Modelling the combined effect of salt,sorbic acid and nisin on the probability of growth of Clostridium sporogenes in high moisture processed cheese analogue

This study focused on the application of hurdle technology to inhibit the growth of Clostridium sporogenes in high moisture, low salt, ambient shelf-stable processed cheese analogue (PCA). The effectiveness of various combinations of salt, potassium sorbate and nisin in inhibiting C. sporogenes growth was quantified using logistic regression to produce probability-of-growth models. The results of this study demonstrate that it is feasible to produce a high moisture and low salt PCA, if sufficient concentrations of potassium sorbate and nisin are added to the product. Any change in the concentration of one of the selected preservatives must be compensated for by changes in concentrations of the other two preservatives. The models developed enable the level of probability to be set to provide a required level of stringency. The models tend to be fail-safe as they were developed using an inoculum size larger than the expected contamination in processed cheese.     

Reduction of Listeria monocytogenes in cold‐smoked salmon by bacteriophage P100, nisin and lauric arginate,singly or in combinations

Three GRAS antimicrobials including, lauric arginate (LAE), bacteriophage P100 (phage P100) and bacteriocin nisin, were evaluated either singly or in combinations for the reduction of initial load of Listeria monocytogenes in cold‐smoked salmon (CSS). The stability of phage P100 in the presence of LAE (200 ppm) and nisin (500 ppm) or at 10× and 100× of these concentrations was determined at 4 °C or 30 °C for 24 h in a broth model. Phage P100 was found to be highly stable in the presence of these antimicrobial agents as plaque‐forming units (PFU) did not vary between control and antimicrobial‐treated phage. The survival of L. monocytogenes in the presence of phage P100, nisin and LAE showed remarkable reduction within 24 h both at 4 °C or 30 °C in broth. Treatment of CSS containing 3.5 log CFU cm^?2 L. monocytogenes with phage P100 (10⁸ PFU mL^?1), nisin (500 ppm) and LAE (200 ppm) showed strong listericidal action and reduced the L. monocytogenes by 2–3 log CFU cm^?2 after 24 h. Among the combined treatments, phage P100 + LAE or nisin + LAE exhibited the most listericidal action in which L. monocytogenes cells were reduced to undetectable level within 24 h in CSS.     

Effect of Mechanically Separated Pork, Sodium Erythorbate and Sodium Nitrite Combinations on Clostridium sporogenes Survival and Growth in Liver Sausage

Liver sausage formulated with 50 or 75% mechanically separated pork (MSP), 75 or 150 ppm sodium nitrite and 0, 550 or 1100 ppm sodium erythorbate was examined for Clostridium sporogenes PA3679 survival and growth during refrigerated temperature storage (5°C, 6 days) and during subsequent exposure to elevated temperatures (22–24°C, 2 d). Sodium nitrite at 150 ppm inhibited clostridial growth in sausage held at 22–24°C, regardless of MSP content, if 550 ppm sodium erythorbate were also present. Sodium nitrite at 75 ppm combined with erythorbate at 550 and 1100 ppm did not inhibit C. sporogenes growth during simulated temperature mishandling of liver sausage. Residual nitrite decreased (P<0.05) with erythorbate addition but was unaffected by MSP.     

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L⁻¹ nisin or 6.4 mg L⁻¹ reutericyclin for 0–60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L⁻¹ and 1.11 ± 0.48 mg L⁻¹, respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.     

Use of Lysozyme,Nisin, and EDTA Combined Treatments for Maintaining Quality of Packed Ostrich Patties

ABSTRACT: The antimicrobial effectiveness of lysozyme, nisin, and ethylene diamine tetraacetic acid (EDTA) combination treatments (Mix₁: 250 ppm lysozyme, 250 ppm nisin, 5 mM EDTA; Mix₂: 500 ppm lysozyme, 500 ppm nisin, 5 mM EDTA) on bacterial growth of ostrich patties packaged in air, vacuum, and 2 different modified atmospheres (MAP₁: 80% O₂, 20% CO₂; MAP₂: 5% O₂, 30% CO₂, 65% N₂) was evaluated. Moreover, the lipid oxidation was evaluated as well as color and sensory characteristics. The growth of total viable counts and lactic acid bacteria were strongly inhibited by the antimicrobial treatments in all the running time (Inhibition Index >97%) whereas for Enterobacteriaceae and Pseudomonas spp. lower inhibition indices from 12% to about 28% were observed. The lipid oxidation was more pronounced in the control respect to the treated meat patties. Moreover, the mixture at low concentration of lysozyme and nisin showed the best antioxidative effect. High concentrations of lysozyme and nisin showed the greatest color loss. Also, off-odors for the untreated patties developed faster than the treated samples. Practical Application: Great interest is developing in food bio-preservation, because of the ever-increasing needs to protect consumers' health and to valorize the naturalness and safety of food products.     

The ability of zinc to inhibit the sporulation and viability of Clostridium sporogenes and growth of other bacteria

The objective of this study was to investigate the influence of zinc on the sporulation and viability of Clostridium sporogenes and on the growth of other bacteria. When 0.5% ZnCl₂ was added to a sporulation medium, it completely inhibited C. sporogenes (PA 3679) sporulation for up to 3 weeks. At concentrations of 0.5% and 1.0%, ZnCl₂ not only completely inactivated the vegetative cell viability (>7.0 Log reduction) but also significantly reduced the spore viability (<2.1 Log reduction) of C. sporogenes. Taken together, it was concluded that zinc blocks C. sporogenes sporulation by damaging (or killing) vegetative cells and probably by interfering with the biosynthesis of spore components. In addition to the inhibitory effect on the sporulation and viability of C. sporogenes, ZnCl₂ was found to have a broad antimicrobial spectrum against all Gram‐positive and Gram‐negative spoilage and pathogenic bacteria tested. The minimal inhibitory concentration for inhibiting the bacteria ranged between 3.7 and 7.4 mm . Therefore, we expect that this compound or a combination thereof has a potential as a surface‐cleaning agent or disinfectant.     

Effect of Sodium Chloride Levels in Frankfurters on the Growth of Clostridium sporogenes and Staphylococcus aureus

Frankfurters were made with 100%, 80%, and 60% of the normal 2.5% added NaCl, plus 150 ppm NaNO₂ and 430 ppm ascorbate. They were either untreated or inoculated with Staphylococcus aureus or Clostridium sporogenes and incubated at a moderate abuse temperature of 11°C for up to 11 days. A slightly more rapid growth of the natural flora was noted in the frankfurters containing 60% of the normal salt compared to those containing higher levels of salt. No outgrowth of clostridial spores occurred in any inoculated sample. Staphylococcal growth was slow and inconsistent, with no effect related to salt levels. Incubating frankfurters at higher tern. peratures showed that abuse temperature had a greater effect on total aerobic and staphylococcal growth than did the salt levels tested.     

Antimicrobial Activity and Functionality of Reduced Sodium Chloride and Potassium Sorbate in Uncured Poultry Products

Uncured turkey and chicken breast meat products, formulated with NaCl (0.65 and 1.30%) and potassium sorbate (0 and 0.26%), were tested for antimicrobial activity and functional properties. The chopped products were inoculated (Clostridium sporogenes, 10/g) in cans and in packages and abused at 20°C. Microbial growth and gas production were rapid at both NaCl levels tested. Inclusion of potassium sorbate in the formulation delayed initiation and rate of microbial growth and gas production. Potassium sorbate also reduced (P<0.05) weight losses during cooking of the low NaCl turkey product. Rates of gas production were similar in canned and vacuum packaged products. Differences in microbial growth and gas production between chicken and turkey products were minor.     

Reduction of Clostridium sporogenes spore outgrowth in natural sausage casings using nisin

Preservation of natural sausage casings using dry salt or saturated brine is regarded as sufficient to inactivate vegetative pathogenic non-spore-forming bacteria present on the casings. Although the outgrowth of bacterial spores is prevented by salt or saturated brine preservation, these spores will remain present and develop into vegetative cells when conditions are more favourable. To prevent subsequent outgrowth additional preservation measures should be implemented. In the experiments described the use of nisin was evaluated to reduce outgrowth of spores in desalinated casings. The bacteriocin nisin was chosen because of its known efficacy against spore-forming bacteria and their spores in various foodstuffs. Clostridium spore suspensions (Clostridium sporogenes, ATCC 3584) were used in two concentrations to inoculate three nisin concentrations (10, 50, 100 μg/mL) in water containing gamma-irradiated casings. Additionally, the binding of nisin to casings, using ¹⁴C-labeled nisin Z and subsequent availability of nisin were evaluated. Results demonstrate that nisin is partly reversibly bound to casings and can reduce the outgrowth of Clostridium spores in the model used by approximately 1 log₁₀ (90%). However, the biological relevance of these results needs to be determined further by conducting industrial trials before any recommendation can be made on the practical implementation of nisin in the preservation of natural sausage casings.     

Use of nisin, lysozyme and EDTA for inhibiting microbial growth in chilled buffalo meat

The effect of nisin, lysozyme and lysozyme combined with disodium ethylenediaminetetraacetate salt (EDTA) for inhibiting the growth of spoilage micro‐organisms in buffalo meat was investigated. Prior to packaging, the samples were alternatively dipped in solutions containing different concentrations of nisin, lysozyme and combinations of a pre‐fixed amount of lysozyme and different concentrations of EDTA. The packed meat samples were stored at 4 °C, and the microbial changes were monitored for a period of 8 days. The best results, in terms of microbial shelf‐life extension, were obtained by dipping the meat steaks in a solution containing a combination of 0.5% of lysozyme and 2% of EDTA. This treatment was able to reduce the growth of all the investigated bacteria and showed a bactericidal effect on Brochotrix thermosphacta during the entire period of storage.     

Packaging Film Permeability in Conjunction with Sodium Nitrite, Potassium Sorbate or Lactic Acid Starter Culture for Control of Clostridium sporogenes (PA3679) Growth in Sliced Bologna

A sliced bologna was prepared and inoculated with Clostridium sporogenes (PA3679), packaged in films ranging in oxygen permeability from 0.1 cc/m²/24 hr to 120 cc/m²/24 hr and stored at 5°C, 15°C or 25°C. Subsequent bologna preparations included either 156 ppm sodium nitrite, 0.26% potassium sorbate or a lactic acid starter culture. Water activity, pH, TBA number and PA3679 counts were monitored during 28 days of storage. TBA numbers increased in packages with over 60 cc/m²/24 hr permeability but PA3679 counts did not change as a function of packaging film. Nitrite and sorbate were equally effective as inhibitors. At 15°C and 25°C, the lactic acid culture allowed the least PA3679 growth. Oxygen permeability did not alter any inhibitory effects even when increased TBA numbers resulted from using an oxygen-permeable film.     

Effect of nisin‐producing Lactococcus lactis starter cultures on the inhibition of two pathogens in ripened cheeses

The effect of Lactococcus lactis nisin‐producing strains, isolated from Italian fermented foods, on the survival of two foodborne pathogens namely Listeria monocytogenes and Staphylococcus aureus was investigated in experimental cheese production. One of the three Lactobacillus lactis nisin innoculated as starters, Lactobacillus lactis 41FL1 lowered S. aureus count by 1.73 log colony‐forming units (cfu)/g within the first 3 days, reaching the highest reduction, 3.54 log cfu/g, by the end of ripening period of 60 days. There was no effect on L. monocytogenes. The application of L. lactis 41FL1 as bioprotective culture in controlling S. aureus shows considerable promise.     

Effect of Heat Treatment and Selected Antimicrobials on the Shelf-life and Safety of Cooked, Vacuum-Packaged, Refrigerated Pork Chops

The shelf-life of refrigerated (2–4°C) pork chops inoculated with Clostridium sporogenes PA3679 and Staphylococcus aureus Z88 was examined in products sliced from loins cooked to 66°C (150°F), dipped in 5% polyphosphate blend, 2.5% potassium sorbate or 2% acetic acid solutions, vacuum-packaged and stored at 2–4°C. The effect of a second in-the-bag cooking step to 66°C (150°F) after vacuum packaging was also studied. Pork chops not reheated after packaging showed incipient spoilage after 15 days at 2–4°C, depending on surface treatment. The second cooking increased the shelf-life of refrigerated product to more than 60 days and reduced counts of inoculated cultures to undetectable levels. However, on exposure of the chops to simulated mishandling (24–25°C), clostridial growth was detected in all samples except those dipped in polyphosphate or acetic acid solutions.     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Effect of Sodium Erythorbate on Residual Nitrite, pH and Bacterial Growth in Liver Sausage Formulated with Different Levels of Mechanically Separated Pork

Some microbiological, chemical, and physical characteristics of liver sausage formulated with 0, 25, 50, and 75% mechanically separated pork (MSP), 156 ppm sodium nitrite and 0 or 550 ppm sodium erythorbate were studied during refrigerated storage (5°C, 6 days) and subsequent elevated-temperature holding (20–24°C, 48 hr). Growth of inoculated Clostridium sporogenes was inhibited by 156 ppm nitrite in all sausages even after 48 hr at 20–24°C, regardless of MSP level. Growth of aerobic mesophiles and facultative anaerobes at 20–24°C was also inhibited in sausage formulated with 156 ppm nitrite and 550 ppm erythorbate. Erythorbate addition and increased MSP levels resulted in higher Hunter a color values. Residual nitrite, pH values, and total iron content of liver sausage increased with increasing MSP content.     

Effects of Mentha longifolia L. essential oil and Lactobacillus casei on the organoleptic properties and on the growth of Staphylococcus aureus and Listeria monocytogenes during manufacturing,ripening and storage of Iranian white‐brined cheese

This research evaluated the effects of Mentha longifolia L. essential oil (EO) in concentrations 0, 50, 150 and 300 ppm and Lactobacillus casei (10⁸–10⁹ CFU/mL) on the growth of Staphylococcus aureus and Listeria monocytogenes during the manufacturing, ripening and storage of Iranian white‐brined cheese. The growth of the two pathogens was significantly reduced (P < 0.01) by both EO concentrations ≥50 ppm and probiotic and their combination in the standard manufacturing and storage process conditions of the cheese. Furthermore, the treatment containing 150 ppm of this EO combined with probiotic had a favourable inhibitory effect on the growth of two pathogenic micro‐organisms and also was the most appropriate treatment in sensory assessment. The synergistic effects of the above‐mentioned concentration level between the essential oil and probiotic were significant compared to other treatments, including essential oil and probiotic only. Thus, a lower concentration of this EO can be used when it is combined with this probiotic.     

Effect of Nitrite and Sorbate on Total Number of Aerobic Microorganisms in Chicken White and Dark Meat Patties

The effects of nitrite, sorbate and combinations of these two ingredients plus salt on the number of aerobic microorganisms in chicken white and dark meat patties were examined. All patties were stored at 4–5°C for 12 days. Nitrite concentrations of 400 and 2500 ppm were effective in preventing bacterial growth in chicken white meat patties while 2500 ppm was required to prevent growth in dark meat. A reduction in bacterial growth (4–10 days) was demonstrated with 100 and 150 ppm nitrite in chicken white meat patties. Four hundred ppm nitrite reduced bacterial growth in dark meat patties for 6 days. In white meat patties, the use of sorbate (0.26%) in combination with nitrite (40 ppm) was as effective in reducing bacterial growth as higher concentrations of nitrite, salt (2.5%) alone or in combination with sorbate (0.26%) or nitrite (40 ppm) inhibited bacterial growth in both white and dark meat patties during the first 4–6 days of storage.     

Inhibition of Clostridium sporogenes growth in mascarpone cheese by co-inoculation with Streptococcus thermophilus under conditions of temperature abuse

The ability of a biological control system to inhibit the outgrowth of Clostridium sporogenes spores during storage of mascarpone cheese under temperature-abuse conditions was investigated. Challenge studies were carried out on mascarpone cheese artificially contaminated with spores of C. sporogenes (10 cfu g⁻¹), and with or without the coinoculum of a Streptococcus thermophilus strain (10⁵cfu g⁻¹). During storage at 4, 12, and 25°C, the outgrowth of clostridia spores, the growth of S. thermophilus, and the pH changes were evaluated at 10, 20, 30, and 40 days. In mascarpone cheese stored at 4° and 12°C, S. thermophilus and C. sporogenes did not show any growth. The initial pH (6·14) of the product also remained unchanged. During storage at 25°C S. thermophilus grew up to about 10⁷cfu g⁻¹after 10 days, resulting in a pH decrease of mascarpone cheese to values close to 4·5. The cell number decreased progressively during storage reaching values near to 10¹cfu g⁻¹after 40 days, whereas product acidity remained constant. C. sporogenes, when inoculated alone, also grew at 25°C. The cell number increased to levels of about 10⁷cfu g⁻¹after 20–40 days of storage according to the different mascarpone cheese lots used. No growth was found when C. sporogenes was co-inoculated in mascarpone cheese with S. thermophilus and stored at 25°C. The study on the behaviour of C. sporogenes, known as a non-toxigenic variant of Clostridium botulinum, allowed us to obtain useful information for setting up an effective biological control system to inhibit growth of the toxigenic species as well. The use of an additional barrier, besides refrigerated storage, may help to maintain the safety of mascarpone cheese in the event it was exposed to elevated temperatures.     

Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids

The effect of fat on the antibotulinal activity of 11 food preservatives, 12 free fatty acids, and nine lots of enzyme-modified cheese (EMC) was evaluated in a media system. Anhydrous milkfat or soybean oil was added to tubes of Trypticase–peptone–glucose–yeast extract medium (TPGY) supplemented with the additives (final pH adjusted to 5.9). Treatments were inoculated with 3-log₁₀ proteolytic Clostridium botulinum spores/ml (10-strain mixture of serotypes A and B) and incubated anaerobically at 30°C for up to 14 days. For the preservative and fatty acids studies, growth of C. botulinum was determined by measuring optical changes at OD_{640 nm}. Botulinal toxin production was determined in EMC-treatments using the mouse bioassay. Data revealed that the antibotulinal effects of nisin, and free fatty acids caprylic, capric, lauric, myristic, oleic, and linoleic acids were each significantly reduced in treatments supplemented with 20% fat (P<0.05). Similar trends were observed in TPGY supplemented with 20% fat and potassium sorbate, sorbic acid, monolaurin, polyphosphate emulsifier, or EDTA–lysozyme, but the differences were reduced. Fat was also antagonistic to the antibotulinal activity of five EMC-treatments. This study suggests that fat may reduce the efficacy of some antimicrobials added to or found naturally in foods.     

Preparation of alcalase hydrolysed rice bran protein concentrate and its inhibitory effect on soybean lipoxygenase activity

Rice bran protein concentrate (RBPC) was prepared from defatted rice bran and hydrolysed by alcalase at different hydrolysis times. As the hydrolysis times increased, the degree of hydrolysis (DHs) increased. RBPC hydrolysate obtained at 50 min (RBPCH‐50) had the highest inhibitory efficiency on soybean lipoxygenase (LOX) activity (66%). The inhibition kinetics of the reaction analysed by Lineweaver–Burk plots indicates that RBPCH‐50 is a competitive inhibitor. RBPCH‐50 inhibited soybean LOX with an IC₅₀ of 11.73 μg μL^?1 RBPCH‐50, and the obtained K_I was 4.59 μg μL^?1 RBPCH‐50. LOX inhibitory activity of RBPCH‐50 was significantly higher than that of 50 and 100 ppm butylated hydroxyanisole (BHA) and 50, 100, and 200 ppm butylated hydroxytoluene (BHT) (P ≤ 0.05); however, LOX inhibitory activity of RBPCH‐50 was similar to that of 200 ppm BHA (P > 0.05). Therefore, RBPCH might potentially be used as a natural LOX inhibitor.