Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.     

Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods

Many methods and media currently exist for the detection and enumeration of Listeria monocytogenes. However, the suitability of any specific method or media is influenced by the purpose of the analysis and the type of food being analyzed. Food which are likely to contain high populations of contaminating microorganisms require highly selective media for enumeration. Moreover, media which contain indicator systems are also helpful in distinguishing L. monocytogenes colonies. In contrast, less selective media may be adequate for less contaminated foods.     

A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheese

A cold enrichment method and a modified FDA procedure were compared for the isolation of Listeria monocytogenes from raw chickens and soft cheese. L. monocytogenes was isolated from a total of 23 of 222 cheese and 70 of 160 chicken samples by either one or both methods. Neither method alone yielded all isolates from the two food types. Only 12 cheese and 13 chicken samples were shown to be positive by both methods, although the serotypes isolated were not always identical. On some occasions one method yielded L. monocytogenes while the other produced a different Listeria sp. Reasons for differences in the performance of the two procedures and various points of technical interest are discussed.     

High-pressure homogenization of raw and pasteurized milk modifies the yield, composition, and texture of queso fresco cheese

High-pressure homogenization (HPH) of milk was studied as an alternative processing operation in the manufacturing of queso fresco cheese. Raw and pasteurized (65°C for 30 min) milks were subjected to HPH at 0, 100, 200, and 300 MPa and then used to manufacture queso fresco. The cheeses were evaluated for yield, moisture content, titratable acidity, nitrogen content, whey protein content, yield force, yield strain, and tactile texture by instrumental or trained panel analyses. The combination of HPH and thermal processing of milk resulted in cheeses with increased yield and moisture content. The net amount of protein transferred to the cheese per kilogram of milk remained constant for all treatments except raw milk processed at 300 MPa. The highest cheese yield, moisture content, and crumbliness were obtained for thermally processed milk subjected to HPH at 300 MPa. The principal component analysis of all measured variables showed that the variables yield, moisture content, and crumbliness were strongly correlated to each other and negatively correlated to the variables yield strain, protein content (wet basis), and sensory cohesiveness. It is suggested that the combination of thermal processing and HPH promotes thermally induced denaturation of whey protein, together with homogenization-induced dissociation of casein micelles. The combined effect results in queso fresco containing a thin casein-whey matrix that is able to better retain sweet whey. These results indicate that HPH has a strong potential for the manufacture of queso fresco with excellent yield and textural properties.     

Survival of Listeria monocytogenes in commercial cheese brines.

The survival of Listeria monocytogenes was determined in commercial cheese brines collected from cheese factories in Wisconsin and northern Illinois. Survival of L. monocytogenes inoculated into commercial cheese brines ranged from < 7 d to over 259 d. Survival did not correlate with pH, salt content, nitrogen content, mineral content, or inherent microbial populations but was negatively associated with addition of sodium hypochlorite at the dairy plant. The L. monocytogenes generally survived longer in brines held at 4 degrees C than at 12 degrees C. Sodium hypochlorite or hydrogen peroxide inactivated L. monocytogenes when added to commercial brines in the lab at 10 to 100 ppm or 0.001% to 0.02%, respectively. Addition of 1% potassium sorbate or 1% sodium benzoate also decreased survival of L. monocytogenes. Laboratory filtration of commercial brines had a negative effect on survival in one of three brines tested. The L. monocytogenes did not grow during incubation in any of the commercial brine samples tested.     

Processing of soft Hispanic cheese ("queso fresco") using thermo-sonicated milk: a study of physicochemical characteristics and storage life

Queso fresco is a handmade cheese consumed in Latin America and some regions of the United States. However, deficient milk processing has affected its microbial quality and it has an extremely short shelf life and low yield. The objective of this work was to process queso fresco using thermo-sonicated milk; physicochemical parameters were evaluated, including microbial quality during storage (4 °C). An ultrasonic processor (UP400S, 400 W, 24 kHz, 120 μm) was used to sonicate raw milk. Seven milk systems (500 mL each) were evaluated: 1 untreated, and 6 treated at 63 °C/30 min; 63 °C/10 min + sonication; 63 °C/30 min + sonication; 72 °C/15 s; 72 °C/15 s + sonication; and 72 °C/1 min + sonication. A conventional cheese-making process was followed for all systems. The effect of sonication on milk was quite noticeable. Curdling times were reduced considerably, cheese yield (20.6%) was almost doubled, and luminosity of cheese was increased (L*). Textural properties and microstructure images matched very well. Queso fresco processed at 63 °C/120 μm/30 min had the best quality. After storage for 23 d at 4 °C mesophilic count was just 4 log; psychrophilic count, 3.5 log; and enterobacteria count, 3 log. The pH and color remained almost constant and a minor degree of syneresis was observed at end of storage. Due to microstructural rearrangement of the milk components such as fat globules and casein micelles, cheese yield was doubled compared to the traditional handmade product. Shelf life was extended considerably and the product had higher quality.     

Comparison of swab transport media for recovery of Listeria monocytogenes from environmental samples

Environmental monitoring is recognized as an important strategy for controlling Listeria monocytogenes in food processing facilities. Samples are taken by swabbing environmental surfaces, and the swabs are immersed in a medium for transport to the laboratory. In this study, buffered peptone water (BPW), Dey-Engley neutralizing broth (DE), neutralizing buffer (NB), Letheen broth (LE), and newly described MCC buffer (MCC) were evaluated as transport media for recovery of sanitizer-stressed L. monocytogenes from inoculated swabs. After storage at 4°C, the media performed similarly, but at 25°C relative recovery efficiency from the inoculated sponges was DE > LE > BPW > MCC > NB. Recoveries from stainless steel surfaces followed similar trends. MCC, DE, and NB were compared for L. monocytogenes recovery in the presence of Escherichia coli, Enterococcus faecalis, Lactobacillus plantarum, Pseudomonas fluorescens, and Listeria innocua. After 4°C storage, all population levels changed little; after 25°C storage, DE allowed the best growth of L. monocytogenes regardless of other species present. MCC, DE, and NB performed similarly for recovery of L. monocytogenes from an artificial milk biofilm and for recovery of Listeria spp. from swabs obtained from a meat processing facility. Transport medium formulation, time and temperature of swab storage, and coexistence of other species affect recovery of sanitizer-stressed L. monocytogenes from environmental swabs. The study confirms the need to maintain 4°C storage conditions during swab transport.     

Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation

A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out. Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk. The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L. monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars. LMBA proved to be a very useful tool and was able to detect L. monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively. The overall incidence of listeria on equipment was 18.8% (6.3% L. monocytogenes), in the environment was 54.7% (40.6% L. monocytogenes) and in raw milk 44.4% (22.2% L. monocytogenes). On one occasion, L. welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product. The main environmental sources of L. monocytogenes were considered to be a floor drain and stainless steel steps.     

食品中单核细胞增生性李斯特菌的快速分离鉴定

单核细胞增生性李斯特菌(Listeria monocytogenes,简称单增李斯特菌)是一种重要的食源性致病菌,能引起人畜共患李斯杆菌病。本文采用两种不同的增菌分离方法从散装牛奶和猪肉中共分离得到8株单增李斯特菌。通过革兰氏染色、生化鉴定、PCR扩增hlyA基因、16SrDNA测序、血清分型等一系列实验对可疑菌株进行分析鉴定。综合实验结果和其他单增李斯特菌分离标准,探讨开发了一种快速分离鉴定单增李斯特菌的方法,该方法可在4～5d内完成单增李斯特菌的分离检测过程。     

Prevalence and characterization of Listeria monocytogenes isolated from soft cheese

A survey was made in 1995–1996 for Listeria spp. in 63 soft cheeses, made from raw ewe's milk using traditional methods, in the Province of Beira Baixa (Portugal). Listeria spp. were isolated from 47 (75%) of the cheeses, L.monocytogenes was isolated from 29 (46%), and L.innocua but not L.monocytogenes from 18 (29%). Of 24 isolates of L.monocytogenes that were serotyped, 20 were serotype 4b, three were serotype 1/2b and one was serotype 1/2a. Phage typing of isolates of L.monocytogenes and L.innocua showed that in some cases a particular phage type was associated with cheese from a particular source. Twenty four strains of L.monocytogenes tested were able to grow at 30°C in culture medium adjusted with HCl to a pH in the range from 4.4 to 6.0 within 3 days; in the pH range 4.4–6.8 a representative strain grew most rapidly at pH 6.8. The pH range in the cheeses during maturation was between about 5.2–6.4. Whether L.monocytogenes could multiply in the cheeses would depend on factors such as concentration of organic acids and of salt, and storage temperature.     

Comparison of sampling procedures for recovery of Listeria monocytogenes from stainless steel food contact surfaces

A number of techniques exist for microbiological sampling of food processing environments in food industries. In the present study the efficacies of nine sampling procedures for the recovery of Listeria monocytogenes from food contact surfaces, including a new sampling device consisting of a miniroller, were evaluated and compared. A stainless steel table was inoculated with L. monocytogenes strain 935 (serovar 4b, human origin) and L. monocytogenes strain 437/07 (serovar 1/2b, food origin), at 10(5) CFU/100 cm(2). L. monocytogenes strain 935 was best recovered with the minirollers (recovery of up to 6.27%), while poor recoveries (<0.30%) were obtained with the towel (one-ply composite tissue), alginate swab, metallic swab, and Petrifilm methods. In the case of L. monocytogenes strain 437/07 the replicate organism detection and counting (RODAC) ALOA contact plates yielded the best recoveries (4.15%), followed by the minirollers (up to 1.52%). Overall, recovery percentages with the minirollers were higher with stomacher homogenization than with Vibromatic agitation. The recovery percentages obtained for the Listeria strain of human origin were higher than those obtained with the food strain for all sampling procedures except Petrifilm and RODAC ALOA. With the miniroller device coated with wool fiber, the recovery of L. monocytogenes can be improved from 2 to 17 times over recoveries obtained with the sponge and cotton swab. This is the first report of a miniroller device for microbiological sampling in the available literature. The novel sampling procedure is convenient to apply on surfaces, is cost-effective, and results in better recovery of L. monocytogenes than do the conventional methods.     

High incidence of Listeria monocytogenes in European red smear cheese

The incidence of Listeria and Listeria monocytogenes in European red smear cheese was determined in order to assess whether the lack of recent outbreaks of listeriosis associated with cheese is due to improved hygenic conditions in the dairies. Out of European red-smear cheese samples of various types, 15.8% contained organisms of the genus Listeria, 6.4% of the samples were contaminated with L. monocytogenes, 10.6% with L. innocua, and 1.2% with L. seeligeri. Six cheese samples contained two or more Listeria species, including at least one L. monocytogenes isolate. The incidences of L. monocytogenes in cheeses from various countries were: Italy 17.4%, Germany 9.2%, Austria 10%, and France 3.3%. Listeria were found most frequently in soft and semi-soft cheese. Eight samples contained more than 100 L. monocytogenes cfu/cm2 cheese surface, 2 samples had counts above 10(4) cfu/cm2 cheese surface. Surprisingly, a higher incidence of L. monocytogenes was observed in cheeses made from pasteurized milk (8.0%) than in cheeses manufactured from raw milk (4.8%). Phage-typing of isolated Listeria strains clearly confirmed that (i) contaminations within dairy plants were persistent over a period of several weeks to months and (ii) that cross-contamination within the dairy plant is and important factor. Comparison of our data with past surveys seems to indicate that contamination of red smear soft cheese with L. monocytogenes has not decreased sufficiently over the past 15 years. It is therefore strongly recommended that these products are monitored carefully by cheese-making companies.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.     

Evaluation of Listeria innocua as a suitable indicator for replacing Listeria monocytogenes during ripening of Camembert cheese

The suitability of Listeria innocua for use as an indicator for replacing Listeria monocytogenes during the cheese-making and ripening of Camembert cheese was evaluated. Pasteurized whole milk inoculated with either L. innocua or L. monocytogenes was used to make Camembert cheese, which were ripened in three stages. All cheese was ripened in three stages: room temperature (∼20 °C) and relative humidity of 60% for 36 h; 12 °C and relative humidity of 93% for 2 weeks; and 7 °C and relative humidity of 85% for 3 weeks. Results showed that population values of L. innocua and L. monocytogenes on day 1 were 7.16 and 6.11 log₁₀ CFU/g, respectively, which declined to 6.54 and 5.45 log₁₀ CFU/g, respectively, during subsequent 20 days. Thereafter, L. innocua and L. monocytogenes populations increased to 7.38 and 6.06 log₁₀ CFU/g on day 35 of ripening, respectively. During ripening, surface and interior of cheeses were analysed for populations of L. innocua and L. monocytogenes , respectively. The data were collected on day 1, 5, 10, 15, 20, 25, 30, and 35 of ripening. Generally, the growth of L. innocua and L. monocytogenes is faster in surface than in centre. Top centre, bottom centre and bottom surface locations had similar population values during ripening. There were no significant differences ( P  > 0.05) between batch and section of cheese. The ripening time and locations had significant effect ( P  < 0.05) on the survival and growth of L. innocua and L. monocytogenes . The trends of survival and growth of L. innocua and L. monocytogenes were similar. These results indicated that L. innocua can be considered as an indicator for L. monocytogenes during ripening of Camembert cheese.     

Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese

Parmesan cheese was made from a mixture of pasteurized whole and skim milk that was inoculated to contain ca. 10(4) to 10(5) cells of Listeria monocytogenes/ml. Curd was cooked at 51 degrees C (124 degrees F) for ca. 45 min. During cheese making, maximum numbers of L. monocytogenes appeared just before cooking; at this point, the increase over initial numbers was a .61 to 1.0 order of magnitude. During cooking of curd, the average decrease in numbers of L. monocytogenes was a .22 order of magnitude. During cheese ripening, numbers of L. monocytogenes decreased almost linearly and faster than reported for other hard cheeses. Listeria monocytogenes strain California died faster than did strain V7. Listeria monocytogenes were not detected in cheese after 2 to 16 wk of ripening, depending on the strain of the pathogen and the lot of cheese. Parmesan cheese made in this study was not a favorable medium for survival of L. monocytogenes.     

Modeling survival of Listeria monocytogenes in the traditional Greek soft cheese Katiki

In the present work, survival of Listeria monocytogenes in the traditional Greek soft, spreadable cheese Katiki was studied throughout the shelf life of the product. Samples of finished cheese were inoculated with a cocktail of five L. monocytogenes strains (ca. 6 log CFU g(-1)) and stored at 5, 10, 15, and 20 degrees C. Acid-stress adaptation or cross-protection to the same stress was also investigated by inoculation of acid-adapted cells in the product. The results showed that pathogen survival was biphasic. Various mathematical equations (Geeraerd, Cerf, Albert-Mafart, Whiting, Zwietering, and Baranyi models) were fitted to the experimental data. A thorough statistical analysis was performed to choose the best model. The Geeraerd model was finally selected, and the results revealed no acid tolerance acquisition (no significant differences, P > 0.05, in the survival rates of the non-acid-adapted and acid-adapted cells). Secondary modeling (second-order polynomial with a(0) = 0.8453, a(1) = -0.0743, and a(2) = 0.0059) of the survival rate (of sensitive population), and other parameters that were similar at all temperatures (fraction of initial population in the major population = 99.98%, survival rate of resistant population = 0.10 day(-1), and initial population = 6.29 log CFU g(-1)), showed that survival of the pathogen was temperature dependent with bacterial cells surviving for a longer period of time at lower temperatures. Finally, the developed predictive model was successfully validated at two independent temperatures (12 and 17 degrees C). This study underlines the usefulness of predictive modeling as a tool for realistic estimation and control of L. monocytogenes risk in food products. Such data are also useful when conducting risk assessment studies.     

Listeria monocytogenes survival during production and storage of water buffalo Mozzarella cheese

The ability of Listeria monocytogenes to survive during the manufacture of water buffalo Mozzarella and to grow during its shelf life was evaluated. A wild‐type and a reference strain were used to contaminate raw milk. The viable count of the reference strain ATCC 9525 dropped after the stretching process, and in the cheese, it fell to below 100 cfu/g. When the wild‐type strain was used, however, stretching did not appear to have any effect on the pathogen. The artificially contaminated cheeses were stored, for eleven days, at 4, 20 and 30 °C. Pathogen populations increased at 20 (≈2.60 log cfu/g) and 30 °C (≈1.95 log cfu/g).     

Characterization of Listeria monocytogenes isolates from 50 small-scale Austrian cheese factories

One hundred eighty-one small-scale cheese factories (annual production < 100,000 kg) were tested for the presence of Listeria monocytogenes in cheese and smear samples from 1997 to 2000. In total, 2615 samples were drawn. Fifty (27.6%) of 181 enterprises yielded L. monocytogenes. From 14 of the cheese-making facilities, we obtained more than four L. monocytogenes isolates. A total of 182 mostly cheese- and smear-borne L. monocytogenes strains were characterized by serotyping and pulsed-field gel electrophoresis. In 12 of 14 cheese factories, over half of the L. monocytogenes isolates were genetically indistinguishable by pulsetype. On average, genetically indistinguishable isolates were recovered for 11.9 months. Regarding serotypes, 27.3% of the isolates were of serovar 4b. Inadequate personal hygiene could explain the high prevalence of serovar 4b isolates in small-scale cheesemaking facilities. Forty-two percent of the serovar 4b isolates recovered from epidemiologically unlinked facilities (in comparison to 40 and 29% of the 1/2a and 1/2b isolates, respectively) were genetically indistinguishable from at least one other isolate. Indistinguishable serovar 1/2a and 1/2b isolates belonged to five and six different pulsetypes, respectively, whereas serovar 4b isolates belonged to only two pulsetypes. This finding suggested a wide distribution of genetically homologous serovar 4b isolates among the facilities tested in our study.     

Efficacy of direct plating media for recovering Listeria monocytogenes from foods

Studies were done to evaluate 14 direct plating media for their suitability to recover Listeria monocytogenes strain Scott A from pasteurized whole milk, chocolate ice cream mix, Brie cheese and raw cabbage. Healthy cells were inoculated into foods to achieve viable populations of 10(2), 10(4), or 10(6) cells/ml(g). Bind's Acriflavine Agar, Trypaflavine Nalidixic Acid Serum Agar, Listeria Transport Enrichment Agar, Doyle and Schoeni Selective Enrichment Agar (DSSEA) and Modified DSSEA were not suitable for recovering L. monocytogenes from milk and Brie cheese and were therefore not evaluated as direct plating media for recovering the organism from ice cream mix and cabbage. McBride Listeria Agar (MLA), Gum Base Nalidixic Acid Tryptone Soya Agar (GBNTSA), Modified Despierres Agar (MDA) and Modified MLA (MMLA) performed best for recovering all inoculum populations from milk and ice cream mix. Enumeration of L. monocytogenes on several test media was complicated by the growth of large numbers of background microflora present in cabbage and Brie cheese, especially at the lowest test inoculum (10(2)/g). Generally, complete recovery of L. monocytogenes from Brie cheese and cabbage was attained on media when the inoculum population was greater than or equal to 10(4) cells/g. For Brie cheese, MLA, MDA, MMLA and Dominguez Rodriguez Isolation Agar were superior for recovering L. monocytogenes, while GBNTSA, DLEA, MDA and MMLA were best for recovering the organism from cabbage. Results of this experiment indicate that direct-plating procedures, without prior enrichment, can successfully be utilized for recovering L. monocytogenes from foods such as pasteurized milk and ice cream mix which contain low populations of background microflora. However, recovery of L. monocytogenes from foods such as raw cabbage and Brie cheese, which contain high populations of other microorganisms, was not satisfactory using the direct-plating procedures evaluated in this investigation.