EFFECT OF ETHANOL VAPOR ON GROWTH AND TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM IN A HIGH MOISTURE BAKERY PRODUCT

To determine the effect of ethanol vapor on toxin production by Clostridium botulinum, studies were done in English style crumpets (a_w 0.990, pH 6.5) challenged with 500 spores/g C. botulinum types A and proteolytic B and packaged in high gas barrier bags [ethanol transmission rate (ETR) 0.21 g/m²/day @ 25 C]. Crumpets were packaged in air with either commercially available ethanol vapor generators (Ethicap® 2, 4 or 6G) or cotton wool pads saturated with 2, 4 or 6 g of 95% food grade ethanol and stored at 25C. Toxin was detected in all inoculated control crumpets (0% ethanol) after 5 days at ambient temperature (25C). Ethicap® 2G delayed toxicity for 10 days while complete inhibition (>21 days) was observed in all crumpets packaged with 4 or 6G Ethicap® or with 2, 4 or 6 g of ethanol per pad. However, all crumpets were overtly spoiled by this time. Both headspace ethanol and absorption of ethanol by crumpets increased as a function of Ethicap® size/weight of ethanol. Based on these preliminary studies, ethanol vapor would appear to be an effective additional barrier to control the growth and toxin production by C. botulinum in high moisture bakery products and ensure the safety of these products at ambient temperature.     

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.     

Effect of pH and CO2 on growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

The effect of pH and CO2 on both growth of and toxin production by Clostridium botulinum in English-style crumpets, packaged under modified atmospheres was investigated using a 2 x 2 factorial experiment. English-style crumpets (water activity, 0.990; pH 6.5 and 8.3) were inoculated with C. botulinum spores types A and proteolytic B (500 spores/g), packaged in either 60% CO2 (balance N2) or 100% CO2, stored at ambient temperature (25 degrees C), and monitored daily for toxicity. Toxin was detected after 4 days in crumpets packaged in 60% CO2, irrespective of initial product pH. Toxin production was delayed 1.5 to 3 days in crumpets packaged under 100% CO2. Analysis of variance indicated a significant interaction effect of pH and %CO2 on time of earliest toxin detection. Delay of toxin production was greatest for high pH (8.3) crumpets. All products were organoleptically acceptable at the time of toxigenesis, and therefore, high moisture-high pH bakery products, if contaminated with spores of C. botulinum, could become hazardous if packaged in atmospheres containing CO2.     

EFFECT OF FILMS OF DIFFERENT OXYGEN TRANSMISSION RATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM TYPE E IN VACUUM PACKAGED COLD AND HOT SMOKED TROUT FILLETS

Studies were done to determine the effect of film oxygen transmission rate (OTR) on the time to toxicity in vacuum packaged cold and hot smoked rainbow trout fillets challenged with C. botulinum type E (10² spores/g) and stored at refrigerated conditions (4C), and under mild (8C) and moderate (12C) temperature abuse conditions. While no samples were toxic at 4C, toxin was detected within 28 days at 8C for cold smoked trout fillets vacuum packaged in films with high OTR. Toxin was also detected for most vacuum packaged hot smoked trout fillets within 14–28 days at 8C, with the exception of trout fillets packaged in films with an OTR > 10,000 cc/m²/day. In most cases at 8C, spoilage, based on odor/color scores, preceded or occurred simultaneously with toxigenesis. At 12C all cold and hot smoked trout were toxic after 14–21 days and samples packaged in films with an OTR <5000 cc/m²/day became toxic before, or at the same time as, samples became spoiled. This study has shown that vacuum packaging of trout fillets in low gas barrier films, ranging in OTR from approximately 3,000 to approximately 10,000 cc/m²/day at 24C and 0% relative humidity (RH), did not prevent the growth and toxin production by C. botulinum in vacuum packaged cold and hot smoked trout fillets at 12C. Additional barriers, other than the OTR of the packaging film, need to be considered to ensure the safety of vacuum packaged trout fillets, particularly at mild to moderate temperature abuse storage conditions.     

Growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres.

To determine the safety of a high moisture bakery product, packaged under modified atmospheres, challenge studies were done on English-style crumpets (water activity [a(w)] 0.990, pH 6.5) inoculated postbaking with Clostridium botulinum types A and proteolytic B spores (5 X 10(2) spores/g). Products were packaged either in air, in air with an Ageless FX200 oxygen absorbent, or in a CO2/N2 (60:40) gas mixture, stored at ambient temperature (25 degrees C), and monitored for toxicity daily. All inoculated crumpets were toxic within 4 to 6 days and were organoleptically acceptable at the time of toxigenesis. Counts of C. botulinum increased to approximately 10(5) CFU/g at the time of toxicity. To determine the effect of baking on product safety, subsequent challenge studies were done on crumpets inoculated with 5 x 10(2) spores/g (baked weight basis) prior to baking. All crumpets were toxic after only 6 days, irrespective of packaging conditions, and toxigenesis again preceded spoilage. Temperature profile studies showed that the maximum internal temperature reached during baking was 97 degrees C, and the total baking process was equivalent to 0.03 min at 121 degrees C. The actual time to toxin production in both studies (4 to 6 days) correlated well with the predicted time (3.4 days) using the U.S. Department of Agriculture Pathogen Modeling Program (version 5.1) for proteolytic strains of C. botulinum. These studies confirm that high moisture bakery products, if contaminated with C. botulinum spores either pre- or postbaking, could pose a public health hazard, if packaged in air (in a high gas barrier package where O2 was depleted and CO2 was generated during storage) or under modified atmosphere packaging conditions and stored at ambient temperature.     

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.     

Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.     

EFFECT OF HEADSPACE OXYGEN AND FILMS OF DIFFERENT OXYGEN TRANSMISSION RATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM TYPE E IN RAINBOW TROUT FILLETS STORED UNDER MODIFIED ATMOSPHERES

Studies were conducted to determine the effect of various levels of headspace oxygen (0–100%, balance CO₂) or film oxygen transmission rate (OTR) on the time to toxicity in modified atmosphere packaged (MAP) fresh trout fillets challenged with C. botulinum type E (10² spore/g) and stored under moderate temperature abuse conditions (12C). In all cases, trout were toxic within 5 days, irrespective of the initial levels of oxygen in the package headspace. However, spoilage preceded toxigenesis. Packaging of trout fillets in low gas barrier films, with OTRs ranging from 4,000 to 10,000 cc/m²/day at 24C and 0% relative humidity, also had no effect on time to toxicity in all MAP trout fillets. All fillets were toxic within 4–5 days and spoilage again preceded toxigenesis. This study has shown that the addition of headspace O₂, either directly to a package or indirectly by using a low gas barrier film, had no influence on the time to toxigenesis or spoilage. Additional barriers, other than headspace O₂ or film transmission rate, need to be considered to ensure the safety of MAP trout fillets, particularty at moderate temperature abuse conditions.     

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.     

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.     

Model of the Inactivation of Bacterial Spores by Moist Heat and High Pressure

ABSTRACT: Formulae for the prediction of inactivation and accumulated lethality of bacterial spores under moist heat and high pressures were derived on the basis of classic thermodynamic and kinetics principles. The capability of the model to describe the inactivation of bacterial spores was verified using 2 independent data sets corresponding to Clostridium botulinum processed at 60°C to 75°C and Bacillus stearothermophilus processed at 92°C to 110°C. Both sets included pressures between 5 × 10⁸ Pa and 7 × 10⁸ Pa. The equation fit explained more than 86% of the variation of the rate constant data. The developed equations establish a strong foundation on which to compare high-pressure processing treatments of different systems. This is especially useful because most systems have different transient temperature-pressure conditions.     

RELEASE OF PROPYL PARABEN FROM A POLYMER COATING INTO WATER AND FOOD SIMULATING SOLVENTS FOR ANTIMICROBIAL PACKAGING APPLICATIONS

The release phenomena of propyl paraben from a polymer coating to water and three food simulating solvents (10% aqueous ethanol, 50% aqueous ethanol, n-heptane) were studied for antimicrobial packaging applications. The effects of food simulating solvent, initial concentration in the coating and temperature on the propyl paraben release were examined. The initial concentration of propyl paraben in the coating ranged from 1.26 × 10⁴ to 10.52 × 10⁴ g/m³ and the temperature from 5.5 to 30C. For water, the release was controlled by Fickian diffusion with constant diffusion coefficient (7±11 × 10^-11 cm²/s at 30C), and independent of the initial concentration. For 10% ethanol, the release followed again the Fickian model with constant diffusion coefficient (30±40 × 10^-11 cm²/s at 30C). For 50% ethanol and n-heptane, the release was instantaneous and not controlled by Fickian diffusion. For the release into water, the activation energy for diffusion from the Arrhenius relationship was around 88 kJ/mole.     

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.     

Quality of Shredded Carrots as Affected by Packaging Film and Storage Temperature

ABSTRACT: Different packaging films and storage temperatures were used to establish a range of equilibrium modified atmospheres for storage of shredded carrots. Quality and storage-life of the packaged shredded carrots were determined using sensory evaluation, microbial counts, and a range of physical tests. Minimal processing steps such as peeling and shredding caused physical damage, physiological stress, and enhanced microbial growth, leading to a reduced shelf life when compared to the whole vegetable. A P-plus microporous film (CO₂ permeability of 29 X 10³mL.m ^-2.d ^-1.atm ^-1) was the most suitable for the storage of shredded carrots. Findings indicated that deterioration in these products was triggered by the depletion of oxygen more than by the rise in carbon dioxide.     

INACTIVATION OF BACTERIAL SPORES BY COMBINED ACTION OF HYDROSTATIC PRESSURE AND BACTERIOCINS IN ROAST BEEF

Foodborne bacterial spores are normally resistant to high hydrostatic pressure; however, at moderate pressure, they can be induced to germinate and outgrow. At this stage, they can be killed by bacteriocin-based biopreservatives (BP-containing pediocin and nisin at 3:7 ratio; BP_X, BP + 100 μg/mL lysozyme; BP_Y, BP_X+ 500 μg/mL Na-EDTA). Based on this principle, spores of the meat spoilage organism, Clostridium laramie (1–2 × 10² spores/bag) alone or a mixture of four clostridial spores (5 × 10³ spores/bag), Clostridium sporogenes, Clostridium perfringens, Clostridium tertium, and Clostridium laramie, were inoculated in roast beef in the presence of 5000 AU/g of bacteriocin-based biopreservatives. The roast beef samples were subjected to hydrostatic pressure (HP) at 345 MPa for 5 min at 60C and stored at 4 or 12C for 84 days or at 25C for 7 days. The HP treatment of roast beef samples inoculated with a mixture of clostridial spores could be stored for 42 days at 4C. The HP in combination with either BP_X or BP_Y extended the shelf-life of roast beef up to 7 days at 25C. The combined treatment of HP and BP controlled the growth of C. laramie spores and extended the shelf-life of roast beef for 84 days when stored at 4C.     

Physicochemical, Microbiological, and Sensory Evaluation of Minimally Processed Tarocco Clone Oranges Packaged with 3 Different Permeability Films

ABSTRACT: Fruits of 3 Tarocco clones ('Gallo', 'Arcimusa', and 'Scirè') were sliced, packaged with different permeability films, and stored at 4°C for 14 to 15 d to find the best clone for processing and the most suitable packaging conditions to extend the shelf life of these products. Physicochemical parameters of Tarocco oranges slices, packaged with 3 films of different permeability, did not show marked decay phenomena during the storage days. Only a reduction in the ascorbic acid content was observed in almost all the examined clones, especially in products packaged with the most O₂ permeable films. Concerning microbiological contamination, all clones, packaged with the 3 films, showed, until the 12th storage day, a lower number of colony forming units (CFU)/g (≤ 3.6 × 10⁷ CFU/g) for mesophilic viable counts than the 2 microbiological criteria generally used for fresh-cut fruit and vegetables (10⁸ CFU/g for mesophilic viable counts). In fact, for these products it is possible to expect a shelf life longer than 12 to 13 d. Regarding sensory results, it was observed that the minimally processed Tarocco clone slices packaged with the most permeable to O₂ film were the most appreciated.     

PROBABILITY OF GROWTH OF CLOSTRIDIUM BOTULINUM AS AFFECTED BY STRAIN, CELL AND SEROLOGIC TYPE, INOCULUM SIZE AND TEMPERATURE AND TIME OF INCUBATION IN A MODEL BROTH SYSTEM

Using factorial design experiments and MPN methodology, we evaluated the probability (P) of growth initiation of 17 individual proteolytic (A,B,F) and non-proteolytic (B,E,F) C. botulinum strains (spores and cells) in a model broth as it is affected by strain, cell and serologic type, inoculum size (10⁰–10⁶), temperature (4–47°C) and time of incubation (up to 28 days). Regression analyses of the P of growth of the most capable strains for all conditions tested, allowed the development of quantitative equations relating P of growth initiation by one spore or cell to the variables studied. The close agreement between observed and predicted Ps demonstrated the potential usefullness of the modeling approach in studying microbial interactions with food environments.     

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.     

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.     

RADIATION STERILIZATION OF SPICES FOR HOSPITAL FOOD SERVICES AND PATIENT CARE

A survey of the commercial spices used by food services in a typical hospital environment revealed high contamination with microorganisms, i.e., 10⁴ to 10⁷ counts per gram. The predominant microorganisms were as followed (in colony counts/gram): (1) heat-resistant bacterial spores in black pepper, 1 × 10⁷; thyme, 2 × 10⁶; anise, 7 × 10⁴; curry powder, 4 × 10⁵; poultry seasoning, 8 × 10⁴; pickling spice, cardamom, and cumin, 1.5–3 × 10⁴; (2) mixed populations of vegetative cells and bacterial spores in cumin, 1 × 10⁶; (3) molds in cream of tartar, 2 × 10⁴. Sterility of food may be important in a hospital setting, especially in the care of immunocompromised patients. To eliminate the organisms, we recommend radiation treatment, accompanied by appropriate microbiological quality control. On the basis of radiation survival data, the composite natural flora would be reduced to the level of "commercial sterility" (defined as less than 10 organisms per gram((Kiss 1982) by the following minimum radiation doses (in kGy): black pepper, 13; thyme, 13; cumin, 12; anise, 10; curry, 7.3; pickling spice, 7; poultry seasoning, 6; cardamom, 9.4; cream of tartar, 4. For practical purposes, two dose levels can be recommended for treatment of spices in the hospital environment, low = 6–10 kGy and high = 10–15 kGy.