Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Lactose oxidase: An enzymatic approach to inhibit Listeria monocytogenes in milk

Listeria monocytogenes is a ubiquitous pathogen that can cause morbidity and mortality in immunocompromised individuals. Growth of L. monocytogenes is possible at refrigeration temperatures due to its psychrotrophic nature. The use of antimicrobials in dairy products is a potential way to control L. monocytogenes growth in processes with no thermal kill step, thereby enhancing the safety of such products. Microbial-based enzymes offer a clean-label approach for control of L. monocytogenes outgrowth. Lactose oxidase (LO) is a microbial-derived enzyme with antimicrobial properties. It oxidizes lactose into lactobionic acid and reduces oxygen, generating H₂O₂. This study investigated the effects of LO in UHT skim milk using different L. monocytogenes contamination scenarios. These LO treatments were then applied to raw milk with various modifications; higher levels of LO as well as supplementation with thiocyanate were added to activate the lactoperoxidase system, a natural antimicrobial system present in milk. In UHT skim milk, concentrations of 0.0060, 0.012, and 0.12 g/L LO each reduced L. monocytogenes counts to below the limit of detection between 14 and 21 d of refrigerated storage, dependent on the concentration of LO. In the 48-h trials in UHT skim milk, LO treatments were effective in a concentration-dependent fashion. The highest concentration of LO in the 21-d trials, 0.12 g/L, did not show great inhibition over 48 h, so concentrations were increased for these experiments. In the lower inoculum, after 48 h, a 12 g/L LO treatment reached levels of 1.7 log cfu/mL, a reduction of 1.3 log cfu/mL from the initial inoculum, whereas the control grew out to approximately 4 log cfu/mL, an increase of 1 log cfu/mL from the inoculum on d 0. When a higher challenge inoculum of 5 log cfu/mL was used, the 0.12 g/L and 1.2 g/L treatments reduced the levels by 0.2 to 0.3 log cfu/mL below the initial inoculum and the 12 g/L treatment by >1 log cfu/mL below the initial inoculum by hour 48 of storage at refrigeration temperatures. After the efficacy of LO was determined in UHT skim milk, LO treatments were applied to raw milk. Concentrations of LO were increased, and the addition of thiocyanate was investigated to supplement the effect of the lactoperoxidase system against L. monocytogenes. When raw milk was inoculated with 2 log cfu/mL, 1.2 g/L LO alone and combined with sodium thiocyanate reduced ~0.8 log cfu/mL from the initial inoculum on d 7 of storage, whereas the control grew out to >1 log cfu/mL from the initial inoculum. Furthermore, in the higher inoculum, 1.2 g/L LO combined with sodium thiocyanate reduced L. monocytogenes counts from the initial inoculum by >1 log cfu/mL, whereas the control grew out 2 log cfu/mL from the initial inoculum. Results from this study suggest that LO is inhibitory against L. monocytogenes in UHT skim milk and in raw milk. Therefore, LO may be an effective treatment to prevent L. monocytogenes outgrowth, increase the safety of raw milk, and be used as an effective agent to prevent L. monocytogenes proliferation in fresh cheese and other dairy products. This enzymatic approach is a novel application to control the foodborne pathogen L. monocytogenes in dairy products.     

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0 log₁₀ cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0 log₁₀ cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400 MPa. Microbiological analyses were performed 2 h after the UHPH treatments and after 5, 8, and 15 d of storage at 4°C. Maximum lethality values were observed in samples treated at 400 MPa with 15 and 10% fat (7.95 and 7.46 log₁₀ cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4°C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200 MPa but not in the milk samples treated at 300 and 400 MPa. When the load of L. monocytogenes was approximately 3.0 log₁₀ cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.     

Short communication: Nα-Lauroyl-l-arginine ethylester monohydrochloride reduces bacterial growth in pasteurized milk

Effective strategies for extending fluid milk product shelf-life by controlling bacterial growth are of economic interest to the dairy industry. To that end, the effects of addition of l-arginine, Nα-lauroyl ethylester monohydrochloride (LAE) on bacterial numbers in fluid milk products were measured. Specifically, LAE was added (125, 170, or 200 mg/L) to conventionally homogenized and pasteurized 3.25% fat chocolate or unflavored milk products. The treated milks and corresponding untreated controls were held at 6°C and plated on standard plate count agar within 24 h of processing and again at 7, 14, 17, and 21 d of storage. Bacterial counts in all unflavored milk samples treated with LAE remained below the Pasteurized Milk Ordinance limit for grade A pasteurized fluid milk of 4.3 log cfu/mL for the entire 21 d. Bacterial counts in unflavored samples containing 170 and 200 mg/L of LAE were significantly lower than those in the untreated unflavored milk at d 17 and 21 postprocessing. Specifically, bacterial counts in the milk treated with 200 mg/L of LAE were 5.77 log cfu/mL lower than in untreated milk at 21 d postprocessing. Bacterial counts in chocolate milk treated with 200 mg/L of LAE were significantly lower than those in the untreated chocolate milk at d 14, 17, and 21. In chocolate milk treated with 200 mg/L of LAE, bacterial counts were 0.9 log cfu/mL lower than in the untreated milk at 21 d postprocessing. Our results show that addition of LAE to milk can reduce bacterial growth. Addition of LAE is more effective at controlling bacterial growth in unflavored milk than in chocolate milk.     

Effect of modified atmosphere packaging on the growth of spoilage microorganisms and Listeria monocytogenes on fresh cheese

Queso Fresco has a limited shelf life and has been shown to support the rapid growth of Listeria monocytogenes during refrigerated storage. In addition to improving quality and extending shelf life, modified atmosphere packaging (MAP) has been used to control the growth of pathogenic microorganisms in foods. The objectives of this study were to determine the effects of MAP conditions on the survival and growth of spoilage microorganisms and L. monocytogenes during storage of Queso Fresco manufactured without starter cultures. For L. monocytogenes experiments, cheeses were surface inoculated at ～4 log₁₀ cfu/g before packaging. Inoculated and uninoculated (shelf life experiments) cheeses were placed in 75-µm high-barrier pouches, packaged under 1 of 7 conditions including air, vacuum, or combinations of N₂ and CO₂ [100% N₂ (MAP1), 30% CO₂:70% N₂ (MAP2), 50% CO₂:50% N₂ (MAP3), or 70% CO₂:30% N₂ (MAP4), 100% CO₂ (MAP5)], and stored at 7°C. Samples were removed weekly through 35 d of storage. Listeria monocytogenes counts were determined for inoculated samples. Uninoculated samples were assayed for mesophilic and psychrotolerant counts, lactic acid bacteria, coliforms, and yeast and mold. In general, cheeses packaged under conditions consisting of higher contents of CO₂ had lower pH levels during storage compared with those stored in conditions with lower levels or no CO₂ at all. Similarly, the antimicrobial efficacy of MAP in controlling spoilage microorganisms increased with increasing CO₂ content, whereas conditions consisting of 100% N₂, vacuum, or air were less effective. Mean L. monocytogenes counts remained near inoculation levels for all treatments at d 1 but increased ～2 log₁₀ cfu/g on cheeses packaged in air, vacuum, and 100% N₂ (MAP1) conditions at d 7 and an additional ～1.5 log₁₀ cfu/g at d 14 where they remained through 35 d. In contrast, treatments consisting of 70% CO₂ (MAP4) and 100% CO₂ (MAP5) limited increases in mean L. monocytogenes counts to <1 log₁₀ cfu/g through 14 d and ～1.5 log₁₀ cfu/g by d 21. Mean L. monocytogenes counts increased to levels significantly higher than inoculation (d 0) on cheeses stored in MAP2 and MAP3 on d 21, on d 28 for MAP4, and on d 35 for cheeses stored under MAP5 conditions. Overall, significant treatment × time interactions were observed between air, vacuum, and MAP1 when each was compared with MAP2, MAP3, MAP4, and MAP5. These data demonstrate that packaging fresh cheese under modified atmospheres containing CO₂ may be a promising approach to extend shelf life while limiting L. monocytogenes growth during cold storage.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Low pressure CO2 storage of raw milk: microbiological effects

The effects of holding raw milk under carbon dioxide pressures of 68 to 689 kPa at temperatures of 5, 6.1, 10, and 20°C on the indigenous microbiota were investigated. These pressure-temperature combinations did not cause precipitation of proteins from the milk. Standard plate counts from treated milks demonstrated significantly lower growth rate compared with untreated controls at all temperatures, and in some cases, the treatment was microcidal. Raw milk treated with CO₂ and held at 6.1°C for 4 d exhibited reduced bacterial growth rates at pressures of 68, 172, 344, and 516 kPa; and at 689 kPa, demonstrated a significant loss of viability in standard plate count assays. The 689-kPa treatment also reduced gram-negative bacteria and total Lactobacillus spp. The time required for raw milk treated at 689 kPa and held at 4°C to reach 4.30 log₁₀ cfu/mL increased by 4 d compared with untreated controls. Total coliform counts in the treated milk were maintained at 1.95 log₁₀ cfu/mL by d 9 of treatment, whereas counts in the control significantly increased to 2.61 log₁₀ cfu/mL by d 4 and 2.89 log₁₀ cfu/mL by d 9. At d 8, Escherichia coli counts had not significantly changed in treated milk, but significantly increased in the control milk. Thermoduric bacteria counts after 8 d were 1.32 log₁₀ cfu/mL in treated milk and 1.98 log₁₀ cfu/mL in control milk. These data indicated that holding raw milk at low CO₂ pressure reduces bacterial growth rates without causing milk protein precipitation. Combining low CO₂ pressure and refrigeration would improve the microbiological quality and safety of raw milk and may be an effective strategy for shipping raw single strength or concentrated milk over long distances.     

Viability of Listeria monocytogenes on commercially-prepared hams surface treated with acidic calcium sulfate and lauric arginate and stored at 4°C

We demonstrated the effectiveness of delivering an antimicrobial purge/fluid into shrink-wrap bags immediately prior to introducing the product and vacuum sealing, namely the “Sprayed Lethality In Container” (SLIC™) intervention delivery method. The pathogen was Listeria monocytogenes, the antimicrobials were acidic calcium sulfate (ACS; calcium sulfate plus lactic acid; 1:1 or 1:2 in dH₂O) and lauric arginate (LAE; Ethyl-N-dodecanoyl-l-arginate hydrochloride; 5% or 10% in dH₂O), and the product was commercially prepared “table brown” ham (ca. 3 pounds each). Hams were surface inoculated with a five-strain cocktail of L. monocytogenes (ca. 7.0 log₁₀ CFU per ham), added to shrink-wrap bags that already contained ACS or LAE, vacuum-sealed, and stored at 4 °C for 24 h. Pathogen levels decreased by 1.2, 1.6, 2.4, and 3.1 log₁₀ CFU/ham and 0.7, 1.6, 2.2, and 2.6 log₁₀ CFU/ham in samples treated with 2, 4, 6, and 8 mL of a 1:1 and 1:2 solution of ACS, respectively. In samples treated with 2, 4, 6, and 8 mL of a 5% solution of LAE, pathogen levels decreased by 3.3, 6.5, 5.6, and 6.5 log₁₀ CFU/ham, whereas when treated with a 10% solution of LAE pathogen levels decreased ca. 6.5 log₁₀ CFU/ham for all application volumes tested. The efficacy of ACS and LAE were further evaluated in shelf-life studies wherein hams were surface inoculated with either ca. 3.0 or 7.0 log₁₀ CFU of L. monocytogenes, added to shrink-wrap bags that contained 0, 4, 6, or 8 mL of either a 1:2 solution of ACS or a 5% solution of LAE, vacuum-sealed, and stored at 4 °C for 60 days. For hams inoculated with 7.0 log₁₀ CFU, L. monocytogenes levels decreased by ca.1.2, 1.5, and 2.0 log₁₀ CFU/ham and 5.1, 5.4, and 5.5 log₁₀ CFU/ham within 24 h at 4 °C in samples treated with 4, 6, and 8 mL of a 1:2 solution of ACS and a 5% solution of LAE, respectively, compared to control hams that were not treated with either antimicrobial. Thereafter, pathogen levels remained relatively unchanged (±1.0 log₁₀ CFU/ham ) after 60 days at 4 °C in hams treated with 4, 6, and 8 mL of a 1:2 solution of ACS and increased by ca. 2.0–5.0 log₁₀ CFU/ham in samples treated with 4, 6, and 8 mL of a 5% solution of LAE. For hams inoculated with 3.0 log₁₀ CFU, L. monocytogenes levels decreased by 1.3, 1.9, and 1.8 log₁₀ CFU/ham within 24 h at 4 °C in samples treated with 4, 6, and 8 mL of a 1:2 solution of ACS, respectively, compared to control hams that were not treated. Likewise, levels of the pathogen were reduced to below the limit of detection (i.e., 1.48 log₁₀ CFU/ham) in the presence of 4, 6, and 8 mL of a 5% solution of LAE within 24 h at 4 °C. After 60 days at 4 °C, pathogen levels remained relatively unchanged (±0.3 log₁₀ CFU/ham) in hams treated with 4, 6, and 8 mL of a 1:2 solution of ACS. However, levels of L. monocytogenes increased by ca. 2.0 log₁₀ CFU/ham in samples treated with 4 and 6 mL of a 5% LAE solution within 60 days but remained below the detection limit on samples treated with 8 mL of this antimicrobial. These data confirmed that application via SLIC™ of both ACS and LAE, at the concentrations and volumes used in this study, appreciably reduced levels of L. monocytogenes on the surface of hams within 24 h at 4 °C and showed potential for controlling outgrowth of the pathogen over 60 days of refrigerated storage.     

Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in “cuajada” (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10°C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10°C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.     

Microbiological quality of raw milk used for small-scale artisan cheese production in Vermont: Effect of farm characteristics and practices

This study 1) evaluated the overall milk quality and prevalence of 4 target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk used for small-scale artisan cheesemaking and 2) examined specific farm characteristics and practices and their effect on bacterial and somatic cell counts (SCC). Raw milk samples were collected weekly from 21 artisan cheese operations (6 organic) in the state of Vermont that manufactured raw-milk cheese from cow (12), goat (5), or sheep (4) milk during the summer of 2008. Individual samples were examined for standard plate counts (SPC), coliform counts (CC), and SCC. Samples were also screened for target pathogens both quantitatively and qualitatively by direct plating and PCR. Overall, 86% of samples had SPC <10,000 cfu/mL, with 42% <1,000 cfu/mL. Additionally, 68% of samples tested were within pasteurized milk standards for coliform bacteria under the United States’ Grade A Pasteurized Milk Ordinance at <10 cfu/mL. Log₁₀ SPC and CC did not differ significantly among species. Similarly, method of sample delivery (shipped or picked up), farm type (organic or conventional), and duration of milking (year-round or seasonal) did not have significant effects on farm aggregated mean log₁₀ SPC, CC, or SCC. Strong positive correlations were observed between herd size and mean log₁₀ SPC and between log₁₀ SPC and CC as well as SCC when data from all animal species were combined. Although SCC for cow milk were significantly lower than those for goat and sheep milk, 98, 71, and 92% of cow, sheep, and goat milk samples, respectively, were within the compliance limits of the United States’ Grade A Pasteurized Milk Ordinance for SCC. Fourteen of the 21 farms (67%) were positive for Staph. aureus, detected in 38% of samples at an average level of 20 cfu/mL. Neither L. monocytogenes, E. coli O157:H7, or Salmonella spp. were detected or recovered from any of the 101 samples tested. Our results indicate that the majority of raw milk produced for small-scale artisan cheesemaking was of high microbiological quality with no detectable target pathogens despite the repeat sampling of farms. These data will help to inform risk assessments that evaluate the microbiological safety of artisan and farmstead cheeses, particularly those manufactured from raw milk.     

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log₁₀ cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log₁₀ cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log₁₀ cfu/g; ON) or in the curds (ca. 7.0 log₁₀ cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.     

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in milk by caprylic acid and monocaprylin

Listeria monocytogenes and Escherichia coli O157:H7 are major foodborne pathogens implicated in various outbreaks involving pasteurized or unpasteurized milk, and various dairy products. The objective of this study was to determine the antibacterial effect of caprylic acid (CA, C_8:0) and its monoglyceride, monocaprylin (MC) on L. monocytogenes and E. coli O157:H7 in whole milk. A five-strain mixture of E. coli O157:H7 or L. monocytogenes was inoculated in autoclaved milk (10⁶ CFU/ml) containing 0, 25, or 50 mM of CA or MC. At 37°C, all the treatments, excepting 25 mm CA, reduced the population of both pathogens by approximately 5.0 log CFU/ml in 6 h. At 24 h of storage at 8°C, MC at both levels and CA at 50 mM decreased L. monocytogenes and E. coli O157:H7, respectively by >5.0 log CFU/ml. At 48 h of 4°C storage, populations of L. monocytogenes and E. coli O157:H7 were decreased to below detection level (enrichment negative) by 50 mm of MC and CA, respectively. Results indicate that MC could potentially be used to inhibit L. monocytogenes and E. coli O157:H7 in milk and dairy products, but sensory studies need to be conducted before recommending their use.     

Growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham under refrigerated and temperature abuse conditions

This study examined the growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham at refrigerated and abuse temperatures. A five-strain mixture of L. monocytogenes and a native microflora, consisting of Brochothrix spp., isolated from cooked meat were inoculated alone (monocultured) or co-inoculated (co-cultured) onto cooked ham slices. The growth characteristics, lag phase duration (LPD, h), growth rate (GR, log₁₀ cfu/h), and maximum population density (MPD, log₁₀ cfu/g), of L. monocytogenes and the native microflora in vacuum-packed ham slices stored at 4, 6, 8, 10, and 12 °C for up to 5 weeks were determined. At 4-12 °C, the LPDs of co-cultured L. monocytogenes were not significantly different from those of monocultured L. monocytogenes in ham, indicating the LPDs of L. monocytogenes at 4-12 °C were not influenced by the presence of the native microflora. At 4-8 °C, the GRs of co-cultured L. monocytogenes (0.0114-0.0130 log₁₀ cfu/h) were statistically but marginally lower than those of monocultured L. monocytogenes (0.0132-0.0145 log₁₀ cfu/h), indicating the GRs of L. monocytogenes at 4-8 °C were reduced by the presence of the native microflora. The GRs of L. monocytogenes were reduced by 8-7% with the presence of the native microflora at 4-8 °C, whereas there was less influence of the native microflora on the GRs of L. monocytogenes at 10 and 12 °C. The MPDs of L. monocytogenes at 4-8 °C were also reduced by the presence of the native microflora. Data from this study provide additional information regarding the growth suppression of L. monocytogenes by the native microflora for assessing the survival and growth of L. monocytogenes in ready-to-eat meat products.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Fate of Staphylococcus aureus in cheese treated by ultrahigh pressure homogenization and high hydrostatic pressure

We evaluated the influence of ultrahigh pressure homogenization (UHPH) treatment applied to milk containing Staphylococcus aureus CECT 976 before cheese making, and the benefit of applying a further high hydrostatic pressure (HHP) treatment to cheese. The evolution of Staph. aureus counts during 30 d of storage at 8°C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log₁₀ cfu/mL of Staph. aureus was pressurized using a 2-valve UHPH machine, applying 330 and 30 MPa at the primary and the secondary homogenizing valves, respectively. Milk inlet temperatures (T_in) of 6 and 20°C were assayed. Milk was used to elaborate soft-curd cheeses (UHPH cheese), some of which were additionally submitted to 10-min HHP treatments of 400 MPa at 20°C (UHPH+HHP cheese). Counts of Staph. aureus were measured on d 1 (24 h after manufacture or immediately after HHP treatment) and after 2, 15, and 30 d of ripening at 8°C. Counts of control cheeses not pressure-treated were approximately 8.5 log₁₀ cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T_in of 6°C, counts of Staph. aureus were 5.0 ± 0.3 log₁₀ cfu/g at d 1; they decreased significantly to 2.8 ± 0.2 log₁₀ cfu/g on d 15, and were below the detection limit (1 log₁₀ cfu/g) after 30 d of storage. The use of an additional HHP treatment had a synergistic effect, increasing reductions up to 7.0 ± 0.3 log₁₀ cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6°C T_in samples, viable Staph. aureus cells were still recovered. For samples of the 20°C T_in group, complete inactivation of Staph. aureus was reached after 15 d of storage for both UHPH and UHPH+HHP cheese. Staphylococcal enterotoxins were found in controls but not in UHPH or UHPH+HHP treated samples. This study shows a new approach for significantly improving cheese safety by means of using UHPH or its combination with HHP.     

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ～4 log₁₀ spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ～4 log₁₀ cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.     

Predictive model for the behavior of Listeria monocytogenes Scott A in Shrikhand,prepared with a biopreservative,pediocin K7

The limited facilities of hygiene and sanitation during production of traditional dairy foods combined with lack of refrigerated storage throughout the cold chain pose a serious challenge to their microbial safety. The application of biopreservatives like pediocin is promising in controlling the growth of L. monocytogenes in food systems. In the present study, polynomial equations were derived using multivariate analysis, for predicting the survival of L. monocytogenes Scott A, in Shrikhand, a traditional sweetened lactic-fermented milk product with varying parameters of initial Listeria inoculum (3·3, 4·3, 5·3, 6·3 & 7·3 log₁₀cfu g⁻¹), storage temperature (4, 10, 16, 22 & 28°C), storage period (24–120 h) and pediocin K7 levels (11, 14, 17, 20 & 23%). The model predictions indicated the effectiveness of higher pediocin levels, higher storage temperatures and extended periods of storage in inhibiting the growth of L. monocytogenes Scott A in Shrikhand. Further, the comparison of experimental and predicted values of the survival of L. monocytogenes Scott A in Shrikhand, using bias factor, established the adequacy of the derived model to describe the behavioral pattern of L. monocytogenes Scott A in Shrikhand.     

Identification of dairy farm management practices associated with the presence of psychrotolerant sporeformers in bulk tank milk

Some strains of sporeforming bacteria (e.g., Bacillus spp. and Paenibacillus spp.) can survive pasteurization and subsequently grow at refrigeration temperatures, causing pasteurized fluid milk spoilage. To identify farm management practices associated with different levels of sporeformers in raw milk, a bulk tank sample was obtained from and a management and herd health questionnaire was administered to 99 New York State dairy farms. Milk samples were spore pasteurized [80°C (176°F) for 12 min] and subsequently analyzed for most-probable number and for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) at 7, 14, and 21 d after SP. Management practices were analyzed for association with sporeformer counts and bulk tank somatic cell counts. Sixty-two farms had high sporeformer growth (≥3 log cfu/mL at any day after SP), with an average sporeformer count of 5.20 ± 1.41 mean log₁₀ cfu/mL at 21 d after SP. Thirty-seven farms had low sporeformer numbers (<3 log cfu/mL for all days after SP), with an average sporeformer count of 0.75 ± 0.94 mean log₁₀ cfu/mL at 21 d after SP. Farms with >25% of cows with dirty udders in the milking parlor were 3.15 times more likely to be in the high category than farms with ≤10% of milking cows with dirty udders. Farms with <200 cows were 3.61 times more likely to be in the high category than farms with ≥200 cows. Management practices significantly associated with increased bulk tank somatic cell count were a lack of use of the California mastitis test at freshening and >25% of cows with dirty udders observed in the milking parlor. Changes in management practices associated with cow cleanliness may directly ensure longer shelf life and higher quality of pasteurized fluid milk.     

Characterization of cottage cheese using Weissella cibaria D30: Physicochemical,antioxidant, and antilisterial properties

This study aimed to evaluate the potential of Weissella cibaria D30 as an adjunct culture in cottage cheese, including an assessment of antioxidant, antilisterial, and compositional parameters. Cottage cheese samples were manufactured using a commercial starter culture and probiotic strains Lactobacillus rhamnosus GG (GG) or W. cibaria D30 (W) and without probiotic (control). Samples were stored at 4 ± 1°C for 28 d. Bacterial cell counts (log cfu/g) of control, GG, and W samples were counted at 0, 7, 14, 21, and 28 d. Counts of W. cibaria D30 in the W samples remained at 6.85 log cfu/g after 28 d. Total solids, fat, protein, ash, and pH were measured and no significant differences were observed in compositional parameters or pH after 28 d of storage in all cheeses except those inoculated to Listeria monocytogenes. To measure the antilisterial effect, Listeria monocytogenes was inoculated into the cottage cheese samples and bacterial cell counts were obtained at 0, 6, 12, 24, 48, 72, 96, 120, and 144 h. Listeria monocytogenes counts were less than the analytical limit of detection (<10 cfu/g) in the inoculated GG and W samples, whereas the counts of L. monocytogenes in the inoculated control sample remained at 3.0 log cfu/g after 144 h. We used the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activity assays to assess antioxidant activity: GG and W samples exhibited significant increases in antioxidant activity compared with the control sample. These results indicate that W. cibaria D30 has potential as an adjunct culture in the dairy industry.