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.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized, cured meats III. The effect of potassium sorbate

The growth of Clostridium botulinum types A and B spores, at 10¹ or 10³ per container, was studied in a pork slurry system containing nitrite (40 μg/g), sodium chloride (2.5, 3.5, 4.5% w/v) sodium isoascorbate (550 μg/g) at varying pH levels, with or without potassium sorbate (0.26% w/v), without heating and after two heat treatments (80°C for 7 min, and 80°C for 7 min + 70°C for 1 hr) followed by storage at 15, 17.5, 20 or 35°C for up to 6 months. At a given spore inoculum, potassium sorbate significantly decreased toxin production, as did increasing NaCl, decreasing pH or decreasing storage temperature. Heat treatment did not significantly affect spoilage or toxin production overall, but interacted significantly with some factors. The effect of sorbate was greater at 3.5% NaCl than at 2.5%, at pH values below 6.0, and at low storage temperature.     

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.     

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.     

EFFECT OF NITRITE AND STORAGE TEMPERATURE ON THE ORGANOLEPTIC QUALITY AND TOXINOGENESIS BY Clostridium botulinum IN VACUUM-PACKAGED SIDE BACON

The effect of nitrite and storage temperature and toxinogenesis by Clostridium botulinum in vacuum-packed side bacon was investigated. In two series of experiments (A & B) bacon packs were prepared with levels of 0, 50, 100, 150 and 200 ppm nitrite and inoculated with C botulinum at 10² spores/g and 10⁴ spores/g. Packs A were incubated at 20 and 30° C and packs B at 30°C only. Both were held for a maximum of 32 days and analyzed for toxin at intervals of 2, 4, 8, 16 and 32 days. At 20°C none of the controls without nitrite was found to be toxic after 32 days. At 30°C inhibition of toxin formation at the higher nitrite levels was observed at 32 days. Organoleptic evaluation of the bacon packs stored at 30° C showed about one-third of the toxic samples examined were acceptable to the panel.     

Postharvest Microbiology of Farm-reared, Tropical Freshwater Prawn (Macrobrachium Rosenbergii)

ABSTRACT: The microbiological quality of farm-reared, tropical freshwater prawn ( Macrobrachium rosenbergii ) stored at 2 different temperatures was studied. The prawn muscle was found to have the initial bacterial load of 10⁴ cfu/g. The lactics and vibrios were in the range of 10² cfu/g, while the E. coli , aeromonads, staphylococci, anaerobes, and molds were in the level of 10¹ cfu/g. Salmonella and Vibrio cholerae were present in the prawn muscle. The prawn muscle held at room temperature (28 ± 2 °C) was organoleptically acceptable up to 8 h, when the bacterial load was more than 10⁶ cfu/g. However, the prawn muscle stored at freezer temperatures (−10 to −15 °C) was found to be in acceptable condition even after 30 d of storage and the bacterial load was fluctuating in the range of 10³ to 10⁴ cfu/g.     

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 yeast flora of stored ready-to-use carrots and their role in spoilage

Spoilage of ready-to-use grated raw carrots packaged in polymeric films and stored at 10°C was investigated for involvement of yeasts. Cryptococcus albidus was only isolated during the first 3 days of storage, increasing to levels of 10⁵–10⁶g^-1. Candida lambica was more commonly isolated after 3–7 days of storage, and reached 10⁷–10⁸g^-1 after 12 days. Candida sake was present throughout storage, increasing from 10⁵–10⁶ after 3 days to 10⁷–10⁸ after 12 days. In some samples, Candida parapsilosis and Candida tropicalis were also isolated at levels similars to C. sake . All the yeasts isolated at the end of storage were fermentative species and their metabolism was characterized with a Warburg apparatus. Neither the number of yeasts nor the composition of the yeast flora were related to the deterioration of the product. Although Candida lambica inoculated on grated carrots caused spoilage after 12 days at 10°C, the high O₂ permeable film was most effective in reducing exudate.     

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.     

A STUDY OF THE EFFECT OF STORAGE AT 5°C ON THE MICROBIAL FLORA OF HEAT-TREATED MARKET CREAM

Changes in the microbial flora of heat-treated cream due to storage at 5°C for five days were studied. The relatively mixed flora of fresh cream at both 5°C and 30°C incubation temperatures appeared to vary according to source, with few psychotrophic organisms originally. After storage the flora became almost dominated by psychotrophic types with total numbers not less than 10⁶/ml, although the original numbers were very low indeed—in most samples less than 1500/ml.     

The influence of mash pre-aging on the development of the flavour-active compound, 4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF), during soy sauce fermentation

Inoculation of Zygosaccharomyces rouxii into a soy sauce mash free from lactic acid bacteria at 10⁶ cells mL⁻¹ resulted in the formation of the flavour-active compound HEMF over a number of days at 30°C. Delays in inoculation up to 14 days after production of the mash greatly increased the amount of HEMF formed and under some conditions concentrations in the commercial range were achieved without addition of any other microorganisms.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats IV. The effect of pig breed, cut and batch of pork

Pork slurries were prepared from leg or shoulder muscle from three animals from each of three breeds of pig (Pietrain, Gloucester Old Spot and Large White × Landrace cross). Slurries (pork:water, 1:1.5) contained NaNO₂ (100 μ/g), NaCl (2.5, 3.5 or 4.5% w/v on the water), were subjected to one of three heat treatments (unheated, 80°C for 7 min, 80°C for 7 min plus 70°C for 1 hr) and stored at 35, 20, 17.5 or 15°C for up to 6 months to determine the relative effects of the above factors on growth (spoilage) and toxin production by Clostridium botulinum types A and B at 10³ spores per bottle.
Increasing salt or heat treatment, or decreasing storage temperature or inoculum level all reduced spoilage and toxin production. Both 'animal' and 'cut' significantly affected spoilage and toxin production. More spoilage and toxin production occurred in meat from the shoulder cut than from the leg cut. In both cases there was considerable variation between animals within breed, but there was no systematic difference between breeds. There is no obvious explanation for the variation in meat between animals, but it should be borne in mind when planning and assessing results of large multifactor experiments. Although there was more spoilage and toxin production after 6 months' than 3 months' storage, the statistical analyses yielded essentially similar conclusions.     

HEAT PASTEURIZATION OF CRAB AND SHRIMP FROM THE PACIFIC COAST OF THE UNITED STATES: PUBLIC HEALTH ASPECTS

ABSTRACT— A study of the potential public health hazard presented by coagulase-positive staphy-lococci, salmonellae and Clostridium botulinum in the meat of Dungeness crab and Pacific Coast shrimp pasteurized in flexible plastic containers revealed potentially toxinogenic staphylococci on the commercial nonpasteurized product, in 15% of the shrimp and 9% of the crab samples. No salmonellae or Cl. botulinum were isolated from, respectively. 26 and 54 samples of shrimp or 74 samples of crab. Pasteurization for 1 min at 180°F destroyed large inocula (10⁷ and 10⁸ cells) of staphylococci and salmonellae introduced into packages of the products, but processing for 5 min at 180°F allowed some members of an inoculum containing 10³ spores of Cl. botulinum type E to survive. While storage at 40°F prevented the growth on crab and shrimp meat of all staphylococci and salmonellae tested, it permitted growth and toxin formation by Cl. botulinum type E after 30–40 days. No toxin could be detected in packages inoculated with type A and proteolytic B spores and held at 50°F or lower. A 0.1% dip of sodium benzoate, with or without fumaric acid, did not prevent growth and toxinogenesis by Cl. botulinum types A, proteolytic B or E. It was concluded that for complete safety a holding temperature of 36°F or lower at all times would be required, but that it could not be expected to be maintained in commercial channels.     

CHALLENGE STUDIES WITH CLOSTRIDIUM BOTULINUM TYPE E IN A VALUE-ADDED SURIMI PRODUCT STORED UNDER A MODIFIED ATMOSPHERE

Challenge studies were carried out on raw, cooked, and sterilized surimi nuggets, inoculated with 10⁴ spores/g of C. botulinum type E spores. All products were packaged in air and air with an Ageless SS oxygen absorbent and stored at 4, 12 and 25C. Toxin was not detected in any raw product throughout storage (28 days). The absence of toxigenesis was attributed to the low pH (4.1–4.3) due mainly to the growth of lactic acid bacteria (10⁷CFU/g). Toxin was also not detected in any cooked product after 28 days. Product pH did not decrease as previously (due to the absence of LAB), but counts of C. botulinum still decreased throughout storage.
In sterile nuggets , C. botulinum counts increased to 10⁶ cfu/g at both 12 and 25C, respectively, by 28 days. Lactic acid bacteria and Bacillus spp. were not detected throughout the 28 days storage period. Toxin was detected by days 28 and 14 at 12 and 25C, respectively, and toxigenesis preceded spoilage. The absence of toxin in cooked nuggets was attributed to the anti-botulinal role by Bacillus species, the predominant spoilage bacteria in cooked nuggets.     

PURIFICATION AND CHARACTERIZATION OF TWO TOMATO POLYGALACTURONASEISOENZYMES

The properties of tomato polygalacturonases at two ripening stages were investigated. Two isoenzymes, PG I and II, were isolated from underripe fruits with an orange skin color. Fully ripe fruits contained only polygalacturonase II. PG I and II were purified by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and CM-agarose chromatography. PG I had a M_r of 199,500 as determined by Sephacryl S-300 gel filtration and was 50% inactivated at 66.5°C and pH 4.5 after incubation for 5 min. It had an activation energy (E_a) of 16.8 Kcallmol (70.3 times 10³ Jlmol), V_max of 27.7 units/mg protein and K_m value of 7.5 times 10⁻² mM polygalacturonic acid. PG II had a M_r of 45,700 and was 50% inactivated at 58°C under the same conditions. Both isozymes had a pH optimum of 4.6. PG II had an E_a value of 14.8 Kcallmol (61.9 times 10³ Jlmol), V_max value of 58.8 units/mg protein and K_m value of 3.8 times 10⁻² mM polygalacturonic acid. PGI gave rise to only one band during electrophoresis in polyacrylamide gels, whereas PG II showed one major and one minor band both with PG activity. Gel electrophoresis in the presence of sodium dodecyl sulfate resulted in two major bands (M_r= 47,500 and 41,000) for PG I and only one major band (M_r= 47,500) for PG II. PG I is composed of several subunits, all of which are glycoproteins.     

THE SURVIVAL OF MARINE VIBRIOS IN MERCENARIA MERCENARIA, THE HARDSHELL CLAM

A depuration chamber was used to study the persistence of marine vibrios in the hardshell clam, Mercenaria mercenaria. Specimens of M. mercenaria were incubated for two h in artificial seawater containing 10³ cells/ml each of the following bacterial species; Vibrio parahaemolyticus, Vibrio harveyi and Escherichia coli, and then transferred to the depuration chamber (a tank through which U. V.-sterilized artificial seawater was continually flowing). Numbers of the three bacterial species in tissues of M. mercenaira removed from the chamber at various times were determined by differential plating techniques. The number of each species ranged from 10² to 10³ colony-forming units/gram tissues immediately after transfer to the depuration chamber. After 24 h at 25°C the number of E coli cells detected had decreased over 100-fold. Generally, V. parahaemolyticus and V. harveyi were found in increased abundance after 24 h. The abundance of V. parahaemolyticus and V. harveyi in clams that had been incubated in the depuration chamber for 72 h at 25°C was approximately 10% of the abundance of these species immediately after transfer to the chamber. Similar results were obtained when the incubation temperature was 8 or 15°C and when initial cell concentrations were altered. Thus, V. harveyi and the potential human pathogen, V. parahaemolyticus which are both of marine origin were not removed from M. mercenaria at a rate comparable to the rate at which M. mercenaria depurated cells of E. coli.     

Enterobacter sakazakii survives spray drying

Four strains of Enterobacter sakazakii were inoculated into 35% reconstituted skim milk at 10⁷ and 10² cfu/g dry wt. After spray-drying in a Buchi mini spray drier (inlet 160°C and outlet 90°C), the resulting powders were analysed for E. sakazakii by enrichment and enumeration. In all cases, E. sakazakii survived the spray drying process and were detected in the powders with a low inoculum and enumerated in all the powders with the high inoculum for at least 12 weeks. The results emphasize that the controls in place to prevent E. sakazakii from getting to the spray drier are essential.     

The microbial flora of vacuum packed smoked herring fillets

The quantitative and qualitative changes in the microbial flora of vacuum packed smoked herring fillets, held at 10°C over a period of 18 weeks, were examined. Samples were periodically tested by a sensory panel for taste and smell. Initial total counts were in the order of 10⁴/g. After 7 weeks' storage the counts reached 10⁸/g. No significant changes were recorded thereafter. Initially, the incidence of yeasts was high (64%). After 9 weeks of storage these were no longer detected. Homofermentative Lactobacillus sp(p). increased during the keeping period, reaching 84% of the total flora after 12 weeks' storage. Sensory data indicated a keeping quality of at least 3 months at 10°C. No correlation was observed between sensory scores and total microbial counts. It appears that total plate counts have a minor significance in quality assessment of vacuum packed smoked herring fillets.     

Effect of irradiation and modified atmosphere packaging on the microbiological safety of minced pork stored under temperature abuse conditions

The safety of irradiated pork packed in 25% CO₂:75% N₂ and stored at abuse temperature (10 or 15°C) was assessed by inoculation studies involving Salmonella typhimurium, Listeria monocytogenes, Escherichia coli, Yersinia enterocolitica and Clostridium perfringens . Irradiation to a dose of 1.75 kGy reduced pathogen numbers to below the detection limit of 10² cells g^-1. When higher inoculum levels were used (10⁶ cells g^-1) irradiation at 1.75 kGy reduced pathogen numbers by 1 –>5 log₁₀ cycles depending on strain. Clostridium perfringens was the most resistant, and Y. enterocolitica the most sensitive of the pathogens studied.
In all cases when high numbers (10⁶ to 10⁷g^-1) of spoilage and/or pathogenic bacteria were present initially on the pork the meat appeared spoiled, and although irradiation reduced the number of microorganisms, the meat was still unacceptable from a sensory viewpoint after treatment.
It was concluded that the microbiological safety of irradiated, modified atmosphere packaged (MAP) pork is better than that of unirradiated MAP pork.     

DEVELOPMENT OF STRESS AND SURVIVAL OF SALMONELLA TYPHIMURIUM ATCC 7136 IN A TAPIOCA SOYSTARCH PRODUCT CONTAINING POTASSIUM SORBATE

The effects of reduced water activity and 0.1% sorbate on Salmonella typhimurium ATCC 7136 in a soy-starch product were examined. The formulated product (final pH 6.7), was inoculated with cells to give a final concentration of approximately 2 × 10⁶ cells/g. Ten gram samples were placed in a series of desiccators over saturated salt solutions and stored at either 22°C or 40°C. Water activities ranged from 0.79 to 0.97. Samples were withdrawn at prescribed intervals and appropriate dilutions made. A dual plating procedure was used to monitor growth (tryptic soy agar) and development of injury (Levine's Eosin Methylene Blue Agar supplemented with 2% NaCl and 0.05% sodium desoxycholate). Cells stored at 22°C remained viable longer at an a_w of 0.79 than at a_w values of 0.96 to 0.97. As storage time increased, cells were stressed at a faster rate at higher a_w values. This effect was accelerated at 40°C and as storage time increased.