C

The freezing point of muscle fluid from Newfoundland Atlantic cod held at ambient sea water temperature was as low as - 1.30°C in March and as high as - 0.80°C in July. Muscle fluid from cod held live at 0°C for 3 weeks had a freezing point of - 1.02°C in contrast to a muscle fluid freezing point of - 0.90°C for cod acclimated at 10°C prior to sacrifice. Muscle fluid from cold acclimated cod exhibited 0.40°C thermal hysteresis indicating freezing point depression was influenced by antifreeze substances. The following indices of deterioration were measured in muscle sections stored at 0°C or - 3°C for 21 days: extractable protein (EP), free drip (FD), extracellular area (EA), trimethylamineoxide (TMAO), trimethylamine (TMA), dimethylamine (DMA), free amino acids (AA), and pH. Muscle sections at the anterior end of fillets, from myotomes 9–20, prepared using aseptic technique and treated with antibiotic showed less evidence of biochemical deterioration: (a) when stored at - 3°C compared to 0°C with respect to EP, AA, EA; (b) when prepared from fish acclimated at 0°C compared to at 10°C and stored at 0°C or - 3°C with respect to EP, EA, FD, AA. Negligible changes in pH, TMA and DMA occurred during 21 days storage at either temperature. TMAO decreased more during storage at-3°C than at 0°C.     

Kinetic Study of Antibrowning Agents and Pressure Inactivation of Avocado Polyphenoloxidase

Pressure inactivation at room temperature (25 °C) of avocado polyphenoloxidase (pH 5.0) in absence or presence of 10 mM ethylene diaminetetraacetic acid (EDTA), NaCl, or benzoic acid or 0.05 mM 4-hexylresorcinol or glutathione could be described by a fractional conversion model. There appeared to be a resistant enzyme fraction (∼10% to 20%), necessitating about 825 MPa for inactivation in absence of antibrowning agents. EDTA addition resulted in sensitization of the pressure-sensitive enzyme fraction, which was attributed to the acid effect. Benzoic acid or NaCl addition resulted in a marked stabilization, whereas addition of glutathione resulted only in a minor stabilization of the pressure-sensitive enzyme fraction. 4-Hexylresorcinol displayed a sensitizing effect below and a stabilizing effect above 700 MPa. The pressure dependency of the inactivation rate constant was altered (p < 0.05) by addition of 4-hexylresorcinol or benzoic acid.     

Comparison of biochemical properties and thermal inactivation of membrane‐bound polyphenol oxidase from three apple cultivars (Malus domestica Borkh)

A comparative study of the properties of membrane‐bound polyphenol oxidase (mPPO) from three apple cultivars, namely Red Fuji (FJ), Granny Smith (GS) and Golden Delicious (GD), was carried out for the first time. Data indicate that mPPOs from three cultivars exhibit significantly different properties. GS mPPO had the strongest affinity to catechol, but FJ mPPO had the highest maximum velocity. Red Fuji (FJ) mPPO had the significantly higher activity than those of GD and GS mPPOs. Red Fuji (FJ) mPPO had the highest activity at pH 8.00, while GD and GS mPPOs at 4.50 and 7.50–8.00, respectively. Red Fuji (FJ) mPPO was more stable than GD and GS mPPOs over the pH range of 5.0–8.5. The optimal temperature for GS mPPO was within 70–75 °C, which is higher than those for mPPOs from FJ and GD. Thermal inactivation of the three mPPOs followed a first‐order kinetic model with different inactivation kinetic parameters.     

Microbial Safety in Radio-frequency Processing of Packaged Foods

ABSTRACT: Thermal resistance of Clostridium sporogenes (PA 3679) was determined at 115.6 °C, 118.3 °C, and 121.1 °C (240 °F, 245 °F, and 250 °F, respectively) in phosphate buffer (pH 7.0) and mashed potatoes (pH 6.3) using aluminum thermal-death-time (TDT) tubes developed at Washington State Univ. D-values were 1.8, 1.1, and 0.62 min in phosphate buffer and 2.2, 1.1, and 0.61 min in mashed potatoes at 115.6 °C, 118.3 °C, and 121.1 °C, respectively. Z-values were 12 °C and 10 °C in phosphate buffer and mashed potatoes, respectively. The thermal inactivation kinetic results were then used to validate a novel thermal process based on 27.12 MHz radio frequency (RF) energy. Trays of mashed potatoes inoculated with PA 3679 were subjected to 3 processing levels: target process (F₀∼4.3), under-target process (F₀∼2.4), and over-target process (F₀∼7.3). The microbial challenge test data showed that microbial destruction from the RF process agreed with the calculated sterilization values. This study suggests that thermal processes based on RF energy can produce safe and shelf-stable packaged foods.     

The aqueous extraction of glucosinolates from rapeseed

The losses of seed constituents from Brassica campestris var. Toria for various conditions of the leaching process developed at the Overseas Development Natural Resources Institute (ODNRI) were studied. Boiling seeds for 3 min at a seed/water ratio of 1:3 was sufficient to allow inactivation of the enzyme myrosinase; higher ratios did not increase losses in any of the constituents studied.
Heat treatment of the seeds (5 min in boiling water) reduced the nitrogen solubility at the native pH (6.5) from 28 to 8%. The pH had little effect on the extent of glucosinolate leaching from coarsely ground seeds and the minimum protein loss occurred close to the native pH. Increased water temperatures (40, 50 and 80°C) did not lead to an increased leaching efficiency over ambient temperature (20°C). Seed/water ratio was found to be the most important factor during leaching. Cross-current extraction over three stages at a seed/water ratio of 1:10 reduced the glucosinolate content by 98% while the crude protein loss was about 8.6%.     

COLOR STABILITY OF BETANIN

Some chemical and physical factors were evaluated for their effects on stability of betanin in model systems and in beet juice. Results indicate that the stability of betanin color is pH sensitive and generally is less than that of artificial dyes. The color of betanin is most stable between pH 4.0 and 6.0. Thermostability of betanin was pH dependent and was greatest between pH 4.0 and 5.0. The half-life of betanin at pH 5.0 and 100°C was 14.5 min, and increased in an unprotected model system to 1150 min at 25°C. The rate at which betanin degraded in model systems was affected by air and/or light. The cumulative effect of these two conditions was a reduction in the half-life of betanin by 28.6%. The half-life of betanin in beet juice at pH 5.0 was greater than in model systems. Despite limited stability of betanin in model systems, there are many foods in which betanin could be used as a colorant. Foods to be colored with betanin should have a pH between 4.0 and 7.0, be exposed to low temperatures and be protected from air and/or light to achieve maximum color stability.