PERTURBATION OF THE STRUCTURE OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN BY SODIUM DODECYL SULFATE, GUANIDINE HYDROCHLORIDE, pH AND TEMPERATURE

The effects of sodium dodecyl sulfate, guanidine · HCl, heat and pH on the tertiary structure of Clostridium perfringens type A enterotoxin were determined by UV difference spectroscopy and by accessibility of the amino groups to 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) and the one sulfhydryl group to 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB). Biological stability of enterotoxin at several pH's at 55°C in the presence and absence of borate and dipicolinate ions was determined by the guinea pig skin test. Sodium dodecyl sulfate and heat did not unfold the molecule while treatment with >4.5 M guanidine · HCl at ≥25°C completely unfolded the molecule based on UV difference spectral changes and the accessibility of the amino groups to reaction with TNBS. Sodium dodecyl sulfate, at 0.2%, made the sulfhydryl group accessible to DTNB but did not affect the accessibility of the amino groups (7.89 ± 0.15 groups in the native molecule). Guanidine · HCl, at 6.5 M for 30 min and ≥25°C, made all 17 e-amino groups and the one α-amino group accessible to TNBS. At pH's above 8 spectral differences, in relation to the spectrum at pH 6.8, were associated primarily with ionization of the tyrosine residues while at pH's below 5.5 the spectral changes were probably a result of protonation of carboxyl groups in the vicinity of tyrosine and tryptophan residues, and of aggregation.     

MODELING OF THE GERMINATION OF SPORES FROM CLOSTRIDIUM PERFRINGENS FOOD POISONING ISOLATES

Clostridium perfringens type A isolates carrying a chromosomal gene for enterotoxin production (chromosomal c. perfringens enterotoxin [ C- cpe] strains) are frequent causative agents of food poisoning. Predictive models for their growth in meat products have been published; however, the development of germination models needs further research. In this study, the Weibull function was used to describe the germination of C- cpe food poisoning strains in phosphate and Tris–HCl buffer as affected by pH (5.8–8.5), germinant concentration (1–100 mM KCl) and germination temperature (23–60C). As indicated by estimators of model accuracy and bias, an empirical model for spore germination as a function of germination temperature only, predicted accurately the germination potential of 4 C- cpe food poisoning strains in buffer and in laboratory media in the 23–50C range.

PRACTICAL APPLICATIONS

Thermal processing models based on the inactivation of a heat-resistant spore ignore the potential germination and outgrowth of spore survivors as recommended by new food safety guidelines. Germination models for spore survivors to be used in conjunction with outgrowth models should be developed using as samples the product of interest because the bioavailability and concentration of germinants such as ions and free amino acids vary with the product and its manufacturing process. Another germination model application is in conjunction with new processing technologies such as high pressure processing (HPP). The inactivation of bacterial spores has been a major challenge to HPP process developers. Since germinated spores are sensitive to pressure, germination models would be useful to design combined strategies of food thermal treatment and HPP.     

Effects of buffer and temperature on formation of furan, acetic acid and formic acid from carbohydrate model systems

Effects of temperature, pH and phosphate buffer on volatiles formed by heating model solutions of fructose, glucose, sucrose, lactose or starch were investigated by selected ion flow tube-mass spectrometry (SIFT-MS). Among the carbohydrates studied, the monosaccharide reducing sugars fructose and glucose were more reactive than macro molecular starch, lactose and sucrose, which are not reducing sugars. Furan was formed from fructose heated at 120, 100 and 80 °C at pH 5-7 in phosphate buffer and at 100 °C in unbuffered solution. Glucose did not yield furan in unbuffered solution at 100 °C but glucose did yield furan in phosphate buffer solution at a similar pH. Furan formation increased as temperature and pH increased. As temperature, pH value, and buffer components changed, furan formation may occur by different reactive pathways. Both of the buffer components, NaH₂PO₄ and citric acid, enhanced the formation of thermal degradation products from fructose. The enhancing effect of NaH₂PO₄ was stronger than citric acid. Formic and acetic acids were concomitantly formed with furan. More formic acid was produced from fructose than glucose, and more acetic acid was produced from glucose than fructose.     

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.     

Reversed Micellar Extraction of Hen Egg Lysozyme

Egg white was diluted to 10 times its original volume with 50 mM phosphate buffer (pH 9.2) containing 0.1 M potassium chloride. The aqueous solution was mixed with an equal volume of isooctane containing 50 mM bis-(2-ethylhexyl) sodium sulfosuccinate at 10°C for 50 min. After extraction, the organic phase containing lysozyme was separated from the aqueous phase and mixed with an equal volume of 50 mM phosphate buffer (pH 11.8) containing 1M potassium chloride. Backward extraction was then performed at 30°C for 45 min. The procedures recovered 90% lysozyme from the eggwhite. The specific activity of the extract was near 73,000 units/mg.     

A CIRCULAR-DICHROISM STUDY OF THE ENTEROTOXIN FROM CLOSTRIDIUM PERFRINGENS TYPE A

Far-UV circular-dichroism spectra of the enterotoxin from Clostridium perfringens type A were recorded under different conditions, and conformational analysis was performed. The native enterotoxin (pH 6.8) was shown to contain about 80% pleated sheet, 20% random coil and no helix structure. By adding small amounts of the detergent sodium dodecyl sulfate (0–261 mol SDS/mol enterotoxin), the β-sheet structure could gradually be changed to a more α-helical structure. Treatment with 8 M urea resulted in limited alterations of the secondary structure of the enterotoxin, while heat treatment (60°C, 30 min), treatment with 6.5 M guanidine hydrochloride or 0.01 M NaOH led to a dramatic increase in the amount of random coil.     

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.     

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.     

IMMOBILIZATION OF PAPAIN ON AN ANION EXCHANGE RESIN BY PHYSICAL ADSORPTION FOLLOWED BY CROSS LINKING WITH GLUTARALDEHYDE

A practical method involving physical adsorption by vacuum drying of papain on Dowex MWA-1 (mesh 20–50), followed by intermolecular cross linking with 1.0% glutaraldehyde under neutral pH conditions was developed. Potassium phosphate buffer (0.1 M, pH 12.5) was better for immobilization of papain than was acetate buffer (0.1 M, pH 4.0). Nitrogen recovery was increased by increasing the reaction time with glutaraldehyde from 0.5 to 15 min and enzyme concentration from 1 to 80 mg/0.3 mL buffer/0.2 g resin. Recovery of absolute and proteolytic activities, using casein as a substrate, was increased when reaction time was increased from 0.5 to 5 min. These activities were increased in a papain concentration range of 5 mg/0.2 mL buffer/0.2 g resin to 5 mg/0.3 mL buffer/0.2 g resin and then decreased with greater volumes of buffer, ranging from 11.2 to 8.6%. Relative activity recovery was decreased with an increase in reaction time and enzyme concentration, ranging from 40.5 to 7.5% and 47.5 to 1.5%, respectively. Cysteine (0.08 M) and EDTA (2 mM) enabled more than 95% of the original activity of immobilized papain to be maintained for 2 months at 5°C; NaHSO₃ and ascorbic acid inactivated immobilized papain.     

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 .     

CHARACTERIZATION OF PHYTASE OF BEANS (PHASEOLUS VULGARIS)

Phytase from California Small White beans was extracted, concentrated and enriched by heat treatment and ammonium sulfate fractionation. Crude enzyme fractions tenaciously retained endogenous phytate which could be removed by autolysis at 45°C and pH 5.2. Characterization of the phytase preparation showed: (1) increase in activity with increasing temperature from 37 to 60°C, (2) an activation energy of approximately 9,200 cal/mole for the hydrolysis of inositol hexaphosphate; (3) pH optimum of 5.2; (4) a K_m value of 2.22 × 10^-4M; (5) apparently partial inhibition at high substrate concentrations; (6) fully competitive inhibition by one of the reaction products, inorganic phosphate, with K_i=3.1 × 10⁴M. These properties and kinetic constants are comparable to those reported for phytase preparations from similar sources and they adequately account for in situ autolytic loss of phytic acid from these beans.     

INFLUENCE OF SODIUM PHOSPHATE ON THE HEAT STABILITY OF BUFFALO MILK AND ITS CONCENTRATE

The influence of three different concentrations, 0.05%, 0.10% and 0.15% of monobasic sodium phosphate on the heat stability (at 130°C) and pH of buffalo milk and its 2:1 concentrate was determined. It was observed that sodium phosphate caused a considerable increase in the heat stability, determined as heat coagulation time (HCT) of concentrated buffalo milk. The optimum concentration of sodium phosphate for imparting maximum stability to the concentrate was different for different samples. However, with the addition of an appropriate concentration of sodium phosphate it was possible to manufacture evaporated milk up to 36% total solids. Depending on the HCT/pH profile, some of the samples of fluid (unconcentrated) milk were stabilized while the others were destabilized due to the addition of sodium phosphate. Addition of monobasic sodium phosphate caused a decrease in the pH of fluid milk and its concentrate.     

EFFECT OF ULTIMATE pH UPON THE WATER-HOLDING CAPACITY AND TENDERNESS OF MUTTON

SUMMARY— Ultimate pH values in the musculature of sheep ranging from 5.6–7.0 have been obtained by using pre-slaughter injections of epinephrine. A high speed centrifugal method was used to measure the water-holding capacity of raw M. semitendinosus (ST), semimembranosus (SM) and biceps femoris (BF). Results showed a high correlation with ultimate pH. Cooking losses end the amounts of centrifugally expressed juice were determined for the SM and BF cooked for 1 hr at either 65°C or 90°C. Cooking losses at 65°C decreased linearly with increasing pH while the losses at 90°C showed little change up to o raw meat pH of ca. 5.9, then decreased linearly with increasing pH. The amount of juice centrifugally expressed from the cooked meat, which has a high positive correlations with organoleptic juiciness, increased linearly with pH. Tenderness of the cooked SM and BF muscles was measured using o Warner-Bratzler shearing device and an Instron Universal Testing Machine: both gave high objective-subjective correlations. Instron measurements have high negative linear correlations with ultimate pH for both the 65°C and 90°C cooked samples. Hardness of these muscles, cooked at 65°C or 90°C. decreased approximately three-fold as ultimate pH increased from 5.6–6.9. Results obtained using the Warner-Bratzler device showed linear regressions with a significant quadratic component for one muscle at both 65°C and 90°C.     

GINGER RHIZOME: A NEW SOURCE OF PROTEOLYTIC ENZYME

The proteolytic activity of ginger rhizome was studied with bovine serum albumin (BSA), collagen and actomyosin as substrates. A semipurified, powdered enzyme preparation was prepared by buffer extraction of an acetone powder of ginger rhizome and subsequent acetone precipitation of the proteolytic principle from the buffer extract. With 3% BSA as substrate, a relatively high proteolytic activity occurred over a pH range of 4.5–6.0, with an optimum pH of 5.0. The optimum temperature for proteolysis of BSA was 60°C during a 10 min reaction time, with rapid denaturation of the enzyme occurring at 70° C. NaCl in cone up to 10% produced about a 20 and 50% reduction in proteolysis of collagen and BSA, respectively. The ginger protease was protected by dithiothreitol during extraction and reaction, indicating the involvement of −SH groups at the active site. The analyses of soluble peptide amino acids or terminal amino acids suggest that the proteolysis of collagen is many fold greater than that of actomyosin. The combined proteolysis of these two muscle protein fractions by the ginger protease resulted in significantly more tender meat. According to conventional nomenclature, "Zingibain" is the proper name for this proteolytically active principle in Zingiber officinale roscoe or ginger rhizome which is commonly referred to as gingerroot. For meat applications, a possible advantage of zingibain over papain and ficin is the greater proteolysis of collagen in comparison to actomyosin. When compared to reported values for bromelain, zingibain has a higher optimum activity temperature, which is desirable in some applications     

SURVEY OF FOOD INGREDIENT-DDT REACTIONS UNDER THERMAL PROCESSING CONDITIONS

Aqueous solutions or suspensions of single or multiple food ingredients were heated at 100°C for 2 hr with carbon–14 labeled DDT. Thin-layer chromatography on alumina plates resolved the resulting mixtures into regions corresponding to R_f values for five known transformation products or analogs of DDT. The radioactivity of each plate region was related to the quantity of each product formed. DDT is relatively stable in heated water or buffer solutions of pH 3 to 8 as shown by recoveries of DDT (98–100%); at pH 8.1, approximately 4% of DDE was formed. The compositional changes in radioactive DDT due to heating with water-soluble vitamins, disphosphopyridine nucleotide, yeast nucleic acid, amino acids, peptides and casein were determined. The maximum reduction in DDT content of a suspension heated with a single component (cysteine hydrochloride) was 10%. A mixture of DDT with nine components heated for 2 hr produced a 20% reduction in DDT content.     

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.     

In Vitro and Mouse Evaluation of Methods for Protecting Whey Protein and Casein from Ruminal Degradation

A series of in vitro buffer (protein solubility) and rumen fermentation (ammonia production) studies were conducted to evaluate varying formaldehyde (.25, .5, 1, and 3%) and tannic acid (.5, 1, 2, 3, and 6%), and three heating times (1, 2, and 3 h at 104 C) on protection of whey protein concentrate and casein from ruminal degradation. Subsequent trials of mouse growth were conducted to evaluate the effectiveness of protection of treated proteins. Formaldehyde (.25 to 3%) reduced solubility of whey protein and casein to less than 10% of the untreated in pH 6.8 buffer and approximately 30% of the untreated in pH 2.5 buffer plus pepsin. Formaldehyde reduced ammonia production, indicating protection and reduced solubility of whey protein and casein under rumen conditions in vitro. Tannic acid (.5 to 6%) did not greatly reduce protein solubility and ammonia production. Heat treatment reduced protein solubility to less than 10% of the untreated in pH 6.8 buffer and to 50% of the untreated in pH 2.5 buffer and reduced ammonia production to approximately 20% of the untreated. Growth and feed intake of mice were decreased with 1% formaldehyde treated diets (17% protein), indicating overprotection of protein. Growth decreased as tannic acid increased. Mouse growth also decreased as the length of heat treatment increased. Diets with .5% formaldehydecasein depressed gains. Based on these in vitro and mouse-evaluation studies, formaldehyde less than .5% of the dry weight is required to protect whey protein and casein from ruminal degradation and permit solubilization in the lower intestinal tract.     

RELEASE OF ENTEROTOXIN A AND THERMONUCLEASE FROM GROWING AND NON-GROWING CELLS OF STAPHYLOCOCCUS AUREUS

Enterotoxin A and thermonuclease were produced by Staphylococcus aureus cell populations in all growth phases. Synthesis of entero-toxin was more rapid than that of thermonuclease early in the exponential phase, whereas later in the exponential phase the thermonuclease synthesis was more rapid than that of enterotoxin. The optimum temperature for enterotoxin production was 42°C, while that for thermonuclease production was in the range 27–33°C. Small amounts of enterotoxin and thermonuclease were released at temperatures up to 45°C.
During heat treatment of staphylococcal cells, enterotoxin production was not observed at temperatures in excess of those permitting growth, although small amounts of thermonuclease were produced. Intracellular enterotoxin was not detected after disintegration of cells under pressure at –30°C. Enterotoxin at detectable concentration, was not bound to the cell membranes. All the results obtained indicate that the synthesis of enterotoxin by S. aureus is intimately associated with cell growth.     

Species-Specific Effects of Sarcoplasmic Extracts on Lipid Oxidation in vitro

ABSTRACT:  The degree to which lipid and myoglobin (Mb) oxidation processes interact in meat can be species-specific. We investigated the effects of beef and pork sarcoplasmic extracts containing different Mb concentrations on lipid oxidation in a liposome system. Sarcoplasm was extracted from beef and pork longissimus dorsi and psoas major muscles. Beef sarcoplasm was diluted with 0.1 M phosphate buffer to obtain a Mb concentration equivalent to that in pork sarcoplasm. Conversely, equine heart Mb was added to pork sarcoplasm to match the myoglobin concentration of beef sarcoplasm. This resulted in beef and pork sarcoplasms, each with 2 different Mb concentrations for the longissimus (0.02 mM and 0.07 mM) and psoas (0.05 and 0.12 mM). Sarcoplasm (or phosphate buffer control) was incorporated within a phosphatidylcholine liposome preparation and incubated at 25°C. Thiobarbituric acid reactive substances (TBARS) were measured at 0, 30, 60, 90, and 120 min of incubation. Regardless of species, greater Mb concentration within the sarcoplasm increased lipid oxidation ( P < 0.05). Across muscles, pork sarcoplasm had lower TBARS values than beef sarcoplasm ( P < 0.05). Our results suggest that pork sarcoplasm has a lesser effect on lipid oxidation than beef sarcoplasm for a common Mb concentration. However, increased myoglobin concentration within sarcoplasm promotes lipid oxidation regardless of species.     

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.