EFFECT OF SEVERAL EXPERIMENTAL PARAMETERS ON COMBINATION OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR WITH PORCINE PANCREATIC α-AMYLASE

Purified red kidney bean (Phaseolus vulgaris) amylase inhibitor forms a 1:1 stoichiometric complex with porcine pancreatic α-amylase leading to complete loss of enzyme activity on starch. Rate of complex formation is pH dependent and is maximal at pH 5. The rate constants for complex formation, as measured by loss of amylase activity, were 2.85 × 10⁴ M^-1 sec^-1 at pH 6.9 (ionic strength of 0.918) and 2.55 × 10⁵ M^-1 sec^-1 at pH 5 at 30°C. At pH 6.9, rate of complex formation was 4.8 times faster at 0.918 ionic strength as compared with the rate at 0.138 ionic strength. At 30°C, pH 6.9 and ionic strength of 0.168 the dissociation constant of the enzyme-inhibitor complex was determined to be 3.5 × 10^-11 M. The rate constant for dissociation of the complex was calculated to be 8.7 × 10^-8 sec^-1 under the same conditions. The rate constant for complex formation, at ionic strength of 0.168, was 1.1 × 10⁴ M^-1 sec^-1 at 37⁰ and 9.77 × 10² M^-1 sec^-1 at 25.7°C. The calculated activation energy for complex formation is 39.5 kcal/mole suggesting a rate-controlling conformational change. Oxidation of the carbohydrate moiety of the glycoprotein inhibitor caused complete loss of activity. Maltose, a competitive inhibitor of α-amylase, bound as readily to the enzyme-inhibitor complex as to free α-amylase. Trypsinized α-amylase, although still able to bind to Sephadex, did not bind inhibitor. The experiments with maltose and trypsinized amylase suggest the inhibitor may not bind at the active site of α-amylase.     

The effect of post-mortem conditions on the extractability and adenosine triphosphatase activity of myofibrillar proteins of rabbit muscle

Summary. 1. Washed myofibrils from rabbit muscle have been heated at pH values between 4.8 and 5.6 and temperatures between 35°C and 42°C. It has been found that, under these conditions, myofibrils lose their Ca²⁺ activated adenosine triphosphatase, their Mg²⁺ activated adenosine triphosphatase and also become less extractable in M KCl–30 mM sodium glycerophosphate, pH 6.2.
2. The reactions follow first-order kinetics and the rates are dependent on pH and temperature. The first order rate constants, enthalpies and entropies for the three reactions are sufficiently near each other to suggest that all three reactions are occurring simultaneously.
3. When a muscle is allowed to go into rigor at 37°C the extractability in M KCl–30 mM sodium glycerophosphate is reduced after 4 hr at 37°C when the pH of the muscle has reached 5.55. At the same time the Ca²⁺ adenosine triphosphatase activity falls but the Mg²⁺ adenosine triphosphatase does not. The latter is reduced by prolonging the period at 37°C to 6 hr.
4. It is suggested that there is present in muscle, undergoing rigor at 37°C, myosin which does not bind to actin and is readily denatured. When bound to actin, myosin in the myofibril is more resistant and denatures only after long exposure to a temperature of 37°C.     

Production of Enterotoxin-B in Cured Meats

SUMMARY— A variety of laboratory cured hams were inoculated with 10³−10⁶ cells of S. aureus strain S-6 and incubated at 10, 22 and 30°C anaerobically for up to 16 weeks. Enterotoxin-B was detected by gel-diffusion in hams with original pH over 5.30, up to 9.2% NaCl (brine) and 0.54 ppm undissociated nitrous acid. There was better toxin production at 30° than at 22° or 10°C. Toxin was detected at 10°C after at least 2 weeks incubation and in most samples after 8 weeks when pH was greater than 5.6. Toxic hams had more than 4 × 10⁶ cells/g. Contaminants were always less than 10⁵/g. Tween 80 inhibited toxin production at 30° but not at 10°C. Toxic hams looked normal even after 2 months incubation at 10°C.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

The effects of different incubation temperatures on the acetaldehyde content and viable bacteria counts of bio-yogurt made from ewe's milk

Yogurt and bio-yogurt were manufactured from ewe's milk using a starter culture and a probiotic culture. Incubation was carried out at 37°C and 42°C until pH 4.6 was reached and the yogurts were stored at 4 +1°C for 14 days. Analysis after 1, 7 and 14 days showed that incubation temperature and storage time significantly influenced overall properties of the samples. During the storage, whey separation and pH decreased, but titratable acidity, lactic acid and volatile fatty acid contents increased. Viable bacterial counts in all bio-yogurts were above 10⁷ cfu g⁻¹ at the end of storage.     

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.     

Antioxidant Activity of Soy Protein Hydrolysates in a Liposomal System

ABSTRACT: Native and heated soy protein isolate was hydrolyzed with 3 purified (pepsin, papain, and chymotrypsin) and 3 crude (Alcalase®, Protamex^TM, and Flavourzyme^TM) proteases. The hydrolysates were incubated (37 °C, 1 h) with a liposome-oxidizing system (50 μM FeCl₃/0.1 mM ascorbate, pH 7.0) to test antioxidant activities by determining the concentrations of TBARS. Degree of hydrolysis of SPI hydrolysates ranged from 1.7 to 20.6%. Both hydrolyzed and nonhydrolyzed SPI decreased TBARS (by 28 to 65%), except for papain-hydrolyzed samples. Samples of chymotrypsin- and Flavourzyme-hydrolyzed (0.5 h) preheated SPI had the greatest inhibitory effect on lipid oxidation.     

CHARACTERIZATION OF DYE-SENSITIZED PHOTOOXIDATION OF MUSHROOM TYROSINASE

Dye-sensitized photooxidation was investigated as a nonthermal means to inactivate food quality-related enzymes, using mushroom tyrosinase as a model. Illumination of tyrosinase in the presence of either rose bengal or riboflavin resulted in an apparent first-order destruction of enzyme activity. Both dye and light were required, and photoinactivation was favored by increasing levels of dissolved oxygen. The rose bengal-sensitized photooxidation was generally more rapid than that caused by riboflavin (at 0.01% dye), with k_i values ranging from 0.74 to 1.66 h⁻¹ and 0.25 to 1.23 h⁻¹, respectively. First-order rate constants for photoinactivation decreased with decreasing temperatures (E_a was 8.4 to 12.2 kcal mol⁻¹ between 20° and 50°C), increasing protein concentration (0.175 to 1.40 mg⁻¹ ml), increasing sodium phosphate buffer concentration (10 to 200 m M) and increasing ionic strength (0.02 to 0.20). The dependence of enzyme photoinactivation rates on pH (between 6.0 and 9.0) resembled a titration curve with a pK of 7.5, and maximal rates were observed at pH 8.0–9.0.     

Osmotic Dehydration of Tomato in Sucrose Solutions: Fick's Law Classical Modeling

ABSTRACT:  Osmotic dehydration of tomato was modeled by the classical Fick's law including shrinkage, convective resistance at the interface and the presence of water bulk flow. Tomato slices having 8 mm thickness were osmotically dehydrated in sucrose solutions at 50, 60, and 70 °Brix and at 35, 45, and 55 °C. Other experiments were done in a 70 °Brix sucrose solution at 35 °C with tomato slices of 4, 6, and 8 mm thickness and at different motion levels (velocities 0, 0.053, and 0.107 m/s). Tomato weight, water content, and °Brix of the products were measured as a function of processing time (20, 40, 80, 160, and 320 min). Results showed that temperature, concentration, thickness, and solution movement significantly influenced water loss and sucrose gain during the osmotic dehydration of tomato. The model predicted the modifications of soluble solid content and water content as a function of time in close agreement with the experimental data. Experimental Sherwood number correlations for sucrose and water were determined as Sh _s = 1.3 Re ^0.5 Sc _s^0.15 and Sh _w = 0.11 Re ^0.5 Sc _w^0.5, respectively. The effective diffusion coefficients of water (4.97 10⁻¹¹– 2.10 10⁻¹⁰ m²/s) and sucrose (3.18 10⁻¹¹– 1.69 10⁻¹⁰ m²/s) depended only on temperature through an Arrhenius-type relationship.     

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.     

GROWTH OF STAPHYLOCOCCUS AUREUS AND ENTEROTOXIN A PRODUCTION IN FOODS CONTAINING POLYPHOSPHATES

Effects of pyro-, tripoly- and hexametaphosphates (0.5 and 1%, w/w) on growth of Staphylococcus aureus strain 196E and enterotoxin A (SEA) production were studied in cooked custard and beef at 22 and 30°C. No effect was observed in custard, where cell numbers/g increased from 10³ to 10⁸ and SEA reached 4.2 ng/g after 48 h at 22°C, irrespective of treatment. Cell numbers in cooked beef were ca. 10⁹/g after 48 h, and reduced numbers (by 1.5–2 log cycles) were found in samples containing 0.5 and 1% pyrophosphate during incubation at 22%, but not at 30°C. SEA concentrations in beef were 28 ng/g after 48 h at 22°C, and 93 and 184 ng/g after 24 and 48 h, respectively, at 30°C. SEA concentration correlated with amount of growth, and was nondetectable when cell numbers were ± 10⁶/g. Reduction of the meat pH by sodium acid pyrophosphate contributed to the observed inhibitory effect.     

ENTEROTOXIN FORMATION IN FOODS BY CLOSTRIDIUM PERFRINGENS TYPE A

Growth, sporulation and enterotoxin formation in various foods inoculated with a Clostridium perfringens type A enterotoxin-producing strain were studied. Good vegetative growth, 10⁷–10⁸ cells/g, was obtained after 4 hr of anaerobic growth and remained almost the same throughout the 20–24 hr observation in most of the foods. A gradual increase in spore count to the level of 10⁴–10⁵/g was observed with an increase in the incubation time. Enterotoxin was detected in moist cooked chuck roast, ground beef and turkey as well as in moist cooked and dry roasted chicken at levels up to 0.125μg/g. The earliest time at which enterotoxin was detected was after 10 hr of anaerobic growth in moist cooked turkey at 37°C. Although growth and some sporulation occurred, enterotoxin was not detected in dry roasted beef or turkey with or without gravy, or in moist cooked pork or lamb. Poor growth and sporulation also were obtained with chicken broth, chicken gravy and beef gravy. In moist cooked turkey that had been temperature abused for 6 hr at 37°C, held cold for 15 hr and reheated to 37°C, toxin could be detected after only 5 hr of holding at 37°C. The ability of certain foods to support sporulation and enterotoxin formation indicates that such preformed enterotoxin may contribute to early onset of symptoms in some cases of C. perfringens food poisoning.     

Portable Electronic Nose for Detection of Spoiling Alaska Pink Salmon (Oncorhynchus gorbuscha)

ABSTRACT:  The ability of a portable hand-held electronic nose (EN) in detecting spoilage of whole Alaska pink salmon ( Oncorhynchus gorbuscha ) stored at 14 °C and in slush ice (1 °C) was investigated. Fish were sampled daily at 14 °C for up to 3 d, while fish stored in slush ice were sampled at various intervals up to 16 d. Sensory evaluations indicated that fish were rejected at day 3 when stored at 14 °C and at day 12 when stored in slush ice. Aerobic bacteria counts for fish skin at 14 °C ranged from 3.4 log₁₀ colony-forming units (CFU)/cm² (day 0) to 4.8 log₁₀ CFU/cm² (day 3) and for fish stored in slush ice ranged from 3.4 log₁₀ CFU/cm² (day 0) to 5.5 log₁₀ CFU/cm² (day 16). The correct classification rate using forward stepwise general discriminate analysis was 85% and 92% for EN analysis of belly cavity volatiles for fish held at 14 °C and in slush ice, respectively. A predictive model may be developed for spoilage of whole Alaska pink salmon by analyzing belly cavity odors using the EN.     

Characterization of a thermostable and acidic-tolerable beta-glucanase from aerobic fungi Trichoderma koningii ZJU-T

ABSTRACT:  An extreme thermostable and acidic tolerable β-glucanase was isolated and characterized from aerobic fungi Trichoderma koningii ZJU-T. The optimal reaction temperature and pH for the β-glucanase were 100 °C and pH 2.0, respectively. The β-glucanase showed increased stability at higher temperatures and lower pH values when compared to other β-glucanases. The optimum conditions for the β-glucanase stability were found to be pH 4.0 and 80 °C. Even subjected to 100 °C for 3 h, β-glucanase activity did not show significant reduction. Moreover, K⁺ significantly enhanced β-glucanase activity at the concentration of 1 mM, while EDTA and other metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, Ca²⁺, Fe²⁺, Pb²⁺, and Fe³⁺ inhibited β-glucanase activity. Denaturants, including sodium dodecyl sulfate (SDS) and mercaptoethanol, also inhibited β-glucanase activity at a concentration of 5%. However, in the presence of 7 M urea, residual activity of the β-glucanase still remained 14.5%.     

Temperature-Sensitive Microcapsules Containing Lactoferrin and Their Action Against Carnobacterium viridans on Bologna

ABSTRACT:  Lactoferrin (LF) was encapsulated in 2 types of emulsion to protect it from contact with agents like divalent cations, which interfere with its antimicrobial activity. First, paste-like microcapsules were prepared as water-in-oil (W₁/O) emulsions from mixtures of 20% w/v LF in distilled water, 20% w/v LF in 3% w/v sodium lactate or in 20 mM sodium bicarbonate, which were emulsified with an oil mixture of 22% butter fat plus 78% corn oil and 0.1% polyglycerol polyricinoleate. Second, freeze-dried double emulsion (W₁/O/W₂), powdered microcapsules were produced following emulsification of paste-like microcapsules in an external aqueous phase (W₂) consisting of a denatured whey protein isolate (WPI) solution. The release of LF from the W₁/O microcapsules was dependent on temperature and NaCl concentration. LF was not released from the W₁/O emulsion at <5.5 °C. Its release was greater from W₁/O microcapsules when suspended in 5% aqueous NaCl than in water at ≥10 °C, whereas LF release from freeze-dried microcapsules was not controlled by temperature change. Paste-like microcapsules were incorporated in edible WPI packaging film to test the antimicrobial activity of LF against a meat spoilage organism Carnobacterium viridans . The film was applied to the surface of bologna after its inoculation with the organism and stored under vacuum at 4 or 10 °C for 28 d. The growth of C. viridans was delayed at both temperatures and microencapsulated LF had greater antimicrobial activity than when unencapsulated. The temperature-sensitive property of the W₁/O microcapsules was reduced when they were incorporated into a WPI film.     

EARLY DETECTION OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN AND ITS RELATIONSHIP TO SENSORY QUALITY OF COOKED CHICKEN

Growth, sporulation, and enterotoxin formation by Clostridium perfringens NCTC 8239 were determined in chicken thigh meat incubated at 45°C for 1.5 h and 37°C for up to 12.5 h. With an inoculum of 10⁶ vegetative cells per g, the cell counts reached mean log ₁₀ 7.32/g after 6 h of incubation and remained in that range through 14 h. Heat-resistant spores (log₁₀ 2.48/g) were first detected at 4 h, and the number increased to log₁₀ 5.19/g at 14 h. Enterotoxin (0.19 μg/g) was first detected after 2 h of incubation (1.5 h at 45°C and 0.5 h at 37°C) in the absence of detectable sporulation, and the enterotoxin concentration increased to 0.76 μg/g after 14 h. Significant differences (p < 0.01) in the odor, color, and texture scores for inoculated versus uninoculated cooked chicken following 2 h incubation correlated with the production of enterotoxin and suggested that these parameters could be used as indices of chicken spoilage by C. perfringens.     

Alicyclobacillus acidoterrestris in fruit juices and its control by nisin

Summary The acid-tolerant and heat-resistant bacterium Alicyclobacillus acidoterrestris is a spoilage problem in pasteurized and heat-treated fruit juices. In this study it was shown to grow in orange juice, grapefruit juice and apple juice to produce detectable taint at levels of about 10⁴–10⁵ c.f.u. ml⁻¹. Decimal reduction times were determined at 80 °, 90 ° and 95 °C in each juice and confirmed the heat-resistant nature of the spores under normal juice pasteurization conditions. They also confirmed that heat sensitivity increased with decreasing pH but that this effect was less pronounced at higher temperatures. The organism was, however, sensitive to the bacteriocin food preservative nisin. The presence of nisin during heating decreased the D value by up to 40% and the MIC for nisin against spores at 25 °C was only 5 International Units (IU) ml⁻¹. The results indicate that use of nisin is a potentially useful way of controlling this organism in fruit juices and fruit juice-containing products.     

Mechanisms of gelation of sardine proteins: influence of thermal processing and of various additives on the texture and protein solubility of kamaboko gels

Surimi prepared from freshly caught sardines was mixed with NaCl and other additives and used to prepare kamaboko gels. Protein-protein interactions involved in the setting (at 4 or 37°C) and/or the cooking (at 90°C) gelation steps were investigated (i) by assessment of kamaboko texture as a result of the type and concentration of additive added; (ii) by partial solubilization of kamaboko gels in buffers containing mercaptoethanol, sodium dodecyl sulphate (SDS) and/or urea, followed by determination of the soluble protein constituents by polyacrylamide gel electrophoresis. Cooked gels of high elasticity and of varying rigidity and gel strength were obtained in the 73–80% water range. Adequate gel texture required a NaCl content of 1.7–3.5% and a pH range of 6.4–8.4. Low concentrations of reducing agents (mercaptoethanol, dithiothreitol, cysteine) or of divalent cations (Ca²⁺, Mg²⁺) improved the texture of gels obtained by setting at 37°C with and without subsequent cooking at 90°C. On the other hand, the addition of N -ethyl maleimide or of ethylene diamine tetra-acetate led to texture deterioration after cooking. These data demonstrate the involvement of disulphide bonds and of electrostatic interactions in surimi gelation. Gel solubilization experiments indicate that the aggregation of myosin heavy chains through various types of protein-protein interactions may be responsible for the elastic gel network formed during setting at 37°C (30 min) or 4°C (24h). Strengthening of the gel network after cooking appears to be due to additional disulphide and hydrophobic interactions.     

Quenching Mechanism and Kinetics of Ascorbic Acid on the Photosensitizing Effects of Synthetic Food Colorant FD&C Red Nr 3

ABSTRACT:  The pH effect on the oxidative stability of ascorbic acid in the presence of food colorant FD&C Red Nr 3 during storage with or without light was investigated. The quenching mechanism and kinetics of ascorbic acid on the FD&C Red Nr 3 photosensitized oxidation in an aqueous system at 25 °C were also studied by measuring the degradation of ascorbic acid or depletion of headspace oxygen. Red Nr 3 had no influence on the oxidation of ascorbic acid under dark storage, but accelerated its oxidation rate under light storage. The oxidative stability of ascorbic acid decreased as the pH increased from 4 to 7 under light without FD&C Red Nr 3. The quenching rates of ascorbic acid on the singlet oxygen by measuring the degradation of ascorbic acid in the presence of Red Nr 3 under light storage were 1.53 ± 0.15 × 10⁸, 1.86 ± 0.25 × 10⁸, and 1.19 ± 0.12 × 10⁸ M⁻¹S⁻¹ at pH 4, 5.6, and 7, respectively.     

The air drying behaviour of fresh and osmotically dehydrated banana slices

Ripe banana, cut to 10mm thick slabs were osmotically treated in sugar solutions of 35, 50 and 65° Brix for 36h. The initial moisture content fell from a value of 3.13kg H₂O DM to 2.19, 1.63 and 1.16kg H₂O kg⁻¹ for treatment in the three solutions, respectively. These slabs, with Total Soluble Solids (TSS) contents of 26, 34 and 39° Brix, respectively, as well as freshly cut but untreated slabs (15° Brix) were air dried in a cabinet type tray drier to near equilibrium conditions at fixed temperatures from 40 to 80°C and at a constant air speed of 0.62m s⁻¹. Drying was found to occur in the falling rate period only for both banana types and two drying constants K₁ and K₂ were established for a first and second falling rate period of drying. Increasing the drying air temperature significantly enhanced the drying rate and the K-values, except at 80°C when the rates fell, possibly because of case hardening of the slabs. Reducing the slab thickness also improved the drying rate, but increasing the air speed to 1.03m s⁻¹ did not have any profound effect. As the sugar content of the banana slabs increased through the osmotic treatment, drying rates fell. Calculated apparent moisture diffusivities at 60°C ranged from 34.8× 10⁻¹⁰ m² s⁻¹ (fresh slab) to 8.8×10⁻¹⁰ m² s⁻¹ for dried (39° Brix) slabs. The moisture diffusivity was significantly lowered as the moisture content dropped in drying and with increased levels of sugar. Previously osmosed and then air dried banana slabs showed appealing colour and texture compared to the fresh banana.